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Autolab Twingle-Springle SPR user manual 4.4.0-6

1. 98 Polymer 153 154 160 161 173 Pump control 100 PVDF 24 Regeneration Recommended solutions 22 Save data Loop Save 125 Measurement Save 125 Sampler Save 125 Save procedure 91 semi automatic sequence 134 sensor 165 Sensor 2 146 153 154 Definition 146 sensor surfaces
2. 165 9 Maintenance 165 9 1 Index 165 9 2 Introduction 166 9 3 Storage of SPR disk and sensor chip 166 9 4 Optics 166 9 5 Routine inspections 167 9 6 Replacing syringe and piston 167 Maintenance 166 9 2 9 2 9 2 9 2 Introduction Introduction Introduction Introduction In this chapter the regular maintenance procedure is described During maintenance wear gloves use clean lens paper and ultra pure cleaning solutions If the instrument is contaminated with biohazards like bacteria or viruses disconnect all devices of the instrument that are exposed to the biohazard and clean them with the right cleaning agents for that biohazard If any doubts exist about the cleaning procedure please contact the local distributor 9 3 9 3 9 3 9 3 Storage of SPR disk Storage of SPR d
3. 111 5 8 2 Automation 111 5 8 3 Scope mode 111 5 8 4 Scanner 111 5 8 5 Customize 111 5 9 Communications menu 113 5 10 User menu optional 114 5 11 Window menu 115 5 12 Help menu 115 5 13 Event Log 116 Data Acquisition software 88 The screen contains top bottom the Title bar 1 Menu bar 2 Tool bar 3 Binding Curve Plot 4 SPR 1 and 2 Plot 5 System Monitor bar 6 Event Log 7 and Status Bar 8 This chapter will provide in depth coverage of the TWINGLE SPRINGLE software 5 2 5 2 5 2 5 2 Overview of the functions Overview of the functions Overview of the functions Overview of the functions The Menus The Menus
4. 26 2 Software Installation 26 2 1 Index 26 2 2 Introduction 27 2 3 Installation of the Autolab SPR software 27 2 4 Autolab SPR software setup on the hard disk after installation 34 2 4 1 Folder structure 34 2 4 2 Files in C Autolab SPR 34 2 4 3 Examples of data files in the subfolder C Autolab SPR Data 35 2 4 4 Softcopy manuals in the subdirectory MANUALS 36 2 4 5 Examples of KE models in the subdirectory MODELS 36 2 4 6 Examples of KE PROJECT files in the subdirectory Data 36 2 4 7 Autolab TWINGLE sequence files in subdirectory SEQUENCES 37 2 4 8 Autolab SPRINGLE sequence files in subdirectory SEQUENCES 38 Chapter 3 39 3
5. 109 5 7 13 Lift Calibration 109 5 7 14 System Parameters 110 5 8 Options menu 111 5 8 1 Sequencer 111 5 8 2 Automation 111 5 8 3 Scope mode 111 5 8 4 Scanner 111 5 8 5 Customize 111 5 9 Communications menu 113 5 10 User menu optional 114 5 11 Window menu 115 5 12 Help menu 115 5 13 Event Log 116 Chapter 6
6. 18 Electromagnetic wave 147 150 Equilibrium dialysis 146 ESPR optical configuration 153 Event Log 115 Expand one level 92 File menu 88 Graph Properties 97 hardware requirements 12 Help menu 89 90 91 Hydrodynamic parameters of the cuvette 158 Immobilization 153 154 Immunoassay Affinity chromatography 146 Equilibrium dialysis 146 Spectrosc
7. 104 Figure 5 14 Two TWINGLE DA screens showing two ways to get quick access to customize and to sequences 104 Figure 5 15 Example of a Customize window to change a linked sequence shown in the Menu_Twingle Inject 105 Figure 5 16 An example of a TWINGLE inject sequence 106 Figure 5 17 Two TWINGLE DA screens showing two ways to be able to get quick access to customize and to sequences 106 Figure 5 18 Example of the Customize window to change a linked sequence shown in the Menu_Twingle Wash 107 Figure 5 19 Direct access to start and stop the drain pump 107 Figure 5 20 Update SPR Recording 108 Figure 5 21 Right mouse click on SPR plot 1 or on SPR plot 2 108 Figure 5 22 Lift calibration window 109 Figure 5 23 System settings 110 Tab sheet pump2 for Twingle only 110 Figure 5 24 Options menu 111 Figure 5 25
8. 123 6 4 1 The sequence editor menu and toolbar 123 6 5 Set up of sequence files 124 6 5 1 Include sequence 124 6 5 2 Safety lines 125 6 5 3 Wait command 125 6 5 4 Save data 126 6 5 4 1 Loop Save xxxxxx00 126 6 5 4 2 Measurement Save filename 126 6 5 4 3 Automation Save see automation window 126 6 5 5 Commands with variables 127 6 5 6 Commands for Semi Automatic sequences 127 6 5 6 1 The main automatic kinetic sequence with all of its included sequences 128 6 5 6 2 The interaction plot sequence 131 6 5 6 3 The inject sequence 133 6 5 6 4 The stabilization seque
9. 118 6 Sequencer 118 6 1 Index 118 6 2 Introduction 119 6 3 Sequence editor window 119 6 4 Software Sequence editor 123 6 4 1 The sequence editor menu and toolbar 123 6 5 Set up of sequence files 124 6 5 1 Include sequence 124 6 5 2 Safety lines 125 6 5 3 Wait command 125 6 5 4 Save data 126 6 5 5 Commands with variables 127 6 5 6 Commands for Semi Automatic sequences 127 6 5 7 The semi automatic sequences 135 6 5 8 Writing a sequen
10. 125 Axis zoom 97 Biosensor Definition 146 Specifically SPR 146 Biotinylated macromolecules 161 BNC connectors 17 Buffer Recommended solutions 21 Carbodiimide coupling reaction 161 Chemical resistance 21 Clear measurement plot 92 Collapse one level 92 Connectors 18 Curve a full kinetic plot seq 136 Curve properties 97 Cuvett
11. 160 Sensor chip Biacore 158 Sequence editor 92 110 115 118 132 Setup procedure software 26 Show all links 92 Spectroscopic techniques 146 SPR Advantages 146 Kretschmann configuration 148 Definition 147 SPR minimum dip 110 169 173 174 176 177 Dip 147 Ideal dip 174 No SPR dip 174 SPR curve channel 1
12. 129 Figure 6 8 A Twingle list of interaction experiment sequences 131 Figure 6 8 B Springle list of interaction experiment sequences 131 Figure 6 9 The inject sequence 133 Figure 6 10 List of stabilization sequences 134 Figure 6 11 The imobilization sequences 134 Figure 6 12 Example of the sequence message alert box 136 Figure 6 13 An example of a sequence where the message alert has been used 136 Figure 7 1 The autosampler control window selection 143 Figure 7 2 The automation window with three tab sheets to set up the experiment 143 Figure 7 3 The EDIT button gives access to these settings 144 Figure 7 4 The automation window with the incubation time TAB sheet to set up the experiment 145 Figure 7 5 The automation window with the volumes time TAB sheet to set up the sample volumes of the experiment 14
13. 148 Label less detection 146 Ligand 153 154 155 Link parameters 92 Macromolecular interactions 160 Mass transport 159 Measurement settings 94 100 Menu bar 87 122 Modified gold layer 154 New procedure 91 NHS 154 155 156 Pause measurement 92 Personal precautions 5 Plasma 148 149 Plot menu
14. 146 8 1 Index 146 8 2 Introduction 147 8 3 Surface Plasmon Resonance 148 8 4 AUTOLAB Twingle configuration 153 8 4 1 Optics of the Twingle system 154 8 4 2 Sensor 154 8 4 3 Cuvette 159 8 4 4 Liquid handling 160 8 5 SPR methods 161 8 5 1 Introduction 161 8 5 2 Methods using the SPR disk 161 8 6 References 162 Chapter 8 147 8 2 8 2 8 2 8 2 Introduction Introduction Introduction Introduction A biosensor is a device that incorporates a biological recognition sensing element in clo
15. 24 Temperature plot 98 Thiol 154 156 161 Tools menu 89 Total internal reflection 148 150 Unlink parameters 92 View menu 88 Zoom 97 07 2009 Kanaalweg 29 G 3526 KM Utrecht The Netherlands
16. 30 Figure 2 9 Installation window 9 31 Figure 2 9 Installation window 9 31 Figure 2 10 Installation window 10 32 Figure 2 11 Installation window 11 32 Figure 2 12 Installation window 12 32 Figure 2 14 Installation window 14 33 Figure 2 13 Installation window 13 33 Fig 2 15 The desktop icons shown after the installation of the SPR software 34 Figure 2 16 Folder structure 34 Figure 2 17 Content of C Autolab SPR folder 35 Figure 2 18 Content of C Autolab SPR Data folder 35 Figure 2 19 Manuals installed during the installation of the software 36 Figure 2 20 Examples of kinetic evaluation models installed with the software
17. 34 2 4 2 Files in C Autolab SPR 34 2 4 3 Examples of data files in the subfolder C Autolab SPR Data 35 2 4 4 Softcopy manuals in the subdirectory MANUALS 36 2 4 5 Examples of KE models in the subdirectory MODELS 36 2 4 6 Examples of KE PROJECT files in the subdirectory Data 36 2 4 7 Autolab TWINGLE sequence files in subdirectory SEQUENCES 37 2 4 8 Autolab SPRINGLE sequence files in subdirectory SEQUENCES 38 Chapter 2 27 2 2 2 2 2 2 2 2 Introduction Introduction Introduction Introduction The Autolab SPR installation CD contains the Autolab ESPRIT TWINGLE and SPRINGLE instruments software During the software installation choose the right setup software for the corresponding Autolab SPR instrument The software can be used on the Windows 2000 XP or Vista platforms Insert the CD into the computer and the installation will start automatically see Figure 2 1 2 3 2 3 2 3 2 3 Installation of the Autolab SPR software Installation of the Autolab SPR software Installation of the Autolab SPR software Installation of the Autolab SPR software Start the computer and wait until the Windows start up is finished Close all open
18. 36 Figure 2 21 Examples of kinetic evaluation projects installed with the software 36 Figure 2 22 Sequences for the Autolab TWINGLE instrument 37 Figure 2 23 Sequences for the Autolab SPRINGLE instrument 38 Figure 3 1 Flow chart of the experimental setup 41 Figure 3 2 6 Well Microtiter culture plate 42 Figure 3 3 the back panel of the TWINGLE 43 Figure 3 4 The draining tube from the drain peristaltic pump 43 Figure 3 5 Menu TWINGLE to open Manual Control window 44 Chapter 11 181 Figure 3 6 Open the Lift calibration window 45 Figure 3 7 The lift calibration window 45 Figure 3 8 a The lift calibration procedure 46 Fig 3 8 b The final steps of lift calibration 47 Figure 3 9 A drop of immersion oil on top of the hemi cylinder 47 Figure 3 10 Assembly of a disk 48 Figure 3
19. 65 4 Getting started Autolab SPRINGLE 65 4 1 Index 65 4 2 Introduction 66 4 3 Startup of the Autolab SPRINGLE instrument 68 4 4 Preparation of solutions 68 4 4 1 Chemicals 68 4 4 2 Reagents 69 4 5 Liquid Handling set up Autolab SPRINGLE 70 4 6 Initialization of the SPRINGLE instrument 71 4 6 1 Autolab SPRINGLE lift position calibration 71 4 7 Installation of the gold sensor disk 75 4 7 1 Preparation of a self assembled monolayer of 11MUA on the gold surface 75 4 7 2 Assembling the sensor disk on the hemi cylinder 75 4 7 3 Installation of the cuvette
20. 4 Analysis view 92 Analyte 147 Definition 146 Mass transport 159 Angle of incidence 147 148 150 Angle scan 153 AUTOLAB ESPRIT 3 Autolab SPR 33 35 36 37 68 168 Autolab SPR folder 33 Autolab SPR software 26 Automatic aspirate dispense mixing needle 158 Autosampler 110 168 precaution 5 Sampler Save
21. 75 Figure 4 9 Assembly of a disk 75 Figure 4 10 Different positions on the gold disk 76 Figure 4 11 Installed SPR gold disk 77 Figure 4 12 An overview of the cuvette holder 77 Figure 4 13 The positioning pin of a cuvette 77 Figure 4 14 Check for leakage outside of channel 1 78 Figure 4 15 Two ways to activate the Sequencer 79 Figure 4 16 SPRINGLE The sequence Initialization of Instrument SEQ 79 Figures 182 Figure 4 17 SPR dip 80 Figure 4 18 The optical path cover 80 Figure 4 19 Adjustment of the baseline angle before immobilization 81 Figure 4 20 Stabilization cleaning of the gold disk surface 82 Figure 4 21 The sequence editor 83 Figure 4 22 Sequence Editor Window 84 Figure 4 23 A typical example of an association experiment
22. 101 5 7 2 Lift position in the software 103 5 7 3 Inject 104 5 7 4 Wash 106 5 7 5 Drain 107 5 7 6 Place Event Marker 108 5 7 7 Update SPR recording 108 5 7 8 Start measurement 109 5 7 9 Pause measurement 109 5 7 10 Stop measurement 109 5 7 11 Set Baseline 109 5 7 12 Adjust to zero 109 5 7 13 Lift Calibration 109 5 7 14 System Parameters 110 5 8 Options menu 111 5 8 1 Sequencer
23. 99 SPR curve channel 2 99 SPR dip shift 174 SPR dip shifted left 176 SPR dip shifted up 177 Unsymmetrical SPR dip 177 Update SPR recording 107 SPR1 BNC connector 17 SPR2 BCN connector 17 Start measurement 91 Surface Plasmon Resonance 143 147 152 syringe pumps 168 Syringe pumps 101 Teflon FEP and PFA 24 Tefzel ETFE
24. Fill tubing with buffer Fill tubing with buffer Fill tubing with buffer Fill tubing with buffer Exchange Exchange Exchange Exchange th th th the buffer solution e buffer solution e buffer solution e buffer solution Why should there be liquid in the tubing Why should there be liquid in the tubing Why should there be liquid in the tubing Why should there be liquid in the tubing The solution in the tubing is used for washing the pipette tip Secondly fluid is not compressible like air which results in highly accurate sample volumes and flow rates The The The The general ru general ru general ru general running buffer nning buffer nning buffer nning buffer In particular applications with non specific interaction buffer solutions and samples should contain 0 005 v v Tween 20 to minimize non specific adsorption to the sensor disk Be aware of samples that are detergent sensitive Chemicals Water demineralised demi pro analysis Merck 1 16754 9010 HEPES free acid MW 238 3 Fluka 54457 Preparation 10 mM HEPES 150 mM NaCl 3 mM EDTA 0 005 Tween 20 In 90 ml dissolve 238 mg HEPES free acid in 90 ml demiwater Set pH to 7 with few drops of 10 M NaOH Add 865 mg NaCl 87 6 mg EDTA 50 l Tween 20 10 Adjust volume to 100 ml In a starting situation the tubing can be empty filled with air or filled with a solution Run the sequence Initialization of instrument seq to fill the tubing
25. AS AS AS AS a full a full a full a full kinet kinet kinet kinetic plot SEQ sequence ic plot SEQ sequence ic plot SEQ sequence ic plot SEQ sequence Figure 4 23 Figure 4 23 Figure 4 23 Figure 4 23 A typical example of an association experiment A typical example of an association experiment A typical example of an association experiment A typical example of an association experiment Chapter 4 85 4 11 4 11 4 11 4 11 Cleaning of the Cleaning of the Cleaning of the Cleaning of the SPRINGLE SPRINGLE SPRINGLE SPRINGLE instrument instrument instrument instrument This is a guiding principle for cleaning all part s in the system which are in contact with the solutions used in the experiments Replace the buffer flask solution step by step with cleaning solution after the specific sequence is finished Cleaning Solution 1 0 5 w v SDS 1 w v Triton in water Total cleaning time about 10 min Cleaning Solution 2 0 5 w v SDS Total cleaning time about 10 min Cleaning Solution 3 50 mM Glycine NaOH pH 9 5 Total cleaning time about 10 min Cleaning solution 4 6 M Urea Total cleaning time about 10 min Cleaning solution 5 1 acetic acid Total cleaning time about 20 min Cleaning solution 6 0 2 M NaHCO3 Total cleaning time about 10 min Cleaning solution 7 Hydrochloric acid 0 1 M HCl Total cleaning time about 20 min Cleaning solution 8
26. Installation of the gold Installation of the gold Installation of the gold Installation of the gold sensor sensor sensor sensor disk disk disk disk 4 7 1 4 7 1 4 7 1 4 7 1 Preparation of a self assembled monolayer of 11MUA on the Preparation of a self assembled monolayer of 11MUA on the Preparation of a self assembled monolayer of 11MUA on the Preparation of a self assembled monolayer of 11MUA on the gold surface gold surface gold surface gold surface Incubate a bare gold disk in a well of a 6 wells culture disk in a solution of 11mg 11 MUA in 50ml ethanol or isopropanol To get a reproducible quality thiol layer filter the solution prior to the incubation and perform the incubation overnight Wash the disk three times with ethanol or isopropanol to remove the excess thiol To remove the alcohol rinse three times with dematerialized water Blow the disk dry with compressed air or nitrogen gas The thiol covered gold disk can be stored dry up to 2 months in the original container 4 7 2 4 7 2 4 7 2 4 7 2 Assembling the sens Assembling the sens Assembling the sens Assembling the sensor disk on or disk on or disk on or disk on the the the the hemi hemi hemi hemi cylinder cylinder cylinder cylinder Place a small drop of immersion oil on the outer edge of the hemi cylinder and gently slide the modified gold disk coated with 11 MUA from the start to the end of the hemi cylinder
27. The Automation control window The Automation control window The Automation control window The Automation control window The automation window enables changing parameters incubation times and sample volume outside of the sequence editor Specific sequence commands are linked to a typical box that can be filled out in the Automation window The sequencer command Automation Load Parameter Set 1 4 will look for the parameters settings filled out in the parameters TAB sheet figure 6 2 With the button EDIT a new window System parameters pops up Within this window every item can be adjusted The settings will be used for the experiment and loaded into the sequence whenever the command line Automation Load Parameters Set 1 4 is written When the desired parameters settings are set the incubation times can be adjusted Figure Figure Figure Figure 7777 3333 The The The The EDIT button gives access to the EDIT button gives access to the EDIT button gives access to the EDIT button gives access to these se se se settings settings settings settings Chapter 7 145 Select the Times Tab sheet to address the incubation times of the experiment Eight different times can be edited The box to edit depends on the sequence to be executed So the typical WAIT xxxxxx s 6 5 6 used in the sequence decides which box is necessary to fill out Tables 6 1 and 6 2 show some
28. 60 3 7 The Autolab Twingle data 63 3 8 Cleaning of the TWINGLE instrument 63 40 Getting Started Autolab TWINGLE 3 2 3 2 3 2 3 2 Introduction Introduction Introduction Introduction This getting started document will take you step by step through the initialization of the TWINGLE SPRINGLE instrument and subsequently through an interaction experiment where the antibody anti Insuline will be interacting with the immobilized protein Insulin Throughout this procedure most features of the software will be illustrated It is presumed that the hardware and software have been installed before the cuvette and hemi cylinder are disassembled and all tubing is empty A 60 min warm up time of the Autolab TWINGLE should be taken into account Detection of binding events between Insulin and anti Insulin First the modified gold layer on the sensor disk is coated with mercapto undecanoic acid 11 MUA After being assembled into the instrument the modified surface is stabilized with the immobilization coupling buffer In the immobilization procedure Immobilization the acid group of this molecule is activated by incubation with EDC and NHS Subsequently the Insulin is immobilized onto the modified gold layer The Insulin layer will thereafter be stabilized with association buffer In the inter
29. 77 4 7 4 Check for leakage of the measurement channel 78 4 7 5 Fill tubing with buffer Exchange the buffer solution 78 4 8 The immobilization 80 4 8 1 Sample preparation 80 4 8 2 Set angle position of the sodium acetate buffer B 4 80 4 8 3 Stabilize rehydrate the dry 11 MUA disk 81 4 8 4 Start the immobilization procedure 82 4 9 The interaction 83 4 9 1 Sample preparation 83 4 9 2 Stabilize the surface 83 4 9 3 Start the association procedure 83 4 10 The SPRINGLE Data 84 4 11 Cleaning of the SPRINGLE instrument 85 66 Getting Started Autolab SPRINGLE 4 2 4 2 4 2 4 2 Introduction Introduction Introduction Introduction If this chapter is unclear please read chapter 3 which has a different setup with the same
30. Reage Reage Reage Reagents nts nts nts B 1 Preparation of 1 mM 11 Mercaptoundecanoic acid 11 MUA dissolve 11 mg 11 Mercaptoundecanoic acid Mw 218 36 in 50ml alcohol like ethanol ethanol or propanol A 7 B 2 Preparation of PBS buffer 10mM Dilute PBS buffer in demineralised demi water Pipette 20 ml of concentrated PBS A 2 in a 300 ml flask and add 180 ml of demiwater It is recommended to filter the solution through a 0 22 m filter Degass under vacuum B 3 Preparation of regeneration buffer 0 1M and 1 M hydrochloric acid HCl A 4 Dilute 0 5 ml of the 30 9 46M HCl in 47 ml demi water to get 0 1M HCl Dilute 5 ml of the 30 9 46M HCl in 42 5 ml demi water to get 1 0 M HCl B 4 Preparation of coupling buffer 10 mM Acetate buffer A 11 pH 4 5 Dissolve 68 4 mg NaAc Mw 136 08 in 45 ml demi water Adjust the pH to 4 5 with acetic acid A 12 Adjust volume to 50 ml with demi water The pH of the coupling buffer depends on the pI of the ligand protein The general rule for the pH of coupling buffer is pHbuffer pIligand 0 5 B 4a Preparation of association buffer 10mM HEPES 150mM NaCl 3mM EDTA 0 005 tween 20 In 90 ml dissolve 238mg HEPES free acid in 90 ml demiwater Set pH to 7 with few drops of 10M NaOH Add 865 mg NaCl 87 6 mg EDTA 50 l Tween 20 10 Adjust volume to 100 ml B 5 Preparation of 1mg ml BSA to immobilize on the sensor disk Dissolve10 mg BSA A 5 in 1 ml 10mM Acetate b
31. 16 Figure 1 2 Back panel of the TWINGLE 17 Figure 1 3 A The Electrochemical cuvette and the normal SPR cuvette 20 Figure 1 3 B The Electrochemical cuvette 20 Figure 1 4 Cuvette tubing and fitting 24 Figure 1 5 Peristaltic pump 24 Figure 1 6 Peristaltic pump tubing 24 Figure 1 7 Syringe pump 24 Figure 2 1 Start of the Setup procedure 27 Figure 2 2 Installation window 2 28 Figure 2 3 Installation window 3 Welcome 28 Figure 2 4 Installation window 4 for Autolab TWINGLE 28 Figure 2 5 Installation window 5 29 Figure 2 6 Installation window 6 Twingle 29 Figure 2 7 Installation window 7 30 Figure 2 8 Installation window 8
32. 102 Aspirate Dispense Aspirate Dispense Aspirate Dispense Aspirate Dispense section to aspirate or dispense sample in or out of the cuvette the sample can be positioned in a microtiter plate or a vial which is held manually under the needles Valve options Valve options Valve options Valve options control a three way valve valve to needle connects channel from pump to cuvette Valve to buffer connects channel from buffer flask to pump Flow control Flow control Flow control Flow control enables to select the speed of the pump in l s Corresponds to the aspirating dispensing and mixing speed Mix section Mix section Mix section Mix section for mixing the solution in the cuvette Fill in the mixing volume in l and check the mix box to start mixing This option should only be used when the needles are inside the cuvette and the mix volume should not exceed the cuvette volume Synchronized Pumps Channels 1 amp 2 Twingle only This section is meant for synchronized mixing of pump1 and pump2 Set volume and Flow check the mix box and both pumps will start mixing at the same time with the same flow and with the same volume Measurement box The buttons to start to pause or to stop the measurement Drain is a peristaltic pump Select the pump speed and check the on box to activate the pump the solution is drained from the cuvette Figure Figure Figure Figure 5555 12 12 12 12 A A A A Manual
33. 84 Figure 5 1 Data Acquisition software 88 Figure 5 2 The Data Acquisition menu bar 88 Figure 5 3 The Data Acquisition tool bar 92 Figure 5 4 File menu 94 Figure 5 5 View menu 96 Figure 5 6 The different tab sheets to adjust the curve or graph properties 97 Figure 5 7 The options of adjusting the curve or graph properties 98 Figure 5 9 Right mouse click in DA window 99 Figure 5 8 Plot menu 99 Figure 5 10 SPR curves of channel 1 and SPR curves of channel 2 100 Figure 5 11 TWINGLE menu items 101 Figure 5 12 A Manual control window of the Twingle 102 Figure 5 12 B Manual control window of the SPRINGLE 103 Figure 5 13 Example of two TWINGLE DA screens with Lift Positions choices
34. InInInIndex dex dex dex Chapter 1 11 1 Hardware Installation 11 1 1 Index 11 1 2 Introduction 12 1 3 Computer requirements 12 1 4 Autolab TWINGLE SPRINGLE Hardware 12 1 5 Specifications 13 1 6 Hardware Installation 17 1 7 SPR and ESPR setup 18 1 8 Chemical Resistance 21 1 9 Materials 24 12 Hardware Installation 1 2 1 2 1 2 1 2 Introduction Introduction Introduction Introduction In this chapter the installation of the hardware is described All necessary cables and accessories are supplied with the TWINGLE instrument 1 3 1 3 1 3
35. Customize General tab page 112 Figure 5 26 Customize User directories tab page 112 Figure 5 27 Customize Email configuration tab page 113 Figure 5 28 Communications 113 Figure 5 29 User 114 Figure 5 30 The Administration Control Panel 114 Figure 5 31 The Administration Control Panel 115 Chapter 11 183 Figure 5 32 An example of a kinetic experiment 117 Figure 5 33 A zoom in on the event log 117 Figure 6 1 Two ways to activate the Sequencer 119 Figure 6 3 The menu bar and tool bar buttons 123 Figure 6 4 The sequence menu 123 Figure 6 5 Example of a sequence with include sequences 125 Figure 6 6 A Twingle List of kinetic experiment sequences 128 Figure 6 6 A Springle list of kinetic experiment sequences 129 Figure 6 7 The difference in sample volume
36. Initialization of Instrument SEQ Place all inlet tubings into the buffer flask Run the sequence to clean with solution 1 and 8 C Total clean of the system every two month s Use the sequence Initialization of Instrument SEQ Place all inlet tubing into the buffer flask Run the sequence using every solution 1 3 7 8 9 step by step D Total clean of the system every half year Use the sequence Initialization of Instrument SEQ Place all inlet tubing into the buffer flask Run the sequence using every solution 2 4 5 6 8 step by step Use the routine Use the routine Use the routine Use the routine I I I Initialization of Instrument SEQ nitialization of Instrument SEQ nitialization of Instrument SEQ nitialization of Instrument SEQ to prepare the system to prepare the system to prepare the system to prepare the system before use before use before use before use Running buffer 1 PBS pH 7 4 Or Running buffer 2 10 mM HEPES 150 mM NaCl 3 4 mM EDTA 0 005 Tween P20 Chapter 4 65 Chapter 4 Chapter 4 Chapter 4 Chapter 4 4 4 4 4 Getting started Autolab SPRINGLE Getting started Autolab SPRINGLE Getting started Autolab SPRINGLE Getting started Autolab SPRINGLE 4 1 4 1 4 1 4 1 Index Index Index Index Chapter 4
37. Intensity1 Output of the amplified photodiode signal of channel 1 output impedance is lt 1 the signal varies between 0V total absorption and 10V total reflection Intensity2 Twingle only Output of the amplified photodiode signal of channel 2 output impedance is lt 1 the signal varies between 0V total absorption and 10V total reflection SPR1 This signal is an analogue representation of the SPR angle measured on channel 1 output impedance is lt 1 and the signal varies between 0V and 10V an SPR angle of 0 degrees equals 5V on this output SPR2 Twingle only This signal is an analogue representation of the SPR angle measured on channel 2 output impedance is lt 1 and the signal varies between 0V and 10V an SPR angle of 0 degrees equals 5V on this output This BNC connector is used to connect to the 18 Hardware Installation Potentiostat Galvanostat to record SPR data into the NOVA GPES software Sub D connectors Sub D connectors Sub D connectors Sub D connectors Monitor This output is used for service purposes only It connects to a standard VGA screen to monitor activity and error messages on the internal computer in the TWINGLE SPRINGLE Service For service use only Therm Not in use COM RS232 communication links to the computer Please use the supplied RS232C cable other cables may not operate properly Note Some SERIAL to USB converters
38. 124 Open Sequence Opens a sequence folder determined in the customize user directories tab Figure 5 33 p 122 Save Sequence Saves the sequence Save Sequence As Opens a save as window to save the sequence as SEQ file with a user created filename and directory Print Prints the complete command structure of the opened sequence Delete Delete highlighted command line Numbering Shows or hides the numbering of the command lines Expand Shows all sequence commands lines Collapse Shows only the main sequence command lines Run Sequence Executes the current sequence Close Closes the sequence editor window 6 5 6 5 6 5 6 5 Set Set Set Set up of up of up of up of sequence files sequence files sequence files sequence files 6 5 1 6 5 1 6 5 1 6 5 1 Include Include Include Include sequence sequence sequence sequence All of the basic liquid handling commands are stored in so called include sequence files This reduces the number of lines in a main sequence and gives a better overview on the executed sequence An include sequence executed as a main sequence may cause problems Check the sequence with a buffer to verify if it can be used as a main sequence A sequence should be considered as the folder structure on the hard drive of the PC An include sequence can be described as the function folder in the directory The
39. Immobilization BSA on MUA Baseline Regeneration Association With BSA Dissociation Restore Baseline 68 Getting Started Autolab SPRINGLE 4 3 4 3 4 3 4 3 Startup of the Autolab SP Startup of the Autolab SP Startup of the Autolab SP Startup of the Autolab SPRINGLE instrument RINGLE instrument RINGLE instrument RINGLE instrument Install the power supply cable and connect the com port of the instrument to the computer with the RS232 connector cable Switch on the Autolab SPRINGLE instrument Use the main switch on the back panel Figure 4 2 and the power button situated at the right top position of the front panel The LED in the power button lights up after a few seconds Start the Autolab SPR Data Acquisition software Wait until the instrument has finished initiating the syringe pump 4 4 4 4 4 4 4 4 Preparation of solu Preparation of solu Preparation of solu Preparation of solutions tions tions tions What do I use as a general running buffer What do I use as a general running buffer What do I use as a general running buffer What do I use as a general running buffer A 10mM Phosphate buffered saline PBS solution is recommended as a general wash running buffer In this particular application with proteins buffer solutions and samples should contain 0 005 v v Tween 20 to minimize non specific adsorption to the sensor disk Be aware of samples that are
40. Initialization of the instrument 44 3 4 5 Autolab lift position calibration 44 3 4 6 Installation of the gold sensor disk 47 3 4 6 1 Assembling the sensor disk on the hemi cylinder 47 3 4 6 2 Installation of the cuvette 49 3 4 7 Check for leakage between the two measurement channels 49 3 4 8 Fill tubing with buffer Exchange the buffer solution 51 3 5 D day The immobilization 52 3 5 1 Sample preparation 52 3 5 2 Set angle position of the sodium acetate buffer 54 3 5 3 Stabilize rehydrate the dry 11 MUA disk 55 3 5 4 Start the immobilization procedure 56 3 6 The interaction 59 3 6 1 Sample preparation 59 3 6 2 Stabilize the surface 60 3 6 3 Start the association procedure
41. 6 Move lift to top 7 Enter distance of 38 mm and fine tune with 1 mm steps 8 Set new middle position 9 Move lift to top 10 Close See Figure 3 8 a and b steps 1 to 10 1 2 and 3 and 4 5 6 7 8 Figure 3 8 Figure 3 8 Figure 3 8 Figure 3 8 a a a a The lift calibration procedure The lift calibration procedure The lift calibration procedure The lift calibration procedure Chapter 3 47 3 4 6 3 4 6 3 4 6 3 4 6 Installation of the gold Installation of the gold Installation of the gold Installation of the gold sensor sensor sensor sensor disk disk disk disk 3 4 6 1 3 4 6 1 3 4 6 1 3 4 6 1 Assembling the sensor disk on Assembling the sensor disk on Assembling the sensor disk on Assembling the sensor disk on the the the the hemi hemi hemi hemi cylinder cylinder cylinder cylinder Place a small drop of immersion oil on the outer edge of the hemi cylinder and gently slide the modified gold disk coated with 11 MUA from the start to the end of the hemi cylinder Be sure that the gold layer is facing up A rim of gold can be seen at the gold coated side of the disk when slightly tilting it Make sure that no air bubbles are introduced between the gold disk and the hemi cylinder Check this by looking through the hemi cylinder Recommendations o Do not manipulate the gold sensor disk with bare hands We recomm
42. Connect serial cable between Com port of computer and Com port of Twingle Check communications menu for port settings The minimum of the SPR dip is bad Worse than 10 of the maximum intensity Coatings are too thick or not homogeneous The thin gold layer is not clean or damaged Wafer or sensor chip is out of specs Try a clean wafer or sensor chip Change the coating procedures to higher spinning rates and lower concentrations of polymer in solvent The minimum of the Particles will adhere Filter the sample solution Try Troubleshooting 170 Problem Possible causes Suggested solution SPR dip changes dramatically during a measurement to the surface Inhomogenities in the coating are created during an experiment another coating procedure Signal is too noisy Bad SPR dip Interval time is too small Optics are not clean Clean the surface with SDS in aqua distilled water followed by 96 alcohol and wash with buffer containing SDS Check SPR dip Use an alternate SPR wafer Increase the interval time Clean the hemi cylinder The SPR check has some intensity variations The hemi cylinder is not clean By turning the spindle the intensity variations remain at the same angle clean either the hemi cylinder or optics For a thorough cleaning of the optics and lining out of optics please contact the local distributor Strange behaviour around an angle of zero mdegree when water is r
43. Figure 4 8 Figure 4 8 Figure 4 8 Figure 4 8 A drop of immersion oil on top of the hemi A drop of immersion oil on top of the hemi A drop of immersion oil on top of the hemi A drop of immersion oil on top of the hemi cylinder cylinder cylinder cylinder Figure 4 9 Figure 4 9 Figure 4 9 Figure 4 9 Assembly of a disk Assembly of a disk Assembly of a disk Assembly of a disk 1 2 3 1 2 3 1 2 3 1 2 3 4 5 4 5 4 5 4 5 6 6 6 6 76 Getting Started Autolab SPRINGLE Be sure that the gold layer is facing up A rim of gold can be seen at the gold coated side of the disk when slightly tilting it Make sure that no air bubbles are introduced between the gold disk and the hemi cylinder Check this by looking through the hemi cylinder Recommendations o Do not manipulate the gold sensor disk with bare hands We recommend wearing gloves when handling the disk o Touch only the frosted sides of the hemi cylinder to prevent scratches on the accurately polished round sides of the hemi cylinder o Manipulate the gold sensor disk with a fine tweezers especially when you take it out of the plastic case o The gold sensor disk in the plastic case is oriented with the gold surface facing down Cleaning the hemi Cleaning the hemi Cleaning the hemi Cleaning the hemi cylinder and slider cylinder and slider cylinder and slider cylinder and slider The slider and the hemi cylinder need to be clean
44. In the toolbar find the button lift positions Open the Manual Control window in the SPRINGLE Menu or with the button in the toolbar Figure 4 3 Figure 4 3 Figure 4 3 Figure 4 3 The draining tube from the drain peristaltic pump The draining tube from the drain peristaltic pump The draining tube from the drain peristaltic pump The draining tube from the drain peristaltic pump is is is is inserted into the waste bottle green tubing inserted into the waste bottle green tubing inserted into the waste bottle green tubing inserted into the waste bottle green tubing in reality in reality in reality in reality The sy The sy The sy The syringe tube ringe tube ringe tube ringe tube isisisis inserted into the buffer flask inserted into the buffer flask inserted into the buffer flask inserted into the buffer flask 72 Getting Started Autolab SPRINGLE The lift position has to be calibrated every time you change the pipette tip Figure 4 4 Figure 4 4 Figure 4 4 Figure 4 4 Menu SPRINGLE to open Manual Control window Menu SPRINGLE to open Manual Control window Menu SPRINGLE to open Manual Control window Menu SPRINGLE to open Manual Control window Figure 4 5 Figure 4 5 Figure 4 5 Figure 4 5 Open the Lift calibration window Open the Lift calibration window Open the Lift calibration window Open the Lift calibration window Cha
45. up the experiment using EDIT up the experiment using EDIT up the experiment using EDIT Chapter 3 59 The Coupling Buffer is being used for all washing steps in the immobilization experiment Therefore the buffer flask will be filled with this solution 3 6 3 6 3 6 3 6 The interaction The interaction The interaction The interaction 3 6 1 3 6 1 3 6 1 3 6 1 SSSSample preparation ample preparation ample preparation ample preparation For the interaction procedure prepare the following samples and solutions Chemicals HEPES free acid MW 238 3 Fluka 54457 EDTA Ethylenediaminetetraacetic acid solution MW 292 24 Fluka 03690 pH 8 0 0 5 M in H2O NaCl 58 44 g Mol Sigma Aldrich S7653 Tween 20 10 in water Fluka 93774 Sodium dodecyl sulfate SDS 10 solution Sigma 71736 Preparation of running buffer 10mM HEPES 150 mM NaCl 3 mM EDTA 0 005 Tween 20 In 90 ml dissolve 238 mg HEPES free acid in 90 ml demiwater Set pH to 7 with few drops of 10 M NaOH Add 865 mg NaCl 87 6 mg EDTA 50 l Tween 20 10 Adjust volume to 100 ml Preparation of Regeneration solution 10 mM Acetate buffer 0 1 SDS Preparation of anti Insulin dilutions S stock 1 500 Pipette 2 l of the concentrated stock solution anti insulin in a vial of 1 5 ml dilute this with 1000 l HEPES buffer Mix by pipetting several times up and down This is a 1 500 dilution 1 Dilution 1 1 8000 Pipette 60 l f
46. update Add Event Item line 91 update SPR line 93 incubation time line 94 end the measurement line 96 stop mixing line 97 SPECIALS lines 68 78 82 86 the syringe finish its aspirate or dispense actions before going to the next step in the sequence experiment Chapter 6 139 Line 98 107 The include sequence has been written to be sure that the needle will have no sample left from previous actions Lines 108 to 123 were discussed in figure 5 13 Line 108 The needle pipette tip is accessible to put a vial underneath Line 115 117 Stop the experiment to put the vial underneath the needle pipette tip Line 116 118 Aspirate the sample from the vial Line 119 Stops the experiment to give time to remove the vial from under the needle pipette tip Line 120 122 to drain away any solution on top of the gold before the next sample will be dispensed Lines 127 134 The fixed set of commands to perform the measurement SPECIALS lines 101 107 126 The syringe finish its aspirate or dispense actions before going to the next step in the sequence experiment Lines 150 151 flush 450 l buffer to wash away the analyte Line 154 155 dispense the buffer sample to measure the dissociation Lines 156 157 159 160 161 to put the syringe in the middle position Lines 163 164 170 171 aspirate 50 l air This to prevent contamination of the buffer in the tubi
47. 1 3 Computer requirements Computer requirements Computer requirements Computer requirements The following minimum computer hardware specifications are required An IBM compatible computer 1 GHz Pentium 4 processor preferably from Intel sVGA graphics card with minimal 800 x 600 pixels resolution 512 Mb RAM memory 3 Gb free HDU space Microsoft Windows 2000 or XP Microsoft Vista requires 2 Gb RAM memory remark folder position installation direct in C not in program files folder One free RS232 serial communication port One additional free RS232 com port is required for the optional waterbath control One USB port has to be available if an Autolab with GPES NOVA software has to be installed on the same computer 1 4 1 4 1 4 1 4 Autolab Autolab Autolab Autolab TWINGLE TWINGLE TWINGLE TWINGLE SPRINGLE SPRINGLE SPRINGLE SPRINGLE Hardware Hardware Hardware Hardware Power Supply 100 240V 10 auto select Power Line frequency 47 63 Hz Power consumption 120 VA max Fuses 2 800 mA slow blow Operating Environment 10 C to 40 C ambient temperature lt 80 relative humidity Storage environment 10 C to 40 C ambient temperature Dimensions W x H x D 330mm x 400mm x 360mm Weight 24 kg Warm up time 60 minutes Remote interface RS232 Wave length light 670 nm Chapter 1 13 1 5 1 5 1 5 1 5 Specification Specification Sp
48. B 4 80 4 8 3 Stabilize rehydrate the dry 11 MUA disk 81 4 8 4 Start the immobilization procedure 82 4 9 The interaction 83 4 9 1 Sample preparation 83 4 9 2 Stabilize the surface 83 4 9 3 Start the association procedure 83 4 10 The SPRINGLE Data 84 4 11 Cleaning of the SPRINGLE instrument 85 Chapter 5 87 5 Data Acquisition software 87 5 1 Index 87 5 2 Overview of the functions 88 5 3 File menu 94 5 4 Edit menu 96 5 5 View
49. Figure 2 22 shows typical Autolab TWINGLE sequences to perform double channel SPR experiments The left picture has all stand alone sequences for specific experiments The sequences in the right picture are components to create sequences as shown in the left picture and are called Include Sequences More details about reading adjusting and creating these sequences can be found in Chapter 6 Figure 2 2 Figure 2 2 Figure 2 2 Figure 2 22 2 2 2 S S S Sequences for the Autolab equences for the Autolab equences for the Autolab equences for the Autolab TWINGLE instrument TWINGLE instrument TWINGLE instrument TWINGLE instrument 38 Software Installation 2 4 8 2 4 8 2 4 8 2 4 8 Autolab Autolab Autolab Autolab SPRINGLE sequence SPRINGLE sequence SPRINGLE sequence SPRINGLE sequence files in subdirectory files in subdirectory files in subdirectory files in subdirectory SEQUE SEQUE SEQUE SEQUENNNNCES CES CES CES Figure 2 23 shows typical Autolab SPRINGLE sequences to perform double channel SPR experiments The left picture has all stand alone sequences for specific experiments The sequences in the right picture are components to create sequences as shown in the left picture and are called Include Sequences More details about reading adjusting and creating these sequences can be found in Chapter 6 Figure 2 2 Figure 2 2 Figure 2 2 Figure 2 23 3 3 3 S S S Se
50. Immobilization_SA 50ul mixure EDC NHS for activation step seq starts at command line 91 3 Ligand coupling Read include sequence Tw44 Immobilization_SA Ligand coupling Step seq starts at command line 171 4 Ethanol Amine Deactivation Figure 3 Figure 3 Figure 3 Figure 3 2 2 2 23333 The sequence editor The sequence editor The sequence editor The sequence editor showing the sequence showing the sequence showing the sequence showing the sequence Tw44 Tw44 Tw44 Tw44 IIIImmobilization mmobilization mmobilization mmobilization_SA 50ul samples _SA 50ul samples _SA 50ul samples _SA 50ul samples SEQ SEQ SEQ SEQ Stabilization of the surface is necessary for all kinds of modified gold Stabilization of the surface is necessary for all kinds of modified gold Stabilization of the surface is necessary for all kinds of modified gold Stabilization of the surface is necessary for all kinds of modified gold surfaces surfaces surfaces surfaces Commercially available Dextran surfaces also need extensive cleaning In general change the baseline solution one after the other like 0 1M NaOH and 0 1M HCl alternatively Eventually every solution should show the same SPR angle every time it is dispensed on the surface Chapter 3 57 Read include sequence Tw44 Immobilization_SA Ethanol Amine Deactivation step seq starts at command line 251 5 Regen
51. Saves data Input file name in the Sampler Window Figure 6 6 A Figure 6 6 A Figure 6 6 A Figure 6 6 A Twingle Twingle Twingle Twingle List of kinetic experiment sequences List of kinetic experiment sequences List of kinetic experiment sequences List of kinetic experiment sequences Chapter 6 129 For the SPRINGLE the Sp44 Curve SA_a full kinetic plot_SA 50 l sample seq sequence is the main sequence from which the other sequence is generated For the explanation of the automation the Twingle has been used as an example The Springle has the same setup What and where are the changes to make different sequences is shown below Commands in lines 108 192 shows the Lift up position which indicates that in the next folder include sequence the aspiration of a sample lines 116 118 needs to come from a vial underneath the pipette tip and lines 124 125 are dispensing the sample into the cuvette It is therefore very easy to adjust the sequence to a new volume Even better the right sequence has a link with the automation control window where the volume can be filled out see fig 6 7 Figure 6 6 A Figure 6 6 A Figure 6 6 A Figure 6 6 A Springle l Springle l Springle l Springle list of kinetic experiment sequences ist of kinetic experiment sequences ist of kinetic experiment sequences ist of kinetic experiment sequences Figure Figure Figure Figure 6666 7 7 7 7
52. There is an irreproducible SPR angle shift The needle tubing is not mounted properly on the needles The tubing has a leakage Improve the tube connections on the syringe pump and on the needles Back plate and ground floor in are wet Leakage of pump valve or piston seal syringe Connection of tubing is not fitted properly Tubing is damage Change pump valve piston seal or syringe mounting fittings Check and clean tube connections Change defective tubing Unstable baseline Temperature is not stable air in liquids unstable flow or mixing leakage at tube connections piston wear out View temperature and check stability De gas if air in the liquid is the cause Check the connections on leakage Check the quality of the piston of the pump No response after injection Clogging of the needle or too loosely fitted tubing on the needle Check connections and change the direction of flow Clean the needle The signal has a regular noise with time scale of the interval time The pump frequency is in phase with the interval time Increase the frequency of the pump speed vs pump volume e g twice the interval time Troubleshooting 172 Problem Possible causes Suggested solutions There is a big spike after injection of the sample The bulk refractive indices of the injection and the starting buffer solution are not the same Try to dilute the sample solution with the st
53. Water Total cleaning time about 10 min Cleaning solution 9 70 Ethanol Total cleaning time about 10 min A Clean before shutting down for a weekend Use the sequence Initialization of Instrument SEQ Put the inlet buffer flask tubings out off the flask Run the sequence to empty all tubings It s also OK to replace the solution in the tubings with Solution 7 water B Clean needles cuvette and connected tubings once every two weeks Use the sequence Initialization of Instrument SEQ Place all inlet tubings into the buffer flask Run the sequence to clean with solution 1 and 8 C Total clean of the system every two month s 86 Getting Started Autolab SPRINGLE Use the sequence Initialization of Instrument SEQ Place all inlet tubings into the buffer flask Run the sequence using every solution 1 3 7 8 9 step by step D Total clean of the system every half year Use the sequence Initialization of Instrument SEQ Place all inlet tubings into the buffer flask Run the sequence using every solution 2 4 5 6 8 step by step Use the rout Use the rout Use the rout Use the routine ine ine ine Initialization of Instrument SEQ Initialization of Instrument SEQ Initialization of Instrument SEQ Initialization of Instrument SEQ to prepare the system to prepare the system to prepare the system to prepare the
54. button and chose the position on the data curve This button will mark an event for example the association and dissociation steps in the measurement window and describe this event in the event log window Events indicated by a marker will be recorded in the event log 5 7 7 5 7 7 5 7 7 5 7 7 Update SPR recording Update SPR recording Update SPR recording Update SPR recording Records a SPR dip during the measurement As shown in figure 4 20 there are three different possibilities to have an update SPR recorded A SPR update is necessary to check if there is a gold disk quality problem or an air bubble present on the gold disk during an experiment All updates are automatically stored in the event log The plots are visualized in the SPR 1 plot and SPR 2 Twingle only plot Figure 5 1 Every update can be shown or hidden with a right mouse click In Figure 5 21 all updates are selected but they can also be de selected individually The maximum number is 20 updates Figure Figure Figure Figure 5555 2 2 2 20000 Update SPR Recording Update SPR Recording Update SPR Recording Update SPR Recording Figure Figure Figure Figure 5555 2 2 2 21111 Right Right Right Right mouse click on SPR mouse click on SPR mouse click on SPR mouse click on SPR plot 1 or on SPR plot 2 plot 1 or on SPR plot 2 plot 1 or on SPR plot 2 plot 1 or on SPR plot 2 Chapter 5 109 5 7
55. detergent sensitive 4 4 1 4 4 1 4 4 1 4 4 1 Chemicals Chemicals Chemicals Chemicals A 1 11 Mercapto undecanoic acid 11 MUA Aldrich 450561 A 2 10x PBS buffer Phosphate Buffered Saline 0 1M 9 NaCl Fluka 79383 A 3 Water demineralised demi pro analysis Merck 1 16754 9010 A 4 Hydrochloric Acid HCl 30 9 46M Fluka 17077 A 5 BSA Albumin from Bovine Serum Fluka 05477 A 6 Anti BSA Rabbit anti Cow Albumin DAKO Z0229 or Clone BSA 33 Sigma B2901 Figure 4 2 Figure 4 2 Figure 4 2 Figure 4 2 the back panel of the Autolab SPRINGLE the back panel of the Autolab SPRINGLE the back panel of the Autolab SPRINGLE the back panel of the Autolab SPRINGLE TTTThe black power switch he black power switch he black power switch he black power switch is situated on the left is situated on the left is situated on the left is situated on the left Chapter 4 69 A 7 Alcohol pro analysis 99 5 propanol or ethanol or methanol A 8 NHS N Hydroxy Succinimide Fluka 56480 A 9 EDC EDC HCl Dimethylaminopropyl N Ethylcarbodiimide N 3 hydrochloride Fluka 03449 A 10 Ethanolamine Fluka 02400 A 11 Sodium Acetate trihydrate NaAc 3H2O Fluka 71190 A 12 Acetic Acid Sigma A6283 A 13 Tween 20 10 in water Fluka 93774 A 14 HEPES free acid Fluka 54457 MW 238 3 g Mol A 15 EDTA ethylenediamine tetraacetic acid MW 292 25 g Mol A 16 NaCl 58 44 g Mol 4 4 2 4 4 2 4 4 2 4 4 2
56. include sequence also has many commands that can be read when clicking on the plus box like in widows explorer A list of include sequences can be found under C Autolab SPR Sequencer Include Chapter 6 125 6 5 2 6 5 2 6 5 2 6 5 2 Safety lines Safety lines Safety lines Safety lines To prevent flooding of the cuvette and to prevent stock solution contamination some safety measures are necessary After the Pump1 Mix Stop Pump2 Mix Stop Twingle only or Synchronized Mix Stop Twingle only command the syringe pump will automatically go to its initialization position The initial plunger position is half way the syringe which is the 250 l point Another situation at which the pumps will go to their initial position is after finishing a sequence To prevent mistakes use the Synchronized Mix Stop command as a start of an include sequence From that point on it is clear what the limits are in aspirate and dispense volume Pump1 flow 227 3 l s Pump2 flow 227 3 l s Twingle only Pump1 Valve To buffer Pump2 Valve To buffer Twingle only Synchronized Mix Stop Twingle only Pump Mix Stop Springle only The changes in the syringe position will not affect the buffer and or sample level in the needle and or cuvette 6 5 3 6 5 3 6 5 3 6 5 3 Wait command Wait command Wait command Wait command A Wait command used as an incubation time for a measurement is posit
57. the angular shift is measured for a non coated gold sensor surface with a resolution of approximately 0 05 millidegrees m corresponding to a refractive index resolution of approximately 1 10 5 For a coated gold sensor surface the angular shift is measured with a resolution of approximately 0 1 millidegrees m 8 4 2 8 4 2 8 4 2 8 4 2 Sensor Sensor Sensor Sensor Desirable features of the sensor surface for the study of macromolecular interactions are A rapid simple and reproducible immobilization technique Stability and retained biological activity of the immobilized biomolecules Low non specific interaction Facilities for regeneration after use Flexibility in design of coatings for polymer macromolecule interactions Possibilities to detect particles as viruses bacteria and cells These features cannot be combined in one sensor surface For this reason ESPR measurements can be performed using many different sensor surfaces Measurements can be performed using a disk covered either with a bare gold layer or with a disk covered with one of the many options of modified gold layers Chapter 8 155 The disk contains a gold layer of approximately 50 nm and is used to study interactions of coatings with macromolecules and to study the interactions of large particles as viruses bacteria and cells to coated proteins A modified gold layer disk can be bought but also ma
58. 1988 Surface Plasmon Resonance immunosensors sensitivity considerations Anal Chim Acta 213 213 213 213 35 45 2 Raether H Raether H Raether H Raether H 1977 In Physics of Thin Films 9 9 9 9 145 Eds G Hass M H Francombe R W Hoffman Academic Press New York 3 Liedberg B C Nylander and I Lundstr m Liedberg B C Nylander and I Lundstr m Liedberg B C Nylander and I Lundstr m Liedberg B C Nylander and I Lundstr m 1983 Surface Plasmon Resonance for gas detection and biosensing Sensors and Actuators 4 4 4 4 299 304 4 Welford K Welford K Welford K Welford K 1991 Surface plasmon polaritons and their uses Opt Quant Electronics 23 23 23 23 1 27 5 Stenberg E B Persson H Roos and C Urbaniczky Stenberg E B Persson H Roos and C Urbaniczky Stenberg E B Persson H Roos and C Urbaniczky Stenberg E B Persson H Roos and C Urbaniczky 1991 Quantitative determination of surface concentration of protein with Chapter 8 163 Surface Plasmon Resonance using radio labelled proteins J Coll Interface Sci 143 143 143 143 513 526 6 Kretschmann E Kretschmann E Kretschmann E Kretschmann E 1971 The determination of the optical constants of metals by excitation of surface plasmons Z Physik 241 241 241 241 313 324 7 Len Len Len Lenferink A T M R P H Kooyman and J Greve ferink A T M R
59. 2 association phase analyte 3 dissociation phase buffer 4 regeneration phase regeneration solution 5 baseline buffer Every phase is written as an include sequence A commonly used include sequence is the Parameters seq This sequence defines the measurement variables flow speed mix volume and measurement interval Before starting a measurement it is advised to set the syringes in the home position and to specify all parameters Be careful not to contaminate the buffer in the tubing or syringes with sample always use 50 l of air between buffer in the needle pipette tip tubing and sample Sequence order Define measurement variables PARAMETER seq Measures baseline Tw44 Curve Baseline phase seq Baseline time Wait baseline s Define sample position Sample Move To Sample or Sampler Next If ready then step Inject sample and measure Tw44 Curve_SA Association phase SEQ Association time Wait association s Dissociate complex Tw44 Curve Dissociation phase seq Dissociation time Wait dissociation s Regenerate surface Tw44 Curve_SA Regeneration phase seq Regeneration time Wait regeneration s Wash cuvette Tw44 Curve Back to Baseline phase seq End END seq Conservation gold Tw44 Conservation quality of gold disk The measurement variables can either be defined in the sequence it
60. 7 1 5 7 1 Manual Control Manual Control Manual Control Manual Control of the Autolab of the Autolab of the Autolab of the Autolab Opens the pump control window used for manual control of a measurement Sample identification The Sample identification text box can be used to specify the name and the concentration of the sample Specified text will be shown in the event log when a measurement is started Lift position Move the lift up and down Down is the inject position in the cuvette middle position is just inside the cuvette and the up position is the home position All positions can be calibrated in the lift calibration window After the installation of the software the lift position must be calibrated Measurement settings Measurement Settings box item Interval time allows the user to choose the time between two data points Interval times between 0 1 and 300 seconds are possible Adjust to zero time between 1 to 10 seconds can be used to average the data for calculating the offset to adjust to zero Pump 1 and 2 are syringe pumps Springle has only pump 1 Figure Figure Figure Figure 5555 11 11 11 11 TWINGLE TWINGLE TWINGLE TWINGLE menu items menu items menu items menu items The SPRINGLE has the same list of items The SPRINGLE has the same list of items The SPRINGLE has the same list of items The SPRINGLE has the same list of items Data Acquisition software
61. D H and J Singh Rich D H and J Singh Rich D H and J Singh Rich D H and J Singh 1979 The carbodiimide method The peptides 1 1 1 1 241 261 18 Altschuh D M Altschuh D M Altschuh D M Altschuh D M C Dubs E Weiss G Zeder C Dubs E Weiss G Zeder C Dubs E Weiss G Zeder C Dubs E Weiss G Zeder Lutz and M H V Van Lutz and M H V Van Lutz and M H V Van Lutz and M H V Van Regenmortel Regenmortel Regenmortel Regenmortel 1992 Determination of kinetic constants for the interaction between a monoclonal antibody and peptides using surface plasmon resonance Biochemistry 31 31 31 31 6298 6304 19 Corr M Boyd L F Frankel S R S Kozlowski E A Padlan D H Corr M Boyd L F Frankel S R S Kozlowski E A Padlan D H Corr M Boyd L F Frankel S R S Kozlowski E A Padlan D H Corr M Boyd L F Frankel S R S Kozlowski E A Padlan D H Marulies Marulies Marulies Marulies 1992 Endogenous peptides of a soluble major histocompatibility complex class I molecule H 2L sequence motif quantitative binding and molecular modelling of the complex J Exp Med 176 176 176 176 1681 1692 20 Dubs M Dubs M Dubs M Dubs M C D Altschuh and M H V van Regenmortel C D Altschuh and M H V van Regenmortel C D Altschuh and M H V van Regenmortel C D Altschuh and M H V van Regenmortel 1992 Mappin
62. Getting started Autolab TWINGLE 39 3 1 Index 39 3 2 Introduction 40 3 3 Two days before the experiment 42 3 3 1 Gold surface modification with a 11 MUA layer 42 3 4 One day before the experiment 42 3 4 1 Washing the 11 MUA modified gold surface 42 3 4 2 Startup of the Autolab instrument 42 3 4 3 Liquid Handling set up Autolab 43 3 4 4 Initialization of the instrument 44 3 4 5 Autolab lift position calibration 44 3 4 6 Installation of the gold sensor disk 47 3 4 7 Check for leakage between the two measurement channels 49 3 4 8 Fill tubing with buffer Exchange the buffer solution 51 8 Autolab SPR User manual 3 5 D day The immobilization 52 3 5 1 Sample prepar
63. Preparation of solutions 68 4 4 1 Chemicals 68 4 4 2 Reagents 69 4 5 Liquid Handling set up Autolab SPRINGLE 70 4 6 Initialization of the SPRINGLE instrument 71 4 6 1 Autolab SPRINGLE lift position calibration 71 4 7 Installation of the gold sensor disk 75 4 7 1 Preparation of a self assembled monolayer of 11MUA on the gold surface 75 4 7 2 Assembling the sensor disk on the hemi cylinder 75 4 7 3 Installation of the cuvette 77 4 7 4 Check for leakage of the measurement channel 78 4 7 5 Fill tubing with buffer Exchange the buffer solution 78 4 8 The immobilization 80 4 8 1 Sample preparation 80 4 8 2 Set angle position of the sodium acetate buffer
64. Sensor 154 8 4 3 Cuvette 159 8 4 4 Liquid handling 160 8 5 SPR methods 161 8 5 1 Introduction 161 8 5 2 Methods using the SPR disk 161 8 6 References 162 Chapter 9 165 9 Maintenance 165 9 1 Index 165 9 2 Introduction 166 9 3 Storage of SPR disk and sensor chip 166 9 4 Optics 166 9 5 Routine inspections 167 9
65. The difference in sample volume The difference in sample volume The difference in sample volume The difference in sample volume Automation 130 Table Table Table Table 6666 1 The order of incubation times per sequence 1 The order of incubation times per sequence 1 The order of incubation times per sequence 1 The order of incubation times per sequence Tw44 Curve a full kinetic plot seq Main sequence Tw44 Curve Baseline phase seq Tw44 Curve Association phase seq Tw44 Curve Dissociation phase seq Tw44 Curve Regeneration phase seq Tw44 Curve Back to Baseline phase seq First level of include sequences Tw44 Curve Inject 50 l Buffer Baseline seq Tw44 Curve_SA_Inject 50 l sample Association seq Tw44 Curve Inject 50 l Buffer Dissociation seq Tw44 Curve_SA_Inject 50 l sample Regeneration seq Tw44 Curve Inject 50 l Buffer Back to Baseline seq Second level of include sequences sets the sample volume Sample volume commands are the pump aspirate volume and command pump dispense volume Include sequence Sequence SEQ Initialization TW44 Curve Baseline Phase Tw44 Curve Association phase Tw44 Curve Dissociation phase Tw44 Curve Regeneration phase Tw44 Curve Back to Baseline phase end Tw44 Conserv ation QA gold time time time time time time 35 ul sample 35 ul sample 35 ul sample 35 ul sampl
66. a response unit to quantify the binding of macromolecules to the sensor surface A change of 122 m represents a change in surface protein mass of approximately 1 ng mm 2 MUA coating of the gold disk Stabilization Insulin layer Assembling hemi cylinder gold disk Start up instrument Stabilization of the MUA layer Immobilization Insulin on MUA Baseline Regeneration Association With Insulin Dissociation Restore Baseline Prepare at the latest 2 days before the experiment 3 3 Prepare 1 day before the interaction experiment 3 4 D day the immobilization experiment 3 6 42 Getting Started Autolab TWINGLE 3 3 3 3 3 3 3 3 Two days before the experiment Two days before the experiment Two days before the experiment Two days before the experiment 3 3 1 3 3 1 3 3 1 3 3 1 Gold surface modification with a Gold surface modification with a Gold surface modification with a Gold surface modification with a 11 11 11 11 MUA MUA MUA MUA layer layer layer layer Chemicals 11 Mercapto undecanoic acid 11 MUA Aldrich 450561 Preparation 1 mM 11 Mercaptoundecanoic acid 11 MUA Dissolve 11 mg 11 Mercaptoundecanoic acid Mw 218 36 in 50 ml alcohol like methanol ethanol or iso propanol Incubate a bare gold disk in a 6 well culture disk in a solution of 11mg 11 MUA in 50 ml alcohol This molecule will self assemble a monolayer on
67. and are therefore related to the amount of macromolecules bound to the sensor surface Biomolecular interactions are conventionally studied by techniques as immunoassays ELISA or RIA equilibrium dialysis affinity chromatography and spectroscopic techniques The main advantage of SPR over these techniques to study biomolecular interactions is real time monitoring of binding events and label free detection of macromolecular interactions Advantages of the Twingle instrument are the modular set up which enables a flexible design of experiments and rapid analysis of the interaction plots by the kinetic evaluation software Interaction plots will show binding curves of macromolecular interactions and baseline shifts due to changes in refractive indices of sample solutions Information can be obtained from the binding curves which include properties like Specificity Which molecules interact Concentration How many molecules are there Affinity How strong is the interaction Kinetics How fast is the interaction Cooperativity Are there any steric allosteric effects Biocompatibility coatings How does a molecule interact with a coating SPR Theory 148 8 3 8 3 8 3 8 3 Surface Plasmon Resonance Surface Plasmon Resonance Surface Plasmon Resonance Surface Plasmon Resonance Surface plasmons are created by a consistent longitudinal charge fluctuation at the surface of a metal and typically ha
68. be easily performed semi automatically Open the sequence editor window and select the file immobilization SEQ The lines 67 120s 87 300s 141 900s 195 600s and 249 300s are the incubation times for each different step in the immobilization procedure With a double click on the command line the settings can be edited Select the green button with the arrow to start the immobilization experiment Whenever an action of the user is required a message alert will pop up with information to act upon Figure 4 20 Figure 4 20 Figure 4 20 Figure 4 20 Stabilization cleaning of the gold disk surface Stabilization cleaning of the gold disk surface Stabilization cleaning of the gold disk surface Stabilization cleaning of the gold disk surface with with with with coupling buffer B4 coupling buffer B4 coupling buffer B4 coupling buffer B4 regeneration buffer and 0 1 M NaOH regeneration buffer and 0 1 M NaOH regeneration buffer and 0 1 M NaOH regeneration buffer and 0 1 M NaOH Stabilization of the surface is necessary for all kinds of modified gold Stabilization of the surface is necessary for all kinds of modified gold Stabilization of the surface is necessary for all kinds of modified gold Stabilization of the surface is necessary for all kinds of modified gold surfaces surfaces surfaces surfaces Commercially available Dextran surfaces need extensive cleaning like shown above Chapter 4 8
69. caused by not complying with the following rules and precautions Instrument precautions This instrument was designed for use in laboratories and should not be used in rooms with high air humidity or where there is lot of dust It is not meant to be stored or to be used outdoors High levels of moisture and high concentrations of dust will cause leakage currents in the instrument This can result in a risk of electrical shock and may cause fire The manufacturers warranty is only valid for use in the permitted environments as stated Like most electronic equipment the Autolab TWINGLE SPRINGLE requires air to cool the electronics If the air supply is restricted this may result in a fire Do not cover or block the air vents of the instrument If the instrument is brought from a cold environment into a warm room do not switch on the instrument until it has warmed up Condensed water needs to be able to evaporate Do not expose the instrument to damp or wet conditions Do not place the instrument in direct sunlight or anywhere where it is likely to be exposed to additional external heat sources except a thermostatted water bath Because the instrument works with a scanning mirror and other fixed mirrors place the instrument on a stable levelled table or lab bench Do not lean on the instrument or table during measurements Do not Autolab SPR User manual put the instrument in a position where it is subjecte
70. closing the tool bar Status bar Option for opening or closing the status bar Event log Option for opening or closing the event log The event recorder window records important events from the data acquisition plot Events such as an SPR update or a manually set event marker are recorded here Events can be edited by double clicking the text entry in the log Fixed X scale Fixes the current X axis scale during the measurement Fixed Y scale Fixes the current Y axis scale during the measurement Binding plot properties Possibility to change the curve and or graph settings Figure 5 Figure 5 Figure 5 Figure 5 5 5 5 5 View menu View menu View menu View menu Chapter 5 97 The options to change the curve are based on curve style width size or color The graph changes are text font color change of axis unit grid lines or axis range Figure Figure Figure Figure 5555 6 6 6 6 TTTThe different tab sheets to adjust the curve or graph he different tab sheets to adjust the curve or graph he different tab sheets to adjust the curve or graph he different tab sheets to adjust the curve or graph properties properties properties properties Data Acquisition software 98 The view options of Data Plot Properties is available with the mouse right click See picture 5 9 Zoom Use the left mouse button to zoom in on the plot The available unzoom option see section 5
71. control Manual control Manual control Manual control window window window window of the Twingle of the Twingle of the Twingle of the Twingle Chapter 5 103 Remark Remark Remark Remark In the software the peristaltic pumps are listed as having a selectable range from 1 255 In fact the peristaltic pump has a maximum flow at 40 rpm selectable in 255 steps The SPRINGLE manual control has one extra item the Continuous Flow Continuous Flow The command item will dispense the syringe solution into the pipette tip continuously During refill the pump valve is automatically set to buffer flask and the piston will go its lowest position to aspirate buffer from the flask Then the valve is set to pipette tip to dispense the buffer 5 7 2 5 7 2 5 7 2 5 7 2 Lift position in the software Lift position in the software Lift position in the software Lift position in the software Up position Home position of the lift Middle position Calibrated position in the lift calibration window Normally set at a few millimetres inside the cuvette Down position Inject position calibrated in the lift calibration window Normally set at 1 mm above the gold disk Figure Figure Figure Figure 5 12 5 12 5 12 5 12 B B B B Manual control window Manual control window Manual control window Manual control window of the SPRINGLE of the SPRINGLE of the SPRINGLE of the SPRINGLE Data Acquisition
72. examples Last but not least whenever the commands Automation Pump Aspirate and Automation Pump Dispense are used in the sequence the Tab sheet Volumes needs to be filled out Figure Figure Figure Figure 7777 4444 The The The The automation automation automation automation window with the incubation time TAB sheet window with the incubation time TAB sheet window with the incubation time TAB sheet window with the incubation time TAB sheet to set up the experiment to set up the experiment to set up the experiment to set up the experiment Figure Figure Figure Figure 7777 5 5 5 5 The The The The automation automation automation automation window with the volumes time TAB sheet to window with the volumes time TAB sheet to window with the volumes time TAB sheet to window with the volumes time TAB sheet to set up t set up t set up t set up the sample volumes of the experiment he sample volumes of the experiment he sample volumes of the experiment he sample volumes of the experiment Automation 146 Chapter Chapter Chapter Chapter 8888 8 8 8 8 SPR theory SPR theory SPR theory SPR theory 8 1 8 1 8 1 8 1 Index Index Index Index Chapter 8 146 8 SPR theory
73. frequency 76 Hz Time interval range 0 1 s 300 s Baseline noise 0 1m during a measurement time interval of 1s Sensors Gold coated glass disk Standard supplied cuvette Combined Electrochemistry and SPR Extra cuvette slider Biacore sensor chip adaptor optional Extra cuvette SPR only no electrochemistry optional 16 Hardware Installation Spincoater To spincoat standard gold disks 100 10 000 rpm optional Weight 23 kg Dimensions H x W x D 330mm x 400mm x 360mm Interface RS 232 Power requirements 170 W 100 240 V 50 60 Hz Figure 1 1 Figure 1 1 Figure 1 1 Figure 1 1 B The Autolab SPRINGLE B The Autolab SPRINGLE B The Autolab SPRINGLE B The Autolab SPRINGLE Chapter 1 17 1 6 1 6 1 6 1 6 Hardware Installation Hardware Installation Hardware Installation Hardware Installation The rear panel of the TWINGLE SPRINGLE instruments shows a number of connectors The layout is shown below Figure 1 2 Figure 1 2 Figure 1 2 Figure 1 2 Back panel of the Back panel of the Back panel of the Back panel of the TWINGLE TWINGLE TWINGLE TWINGLE The main entry on the left side holds two fuses both 800mA slow blow and the power switch The functions and signals of the other connectors are described below BNC connectors BNC connectors BNC connectors BNC connectors Trigger A signal output used to trigger an oscilloscope for monitoring the intensity signals
74. have not enough internal memory to handle data transport Digital in out This connector contains 24 free programmable TTL compatible digital inputs and or outputs It can be used to connect third party instruments to SPR for example autosampler FIA instruments or HPLC instruments Automated control of Electrochemical techniques in combination with SPR is performed with a special cable connection between the two systems DIO ports SPR system and PGSTAT Mode For service use only GND This banana socket is connected to the internal instrument analogue ground and indirectly connects to the protective earth This socket is only to be used as a ground terminal for an oscilloscope for service engineers 1 7 1 7 1 7 1 7 SPR and ESPR setup SPR and ESPR setup SPR and ESPR setup SPR and ESPR setup When using the system as a stand alone SPR system connection with the supplied serial cable see figure 1 3 number 3 to the PC is enough In order to perform combined electrochemical and SPR measurements i e ESPR the following setup of the system is required Chapter 1 19 1 Connect the coax cable from the BNC connector SPR 2 Twingle or SPR 1 Springle of the SPR instrument to the ADC 1 or 2 of the PGSTAT 2 Connect the Autolab USB cable to the PC 3 Connect the serial cable from the COM port of the SPR instrument to a free COM port on the PC 4 For automated control of ESPR experiments connect the DIO p
75. manual consists of eleven chapters Chapter 1 describes the hardware installation Chapter 2 describes the software installation Chapter 3 describes a getting started experiment for the TWINGLE Chapter 4 describes a getting started experiment for the SPRINGLE Chapter 5 describes the data acquisition software in detail Chapter 6 provides detailed information regarding the sequence editor Chapter 7 describes detailed information regarding the automation Chapter 8 explains the theoretical background on the Surface Plasmon Resonance technique Chapter 9 discusses the maintenance of the SPR instrument Chapter 10 shows some trouble shooting Chapter 11 has a list of all figures of this document Chapter 12 is the index of this document Important notice The Autolab SPR instruments are developed as a research instrument by Metrohm Autolab B V However Metrohm Autolab B V can never be held responsible for the outcome of results or the interpretation of results measured with Autolab instruments Autolab SPR User manual Safety rules Safety rules Safety rules Safety rules For personal safety and to prevent any unnecessary damage to the Autolab TWINGLE SPRINGLE please read and take note of the following safety rules and precautions Failure to follow these instructions when using the instrument may cause unsafe operation or severe injury Metrohm Autolab B V is not liable for any damage
76. or concentration of primary antibody with antigen to maximize the amount of primary antibody bound Decrease wash volume and time to minimize dissociation of primary antibody Primary antibody reacted poorly with denatured antigen Incubation times with secondary antibody or the streptavidin or avidin conjugate were insufficient Use procedures for retention of the native form of the antigen Increase the incubation times High non specific adsorption Blocking was insufficient Increase concentration of blocking agent Increase incubation time with blocking agent Try to alternate blocking agent Reagents were too concentrated Dilute primary antibody secondary antibody and or streptavidin or avidin conjugate Excessive signal Concentration or amount of reagents used was excessive Excessive incubation times were used Dilute reagents to reduce signal Decrease the amount of antigen employed Decrease incubation times Poor reproducibility of the results Contamination of the cuvette or interfering substances resulted in variable signals During mixing air bubbles are aspirated in the needle Increase wash volumes to remove residual reagents more effectively Use the right cleaning conditions for the cuvette Use lower mixing volumes Prevent evaporation of the sample Troubleshooting 174 10 5 10 5 10 5 10 5 SPR SPR SPR SPR signal problems signal problems signal problems
77. sample 35 ul sample 35 ul sample 35 ul sample Tw44 Curve Interaction plot_SA 35 ul sample Wait Baseline s Wait association s Wait regeneration s Wait interval 1 s Wait s 50 ul sample 50 ul sample 50 ul sample 50 ul sample Tw44 Curve Interaction plot_SA 50 ul sample Wait baseline s Wait association s Wait regeneration s Wait interval 1 s Wait s Flexible Flexible Flexible Flexible sample sample sample sample volume volume volume volume Flexible Flexible Flexible Flexible sample sample sample sample volume volume volume volume Flexible Flexible Flexible Flexible sample sample sample sample volume volume volume volume Flexible Flexible Flexible Flexible sample sample sample sample volume volume volume volume Flexible Flexible Flexible Flexible sample sample sample sample volume volume volume volume Tw44 Curve Interaction plot_SA adjustable volume Wait baseline s Wait association s Wait regeneration s Wait interval 1 s Wait s Chapter 6 133 6 5 6 3 6 5 6 3 6 5 6 3 6 5 6 3 The inject sequence The inject sequence The inject sequence The inject sequence There are two ways to start these sequences by using the sequence editor window and by using the inject button in the toolbar See paragraph 5 7 3 inject It will inject one sample from one position and start th
78. signal problems Signal problems in SPR occur when the minimum of an SPR dip is not determined correctly Before starting a measurement an SPR dip check should be performed to confirm the right conditions Below some SPR dip checks are shown for certain problems that could occur during SPR measurements In case of an abnormal SPR dip please follow the instructions below In practice a SPR dip varies slightly This means that the SPR dips shown in this section may differ slightly from dips produced with your instrument Problem Possible causes Suggested solutions High injection spike The bulk refractive index of the injected sample is too high The temperature of injected sample is different from ambient Dilute the injected sample Add blocking or glucose components to the initial buffer to reduce injection spike Wait 5 minutes before injection of the sample solution and let temperature come to ambient Reaction too slow or too fast Concentration mix frequency volume needle distance Too viscous sample Increase concentration Increase mixing frequency Bad SPR dip The primary coating is too thick or is too irregular or rough Change the spin coat conditions RPM and or concentration of the polymer solvent Shape of SPR dip changes during measurement Particles in the sample solution Agglutination at the surface as a result of denaturation conditions or interacting debris or other compone
79. standard supplied bare gold disk and the sensor chip Biacore supplied gold disk should be installed onto different sliders Figure 8 4 In essence the cuvette is a multi parameter controllable batch reactor in which binding events take place at the bottom at the sensor surface To prevent concentration differences in the cuvette during measurements the instrument is equipped with a controllable automatic aspirate dispense mixing needle A syringe is constantly aspirating and dispensing buffer into the cuvette during measurements to obtain reproducible hydrodynamic conditions The hydrodynamic parameters of the cuvette are mix volume speed or frequency of mixing distance of the pipette tip to the sensor surface volume of the solution in the cuvette diameter of the cuvette geometry of the needle viscosity of the solution temperature of the solution Physical transport phenomena will determine how fast the biomolecular transport from a solution to the surface will be Mass transport limitations Figure Figure Figure Figure 8888 6 6 6 6 The Electrochemical The Electrochemical The Electrochemical The Electrochemical SPR Cuvette SPR Cuvette SPR Cuvette SPR Cuvette With working WE With working WE With working WE With working WE reference reference reference reference RE and counter CE RE and counter CE RE and counter CE RE and counter CE ele
80. system before use before use before use before use Running buffer 1 PBS pH7 4 Or Running buffer 2 10 mM HEPES 150 mM NaCl 3 4 mM EDTA 0 005 Tween P20 Chapter 5 87 Chapter Chapter Chapter Chapter 5555 5 5 5 5 Data Acquisition software Data Acquisition software Data Acquisition software Data Acquisition software 5 1 5 1 5 1 5 1 Index Index Index Index Chapter 5 87 5 Data Acquisition software 87 5 1 Index 87 5 2 Overview of the functions 88 5 3 File menu 94 5 4 Edit menu 96 5 5 View menu 96 5 6 Plot menu 99 5 7 TWINGLE SPRINGLE menu 100 5 7 1 Manual Control of the Autolab
81. the command Loop End thereafter 6 5 7 6 5 7 6 5 7 6 5 7 The semi The semi The semi The semi automatic sequences automatic sequences automatic sequences automatic sequences In a semi automatic sequence the sample is introduced manually to the needles pipette The sample has to be presented manually to the pipette tip at the lift up position In this position a sample vial can be put under the needles pipette The sample can be aspirated from the vial and dispensed into the cuvette The semi automatic sequences can be recognized by the abreviation SA semi automatic in the sequence name for example Tw44 Curve a full kinetic plot_SA 50 l sample seq Two basic commands in the sequence make the experiment semi automated 1 Lift Up in combination with Lift Down 2 Message Alert Tw44 Immobilization_SA 50 l samples SEQ Main sequence Tw44 Immobilization Baseline with Coupling Buffer SEQ Tw44 Immobilization_SA 50 l mixture EDC NHS for activation step SEQ Tw44 Immobilization_SA Ligand Coupling step SEQ Tw44 Immobilization_SA Ethanol Amine Deactivation Step SEQ First level of include sequences Include sequences inside the main sequence Tw44 Immobilization Regeneration Cleaning Step SEQ Tw44 Immobilization inject 50 l Baseline Coupling Buffer SEQ The include sequence inside the above sequence sets the sample volume Automation 136 With the Li
82. wait period Input in the Automation Window time tab Interval 1 Automation time tab Interval 2 Automation time tab Interval 3 Automation time tab Interval 4 Automation time tab Automation 128 6 5 6 1 6 5 6 1 6 5 6 1 6 5 6 1 The main automatic kinetic se The main automatic kinetic se The main automatic kinetic se The main automatic kinetic sequence with all of its quence with all of its quence with all of its quence with all of its included included included included sequences sequences sequences sequences The sequences mentioned below are for double channel synchronized The basic of all the sequences is the same just small changes serve different measurement requirements see fig 6 7 For the TWINGLE the Tw44 Curve a full kinetic plot_SA 50 l sample seq sequence is the main sequence from which all other sequences are generated Loads the parameters settings determined in the automation control window parameters tab Aspirate the sample volume filled out in the volume tab in pump 1 for channel 1 Dispense the sample volume filled out in the volume tab in pump 1 for channel 1 Aspirate the sample volume filled out in the volume tab in pump2 for channel 2 Aspirate the sample volume filled out in the volume tab in pump 2 for channel 2 Pump 2 commands are available in Twingle only
83. 0 ul s Loop Begin Repeat N no limitation Measurement Interval s between 0 1 and 300 Waterbath set temp C between 10 0 70 0 Waterbath set and wait C between 10 0 70 0 Pump 1 or 2 Aspirates Volume l between 1 and 500 Pump 1 or 2 Dispense Volmue l between 1 and 500 Pump 1 or 2 Flow l s between 227 3 and 0 8 in 31 steps Synchronized Mix Flow l s between 227 3 and 0 8 in 31 steps Pump 1 or 2 Mix Volume l between 1 and 100 Synchronized Mix Volume l between 1 and 100 6 5 6 6 5 6 6 5 6 6 5 6 Commands for Commands for Commands for Commands for Semi Semi Semi Semi AAAAuto uto uto utomatic sequences matic sequences matic sequences matic sequences Semi Automatic sequences are generally sequences used in combination with the Automation window A number of commands are linked to the Automation control window Those commands can not be specified in the sequence editor window they are linked to a specific figure filled out in the Automation window menu Options Automation For example the Wait table below is linked with the tab sheet time in the Automation control window See also table 6 1 Baseline wait period Input in the Automation Window time tab Associate wait period Input in the Automation Window time tab Dissociate wait period Input in the Automation Window time tab Regenerate
84. 11 Different positions on the gold disk 48 Figure 3 12 Installed SPR gold disk 49 Figure 3 13 LEFT An overview of the cuvette holder 49 Figure 3 14 RIGHT The positioning pin of a cuvette 49 Figure 3 15 Check for leakage from channel 1 into channel 2 50 Figure 3 16 Check for leakage from channel 1 into channel 2 50 Figure 3 17 Two ways to activate the Sequencer 51 Figure 3 18 TWINGLE The sequence Initialization of Instrument SEQ 52 Figure 3 19 SPR dip 54 Figure 3 20 The optical path cover 54 Figure 3 21 Adjustment of the baseline angle before immobilization 54 Figure 3 22 Stabilization cleaning 55 Figure 3 23 The sequence editor 56 Figure 3 24 The automation window with three tab sheets to set up the experiment 57 Figure 3 25 The automation window with Parameters tab sheets to s
85. 11 Figure 4 11 Installed SPR gold disk Installed SPR gold disk Installed SPR gold disk Installed SPR gold disk Figure 4 12 Figure 4 12 Figure 4 12 Figure 4 12 An overview of the cuvette holder An overview of the cuvette holder An overview of the cuvette holder An overview of the cuvette holder Figure 4 13 Figure 4 13 Figure 4 13 Figure 4 13 The positioning pin of a cuvette The positioning pin of a cuvette The positioning pin of a cuvette The positioning pin of a cuvette 78 Getting Started Autolab SPRINGLE 4 7 4 4 7 4 4 7 4 4 7 4 Check for leakage Check for leakage Check for leakage Check for leakage of the measurement channel of the measurement channel of the measurement channel of the measurement channel To check for leakage pipette 30 l PBS into channel 1 Be sure the fluid reaches the gold disk Check the SPR plot To monitor the dip continuously use the scope mode that is available under the Options menu and on the toolbar Deselect the scope mode by clicking the scope mode button again If there is no leakage in channel 1 a perfect dip will be seen in channel 1 If the cuvette is not correctly assembled the dip will change it s form to become more horizontal Repeat the assembly of the cuvette until it is leakage free Awareness Awareness Awareness Awareness of of of of possible possible possible possible leakage l
86. 3 4 9 4 9 4 9 4 9 The interaction The interaction The interaction The interaction 4 9 1 4 9 1 4 9 1 4 9 1 SSSSample preparat ample preparat ample preparat ample preparation ion ion ion For the interaction procedure prepare the following samples and solutions Running buffer PBS B2 for baseline wash steps and dissociation Anti BSA dilutions for association B9 B10 B11 60 l in a 1 5ml vial Regeneration solution 60 l of 10 mM HCl B3 in a 1 5ml vial 4 9 2 4 9 2 4 9 2 4 9 2 Stabilize the surface Stabilize the surface Stabilize the surface Stabilize the surface Change the coupling buffer in the flask with PBS B2 Stabilize the surface as described in section 4 8 3 of the immobilization paragraph Thiol layers Dextran layers or surfaces with immobilized ligands have to be stabilized to minimize matrix effects that are caused by differences in pH or ionic strength high low salt concentrations of the different buffers used throughout the experiment The matrix effects influence the SPR signal Due to exposure of the layer with the different buffers of the experiment the layer will respond in a more predictive way and will continuously give a SPR signal at the same angle When the desired stability is reached the sequence can be stopped at any time by clicking the Stop measurement button in the tool bar 4 9 3 4 9 3 4 9 3 4 9 3 Start the a
87. 5 Figure 8 4 Slider with hemi cylinder 152 Figure 8 6 The Electrochemical SPR Cuvette 159 Figure 8 7 The normal SPR Cuvette 159 Figure 10 1 Ideal dip 175 Figure 10 3 No SPR dip 175 Figure 10 4 SPR dip shifted right 177 Figure 10 5 SPR dip shifted left 177 Figure 10 7 Consequences of an unsymmetrical SPR dip 178 Figure 10 8 SPR dip shifted up 178 Figure 10 6 Unsymmetrical SPR dip 178 Index 184 Chapter 1 Chapter 1 Chapter 1 Chapter 12222 12 12 12 12 Index Index Index Index Index Index Index Index Abort measurement 92 Affinity chromatography 146 Air vent
88. 5 6 Plot menu Plot menu Plot menu Plot menu This menu contains options to adjust the Data Acquisition plot and the SPR plot All items can also be controlled with a right mouse click in the Data Acquisition window see Figure 5 9 Rescale Undo zoom function also possible with key r case sensitive This will remove all scaling moving and zooming effects Undo Zoom With the left mouse click an area can be zoomed in up to 30 zoom levels can be restored Channel 1 Shows or hides data line of channel 1 in the data acquisition window Channel 2 Shows or hides data line of channel 2 in the data acquisition window Twingle only Differential channel 1 channel 2 Twingle only Shows or hides the differential data of channel 1 minus channel 2 When this option is activated it is not possible to view the temperature as the second y axis Cuvette temperature Shows or hides a second y axis with temperature scale The temperature is always measured even when the temperature plot is not visible in the data acquisition plot Binding curve Shows or hides the data acquisition plot angle versus time Figure Figure Figure Figure 5555 9999 Right m Right m Right m Right mouse ouse ouse ouse click in DA window click in DA window click in DA window click in DA window Figure Figure Figure Figure 5555 8 8 8 8 Plot Plot Plot Plot menu menu menu menu Data Acqu
89. 5 6 5 6 5 6 5 The main immobilization sequence with all of its include The main immobilization sequence with all of its include The main immobilization sequence with all of its include The main immobilization sequence with all of its include sequences sequences sequences sequences The immobilization sequence is used to immobilize a ligand on the modified gold surface The sequence in combination with the automation control Figure Figure Figure Figure 6666 10 10 10 10 List of stabil List of stabil List of stabil List of stabilization sequences ization sequences ization sequences ization sequences Figure 6 Figure 6 Figure 6 Figure 6 11 11 11 11 The The The The i i i imobilization mobilization mobilization mobilization sequence sequence sequence sequencessss Chapter 6 135 window will perform a chemically covalent binding using the EDC NHS strategy For other immobilization techniques new sequences need to be written For the explanation of the automation the Twingle has been used as an example The Springle has the same setup Remark Instead of a long EDC NHS activation time it is better to have multiple incubation times with refreshed EDC NHS solutions For this the sequence needs two extra commands line The include sequence Tw44 Immobilization_SA 50 l mixture EDC NHS for activation step needs a Loop Begin Repeat 3 up front and
90. 6 Replacing syringe and piston 167 Chapter 10 168 10 Troubleshooting 168 10 1 Index 168 10 2 Troubleshoot list general 169 10 3 Troubleshoot list sample handling 171 10 4 Troubleshoot list biochemistry hydrodynamics coatings 172 10 5 SPR signal problems 174 Chapter 11 179 11 Figures 179 Chapter 12 184 12 Index 184 Chapter 1 11 Chapter 1 Chapter 1 Chapter 1 Chapter 1 1 1 1 1 Hardware Hardware Hardware Hardware Installation Installation Installation Installation 1 1 1 1 1 1 1 1
91. 6 can unzoom up to 30 zoom actions Move press Shift and hold down both mouse buttons or middle button on 3 button mouse Move the mouse to change the positioning of the chart Reset Press the F4 function key to remove all scaling moving and zooming effects The r key is used to remove all scaling effects in the active plot DA SPR1 or SPR2 View Right mouse click on the DA window to open a window to change the layout of all components The window contains plot menu options and view menu options Extra options are the Curve and Graph Properties window This window allows to change axis scale for all data lines like angle time temperature and differential It is also possible to put a title on top of the graph set grid lines and change the borderline and background colour See Figure 5 7 and section 5 6 Depending on the second Y axis choice temperature or differential opening the plot view option list with a left mouse click the last tab on the Graph properties window will be either differential or temperature Within the Curve Properties window all data line properties can be changed see Figure 5 6 Figure Figure Figure Figure 5555 7 7 7 7 The The The The options options options options of adjusting the curve or graph properties of adjusting the curve or graph properties of adjusting the curve or graph properties of adjusting the curve or graph properties Chapter 5 99 5 6 5 6 5 6
92. 8 5 7 8 5 7 8 5 7 8 Start measurement Start measurement Start measurement Start measurement Start measurement will plot SPR data in the data acquisition plot angle vs time Each interval time the angle at which the SPR minimum occurs is determined and plotted in the DA window 5 7 9 5 7 9 5 7 9 5 7 9 Pause measurement Pause measurement Pause measurement Pause measurement Pauses plotting SPR minimum in de Binding Curve plot although the time is still proceeding on the background A restart will draw a straight line between the last plotted SPR data point and the first new SPR data point from which the measurement will proceed 5 7 10 5 7 10 5 7 10 5 7 10 Stop measurement Stop measurement Stop measurement Stop measurement Stops recording SPR After measurement start the measurement continues with the last recorded data point 5 7 11 5 7 11 5 7 11 5 7 11 Set Baseline Set Baseline Set Baseline Set Baseline A set of data points which shows a horizontal line in the binding curve can be marked as baseline with this option All following events are recorded with a relative response to this baseline the relative response values are saved in the event log 5 7 12 5 7 12 5 7 12 5 7 12 Adjust to zero Adjust to zero Adjust to zero Adjust to zero Allows user to adjust the measured SPR angle to zero The raw data recorded after adjust to zero i
93. Autolab TWINGLE SPRINGLE Data Acquisition 4 4 User manual SPR Copyright Statement Copyright Statement Copyright Statement Copyright Statement All material in this manual is unless otherwise stated the property of Metrohm Autolab BV Copyright and other intellectual property laws protect these materials Reproduction or retransmission of the materials in whole or in part in any manner without the prior written consent of the copyright holder is a violation of copyright law A single hardcopy and softcopy of the materials is made available solely for personal non commercial use Individuals must preserve any copyright or other notices contained in or associated with them Users may not distribute such copies to others whether or not in electronic form whether or not for a charge or other consideration without prior written consent of the copyright holder of the materials Contact information for requests for permission to reproduce or distribute materials available through this manual are listed below Welcome Welcome Welcome Welcome The Autolab Twingle and Autolab Springle are Surface Plasmon Resonance instruments used for analysis of biomolecular interactions in real time without labelling Our systems also provide the possibility of simultaneous electrochemical measurement with a separate potentiostat galvanostat Surface Plasmon Resonance SPR has become a standard tool in life sciences and pharmaceutical research labora
94. Cleaning of th Cleaning of theeee TWINGLE TWINGLE TWINGLE TWINGLE instrument instrument instrument instrument This is a guiding principle for cleaning all parts in the system which are in contact with the solutions used in the experiments Replace the buffer flask solution step by step with cleaning solution after the specific sequence is finished Cleaning Solution 1 0 5 w v SDS 1 w v Triton in water Total cleaning time about 10 min Cleaning Solution 2 0 5 w v SDS Total cleaning time about 10 min Cleaning Solution 3 50 mM Glycine NaOH pH 9 5 Total cleaning time about 10 min Cleaning solution 4 6 M Urea Total cleaning time about 10 min Cleaning solution 5 1 acetic acid Total cleaning time about 20 min 64 Getting Started Autolab TWINGLE Cleaning solution 6 0 2 M NaHCO3 Total cleaning time about 10 min Cleaning solution 7 Hydrochloric acid 0 1 M HCl Total cleaning time about 20 min Cleaning solution 8 Water Total cleaning time about 10 min Cleaning solution 9 70 Ethanol Total cleaning time about 10 min A Clean before shutting down for a weekend Use the sequence Initialization of Instrument SEQ Put the inlet buffer flask tubings out off the flask Run the sequence to empty all tubings It s also OK to replace the solution in the tubings with Solution 7 water B Clean needles cuvette and connected tubings once every two weeks Use the sequence
95. Figure 2 1 Figure 2 17777 Content of C Content of C Content of C Content of C Autolab SPR folder Autolab SPR folder Autolab SPR folder Autolab SPR folder Figure 2 1 Figure 2 1 Figure 2 1 Figure 2 18888 Content of C Content of C Content of C Content of C Autolab SPR Autolab SPR Autolab SPR Autolab SPR Data folder Data folder Data folder Data folder 36 Software Installation 2 4 4 2 4 4 2 4 4 2 4 4 Softc Softc Softc Softcopy manuals opy manuals opy manuals opy manuals in the subdirectory in the subdirectory in the subdirectory in the subdirectory MANUALS MANUALS MANUALS MANUALS 2 4 5 2 4 5 2 4 5 2 4 5 Examples of KE models Examples of KE models Examples of KE models Examples of KE models in the subdirectory in the subdirectory in the subdirectory in the subdirectory M M M MODELS ODELS ODELS ODELS 2 4 6 2 4 6 2 4 6 2 4 6 Examples of KE PROJECT files Examples of KE PROJECT files Examples of KE PROJECT files Examples of KE PROJECT files in the subdirectory in the subdirectory in the subdirectory in the subdirectory Data Data Data Data C C C C Autolab SPR Autolab SPR Autolab SPR Autolab SPR Manuals Manuals Manuals Manuals Figure 2 1 Figure 2 1 Figure 2 1 Figure 2 19999 Manuals installed during the installation of the software Manuals installed duri
96. Morgan H and D M Taylor Morgan H and D M Taylor 1992b A Surface Plasmon Resonance immunosensor based on the streptavidin biotin complex Biosensors and Bioelectronics 7 7 7 7 405 410 13 Morgan H D M Taylor and C D Silva Morgan H D M Taylor and C D Silva Morgan H D M Taylor and C D Silva Morgan H D M Taylor and C D Silva 1992 a Surface Plasmon Resonance studies of chemisorbed biotin streptavidin multilayers Thin Solid Films 209 209 209 209 122 126 14 Bain C D and G M Whitesides Bain C D and G M Whitesides Bain C D and G M Whitesides Bain C D and G M Whitesides 1987 Angew Chem Int Ed Engl 28 28 28 28 506 512 15 Van den Heuvel D J R P H Kooyman J W Drijfhout and G W Van den Heuvel D J R P H Kooyman J W Drijfhout and G W Van den Heuvel D J R P H Kooyman J W Drijfhout and G W Van den Heuvel D J R P H Kooyman J W Drijfhout and G W Welling Welling Welling Welling 1993 Synthetic Peptides as Receptors in Affinity Sensors A Feasibility Study Anal Biochem 215 215 215 215 223 230 16 Stavros J V R W Wright and D M Single Stavros J V R W Wright and D M Single Stavros J V R W Wright and D M Single Stavros J V R W Wright and D M Single 1986 Enhancement by N hydroxysulfsuccinimide of water soluble carbodiimide mediated coupling reactions Anal Biochem 156 156 156 156 220 222 17 Rich
97. P H Kooyman and J Greve ferink A T M R P H Kooyman and J Greve ferink A T M R P H Kooyman and J Greve 1991 An improved optical method for Surface Plasmon Resonance experiments Sensors and Actuators B 3 3 3 3 261 265 8 Johnson B S Lofas and G Lindquist Johnson B S Lofas and G Lindquist Johnson B S Lofas and G Lindquist Johnson B S Lofas and G Lindquist 1991 Immobilization of proteins to a carboxymethyldextran modified gold surface for biospecific interaction analysis in Surface Plasmon Resonance sensors Anal Biochem 198 198 198 198 268 277 9 O Shannessy D J M Brigham O Shannessy D J M Brigham O Shannessy D J M Brigham O Shannessy D J M Brigham Burke and K Peck Burke and K Peck Burke and K Peck Burke and K Peck 1992 Immobilization chemistries suitable for use in the BIAcore Surface Plasmon Resonance detector Anal Biochem 205 205 205 205 132 136 10 Glaubert M B Glaubert M B Glaubert M B Glaubert M B 1956 The wall jet J Fluid Mech 1 1 1 1 625 643 11 Taylor D M H Morgan and C D Silva Taylor D M H Morgan and C D Silva Taylor D M H Morgan and C D Silva Taylor D M H Morgan and C D Silva Characterisation of chemisorbed monolayers by surface potential measurements 1991 J Phys D Appl Phys 24 24 24 24 1443 1450 12 Morgan H and D M Taylor Morgan H and D M Taylor
98. PR angle every 0 5 seconds Every SPRINGLE instrument is calibrated with water on a bare gold disk The lowest value of the SPR dip is between 0 and 10 percent absolute reflection Figure 4 17 Figure 4 17 Figure 4 17 Figure 4 17 SPR dip SPR dip SPR dip SPR dip Figure 4 18 Figure 4 18 Figure 4 18 Figure 4 18 The optical path cover The optical path cover The optical path cover The optical path cover Chapter 4 81 To change the position of the dip release the retaining screw of the optical path Adjusting the position of the dip is done by turning the micrometer spindle Click on start measurement in the tool bar and follow the change of the angle in real time Set the baseline around 1500 millidegrees m After adjusting the baseline fasten the retaining screw again 4 8 3 4 8 3 4 8 3 4 8 3 Stabilize rehydrate the dry 11 Stabilize rehydrate the dry 11 Stabilize rehydrate the dry 11 Stabilize rehydrate the dry 11 MUA disk MUA disk MUA disk MUA disk Before the modified gold disk can be used for immobilization the baseline must be stabilized Stabilize the surface of the gold disk using one of the sequences Stabilization with buffer from flask SEQ Stabilization with manually injected sample SEQ Stabilization with sample from vial SEQ Figure 4 Figure 4 Figure 4 Figure 4 19 19 19 19 Adjustment of the ba
99. PRINGLE The sequence Initialization of SPRINGLE The sequence Initialization of SPRINGLE The sequence Initialization of SPRINGLE The sequence Initialization of Instrument SEQ Instrument SEQ Instrument SEQ Instrument SEQ 80 Getting Started Autolab SPRINGLE 4 8 4 8 4 8 4 8 The immobilization The immobilization The immobilization The immobilization 4 8 1 4 8 1 4 8 1 4 8 1 Sample preparation Sample preparation Sample preparation Sample preparation For the immobilization procedure prepare the following samples and solutions Coupling buffer B4 for baseline and wash steps EDC NHS activation solution 60 l of 1 1 freshly mixed 0 4M EDC B7 and 0 1 M NHS B6 in a 1 5ml vial Ligand sample 200 l of ligand solution dissolved in coupling buffer B4 in a 1 5ml vial Deactivation solution 200 l of 1M ethanolamine pH 8 5 B8 in a 1 5ml vial Regeneration solution 200 l of 10 mM HCl B3 in a 1 5ml vial 4 8 2 4 8 2 4 8 2 4 8 2 Set angle position of the sodium ac Set angle position of the sodium ac Set angle position of the sodium ac Set angle position of the sodium acetate buffer B 4 etate buffer B 4 etate buffer B 4 etate buffer B 4 Put 50 l of acetate buffer on the gold disk and check the dip by selecting the Scope Mode button To deactivate the scope mode press this button again The scope mode will update the S
100. The Menus The Menus A short overview of the menus is given below The functions for all menu items will be explained in the next sections Figure 5 Figure 5 Figure 5 Figure 5 1 1 1 1 Data Acquisition software Data Acquisition software Data Acquisition software Data Acquisition software A The menu bar of the Springle B The menu bar of the Twingle Figure 5 Figure 5 Figure 5 Figure 5 2222 TTTThe he he he Data Acquisition m Data Acquisition m Data Acquisition m Data Acquisition menu bar enu bar enu bar enu bar Chapter 5 89 File menu File menu File menu File menu shows all the instructions related to old and new experimental data files like open save export and print a data file Some items are only available in the security software version Note Note Note Note some of the instructions have a corresponding button that is present in the toolbar Two commands are only visible in the security software version Edit Edit Edit Edit menu menu menu menu shows all the edit related commands View menu View menu View menu View menu shows all the view related commands Note Note Note Note some of the instructions have a corresponding command using the right click with the mouse Data Acquisition software 90 Plot Plot Plot Plot menu menu menu menu shows all possibilities to organize the data acquisition window plots Not
101. Wait Associate s line 132 3 Wait Dissociate s line 179 4 Wait Regenerate s line 222 5 Wait Interval 1 s line 269 Those times are linked with the number filled out in the automation window Define the analysis time settings Baseline 120s baseline time Association 600s association time Dissociation 60s dissociation time Regeneration 120s regeneration time Back to baseline 120s baseline time The automation window enables to perform the binding experiment semi automatic When the desired interval times are set see fig 3 27 the TAB sheet Parameters can be used to adjust system parameters for the experiment Figure 3 Figure 3 Figure 3 Figure 3 2 2 2 27777 The The The The automation automation automation automation window with three tab sheets to set up the window with three tab sheets to set up the window with three tab sheets to set up the window with three tab sheets to set up the experiment experiment experiment experiment 62 Getting Started Autolab TWINGLE With the button EDIT a new window System parameters pops up Within this window every item can be adjusted The settings will be used for the experiment and loaded into the sequence with the command line 59 Automation Load Parameters Set 1 To finish the Automation window Give the experiment a name under which it will be stored filename example interaction Because of automatic
102. action phase Interaction several dilutions of anti Insulin are used to visualize the Insulin anti Insulin interaction In the reference channel the effect of the plain association buffer is recorded to correct for any a specific interaction factors After the association phase which is used to calculate the association constant Ka the dissociation phase is performed by washing the sample away with association buffer The association buffer is the same buffer as the dilution buffer for the anti Insulin The dissociation phase is used for determining the strength of the interaction After the dissociation phase all bound anti Insulin is removed from the Insulin coated gold disk by diluted SDS Sodium dodecyl sulphate regeneration buffer Then the baseline of the modified disk will be restored by washing the regeneration solution replacing it with the baseline HEPES buffer Insulin Insulin complex K association K dissociation Chapter 3 41 Figure 3 1 Figure 3 1 Figure 3 1 Figure 3 1 Flow chart of the experimental setup Flow chart of the experimental setup Flow chart of the experimental setup Flow chart of the experimental setup After one initial immobilization numerous SPR experiments can be performed The surface plasmon resonance SPR measures angle versus time There is a linear relationship between the amount of bound material and shift in SPR angle The SPR angle shift in millidegrees m is used as
103. ait 10 seconds to measure the SPR signal Lines 287 335 72 repeats of this procedure cover 72 hours of conserving the gold surface Automation 142 Chapter Chapter Chapter Chapter 7777 7 7 7 7 Automation Automation Automation Automation 7 1 7 1 7 1 7 1 Index Index Index Index Chapter 7 142 7 Automation 142 7 1 Index 142 7 2 Introduction 143 7 3 How to open the Automation Control Window 143 7 4 The Automation control window 144 Chapter 7 143 7 2 7 2 7 2 7 2 Introduc Introduc Introduc Introduction tion tion tion The Automation allows the user to perform measurements semi automatically with customized variables such as times volumes and other parameters Every issue of the automation control window will only be used if the sequence to be executed has the commands which will be linked to the automation control window see chapter 6 7 3 7 3 7 3 7 3 How to open the How to ope
104. arate kinetic measurements from concentration measurements Consequently kinetic measurements can be performed with non purified analyte samples These determinations will be explained in chapter 3 and4 together with an explanation of the kinetic evaluation software Methods for SPR measurements of the disk and the sensor chip are different and are therefore summarized separately in the rest of this section 8 5 2 8 5 2 8 5 2 8 5 2 Methods using the SPR disk Methods using the SPR disk Methods using the SPR disk Methods using the SPR disk Measurements can be performed with sensor surfaces of bare gold or polymer coated sensor surfaces Bare gold surfaces Macromolecular interaction measurements can be performed by coating the ligand electrostatically to the surface followed by adding the analyte After coating a blocking compound is usually necessary to prevent a specific interactions This method is especially suitable for detection of large particles as cells and viruses 11 SPR Theory 162 Biomolecular interaction measurements with biotinylated macromolecules 12 13 First the gold sensor surface is coated with biotin followed by binding with streptavidin Then biotinylated molecules are allowed to bind with unoccupied binding sites of streptavidin stoichiometry streptavidin biotin interaction is 1 4 Finally binding of the analyte can be measured Biomolecular interaction measurement
105. arting buffer Look for differences in composition of the solutions Try another injection sequence A big shift of the baseline occurs after washing Especially occurring when a disk with polystyrene coating is applied Wash with the help of the sequencer Add fresh buffer immediately after draining of the sample from the cuvette 10 4 10 4 10 4 10 4 Troubleshoot Troubleshoot Troubleshoot Troubleshoot list list list list biochemistry hydrodynamics coatings biochemistry hydrodynamics coatings biochemistry hydrodynamics coatings biochemistry hydrodynamics coatings Problem Possible causes Suggested solutions No signal or weak signal Reagents were omitted or added in an incorrect order Incorrect reagents were used Use all reagents in the proper sequence Use matched reagents for example a mouse primary antibody with an anti mouse secondary antibody No signal or weak signal Insufficient amounts of antigen were present Increase the primary antibody concentration Increase the interaction time of the primary antibody with the antigen Use more antigen Improper storage of reagents resulted in degradation Store reagents at recom mended conditions Low affinity primary anti body was lost Try higher affinity antibody if available Chapter 10 173 Problem Possible causes Suggested solutions during immuno detection procedure Increase incubation time
106. ation 52 3 5 2 Set angle position of the sodium acetate buffer 54 3 5 3 Stabilize rehydrate the dry 11 MUA disk 55 3 5 4 Start the immobilization procedure 56 3 6 The interaction 59 3 6 1 Sample preparation 59 3 6 2 Stabilize the surface 60 3 6 3 Start the association procedure 60 3 7 The Autolab Twingle data 63 3 8 Cleaning of the TWINGLE instrument 63 Chapter 4 65 4 Getting started Autolab SPRINGLE 65 4 1 Index 65 4 2 Introduction 66 4 3 Startup of the Autolab SPRINGLE instrument 68 4 4
107. ation measurement Diluted antibodies can be stored at 4 C for a few days 4 5 4 5 4 5 4 5 Liquid Handling set up Liquid Handling set up Liquid Handling set up Liquid Handling set up Autolab SPRINGLE Autolab SPRINGLE Autolab SPRINGLE Autolab SPRINGLE Before the system can be used all tubing needs to be filled with buffer Fill buffer flask with PBS buffer and insert the inlet tubing of the syringe pump into the running buffer flask and the green outlet tubing in figure 4 3 shown in red of the peristaltic drain pump into the waste bottle Chapter 4 71 4 6 4 6 4 6 4 6 Initialization of the SPRI Initialization of the SPRI Initialization of the SPRI Initialization of the SPRINGLE NGLE NGLE NGLE instrument instrument instrument instrument Before using the instrument Calibrate lift positions Prepare the gold disk Assemble a new sensor disk Assemble the cuvette new sensor disk Assemble the cuvette 4 6 1 4 6 1 4 6 1 4 6 1 Autolab SPRINGLE lift Autolab SPRINGLE lift Autolab SPRINGLE lift Autolab SPRINGLE lift position calibration position calibration position calibration position calibration There are three ways to find out if the lift is calibrated In the menu SPRINGLE find the lift position item to check the positions up middle down In the menu SPRINGLE find the Manual control item to check the three positions
108. b page Configure for the email address of the user to allow the email message command in the sequencer to send an email message 5 9 5 9 5 9 5 9 Communications menu Communications menu Communications menu Communications menu Serial port The software will automatically check for COM1 up to COM5 Check the correct COM port to start connection with the clear and check option The other COM port can be used for controlling a waterbath Figure Figure Figure Figure 5 27 5 27 5 27 5 27 Customize Customize Customize Customize Email configuration tab page Email configuration tab page Email configuration tab page Email configuration tab page Figure Figure Figure Figure 5555 28 28 28 28 Communications Communications Communications Communications Data Acquisition software 114 Clear and check Re establish the communication between the system and the software In cases where it is necessary to restart the software the clear and check option can be used to reconnect the instrument with the computer 5 10 5 10 5 10 5 10 User menu optional User menu optional User menu optional User menu optional A separate manual specific for the Good Laboratory Practice features is written Here only a few screen shots are shown as examples Administration Control Panel Add or delete User accounts or groups regulates the user access rul
109. be bar item with its commands can be bar item with its commands can be de de de de selected selected selected selected Data Acquisition software 116 5 13 5 13 5 13 5 13 Event Log Event Log Event Log Event Log The Event Log can be selected in the menu VIEW As the name indicates all events during an experiment are stored in this log These events are Update SPR See examples in Figure 4 32 with the lines Update SPR Blue Green Cyan Yellow Black Each event is recorded with time angle temperature relative response and a text line for comments Set Baseline menu Twingle See Figure 4 33 with the lines New baseline value relative response is set to zero m Notice the column angle m and the column Rel ative Response m before and after the New baseline action line Afterwards the angle value in the Rel Response column is set to zero to be able to read the angle shift during the experiment Place marker and update add event item Both actions are used to create an event line in the event log to indicate what happened during the measurement The Update add event item is a command line in the sequence editor see chapter 6 Use Place marker to get a number in the data acquisition plot and in the event log In the event log a remark can be added to this marker If the Update add event item is used as a command in a seq
110. ccording to the application fluids used and quality of maintenance Cleaning the syringe at least once every three months should extend the lifetime of the seal Screw the syringe from the valve port Screw the piston from the manifold Fill a new syringe with distilled water before replacing Carefully eliminate air bubbles in the syringe and replace the syringe Troubleshooting 168 Chapter Chapter Chapter Chapter 10 10 10 10 10 10 10 10 Trouble Trouble Trouble Troublesssshooting hooting hooting hooting 10 1 10 1 10 1 10 1 Index Index Index Index Chapter 10 168 10 Troubleshooting 168 10 1 Index 168 10 2 Troubleshoot list general 169 10 3 Troubleshoot list sample handling 171 10 4 Troubleshoot list biochemistry hydrodynamics coatings 172 10 5 SPR signal problems 174 Chapter 10 169 The following tables address problems that may be encountered with common methods of immuno detection and real time
111. ce 137 Chapter 7 142 7 Automation 142 7 1 Index 142 7 2 Introduction 143 10 Autolab SPR User manual 7 3 How to open the Automation Control Window 143 7 4 The Automation control window 144 Chapter 8 146 8 SPR theory 146 8 1 Index 146 8 2 Introduction 147 8 3 Surface Plasmon Resonance 148 8 4 AUTOLAB Twingle configuration 153 8 4 1 Optics of the Twingle system 154 8 4 2
112. content This getting started document will take you step by step through the initialization of the TWINGLE SPRINGLE instrument and subsequently through an interaction experiment where the antibody anti BSA will be interacting with the immobilized protein BSA Throughout this procedure most features of the software will be illustrated It is presumed that the hardware and software have been installed before the cuvette and hemi cylinder are disassembled and all tubing is empty A 60 min warm up time of the Autolab SPRINGLE should be taken into account Detection of binding events between Insulin and anti Insulin First the modified gold layer on the sensor disk is coated with mercapto undecanoic acid 11 MUA After being assembled into the instrument the modified surface is stabilized with the immobilization coupling buffer In the immobilization procedure Immobilization the acid group of this molecule is activated by incubation with EDC and NHS Subsequently the Insulin is immobilized onto the modified gold layer The Insulin layer will thereafter be stabilized with association buffer In the interaction phase Interaction several dilutions of anti Insulin are used to visualize the BSA anti BSA interaction In the reference channel the effect of the plain association buffer is recorded to correct for any a specific interaction factors After the association phase which is used to calculate the association constant Ka the dis
113. ctrode connections electrode connections electrode connections electrode connections Figure Figure Figure Figure 8888 7 7 7 7 The normal SPR The normal SPR The normal SPR The normal SPR Cuvette Cuvette Cuvette Cuvette Each Each Each Each pipette tip pipette tip pipette tip pipette tip has an has an has an has an individual channel individual channel individual channel individual channel SPR Theory 160 arise when the concentration of analyte at the sensor surface is lower than the total sample concentration In the ESPR cuvette system the mass transport to the surface is highly increased by the aspirate dispense mixing process which is a process according to the dynamic free wall jet principle 10 During a mixing cycle of set volume and set speed the solution is aspirated from the cuvette Normally half the volume that is present in the cuvette will be aspirated followed by a dispense action During the dispense action a jet of the sample solution can be forced to flow into the diffusion layer of the surface As a result the mass transport can be increased enormously The cuvette is connected to a pump to drain the cuvette and to a pump to wash the cuvette 8 4 4 8 4 4 8 4 4 8 4 4 Liquid handling Liquid handling Liquid handling Liquid handling For fully automated measurements the needles connected to the autosampler will aspirate and dispense all solutions The auto
114. ctuations give rise to a surface plasmon wave Electron fluctuations give rise to a surface plasmon wave The generated evan The generated evan The generated evan The generated evanescent field energy is maximum on the surface escent field energy is maximum on the surface escent field energy is maximum on the surface escent field energy is maximum on the surface and decaying exponentially in the Z direction and decaying exponentially in the Z direction and decaying exponentially in the Z direction and decaying exponentially in the Z direction SPR Theory 152 A incoming light beam is being reflected and detected by the detector At a certain angle the reflected light intensity is decreased at this point the Surface Plasmon Resonance effect occurs Figure Figure Figure Figure 8888 4 4 4 4 Slider with hemi Slider with hemi Slider with hemi Slider with hemi cylinder cylinder cylinder cylinder gold layer detector angle glass immersion oil evanescent field sample laser beam SPR hemi cylinder dip Figure Figure Figure Figure 8888 3 3 3 3 Kretschmann configuration Kretschmann configuration Kretschmann configuration Kretschmann configuration special is the oil between special is the oil between special is the oil between special is the oil between hemi hemi hemi hemi cylinder cylinder cylinder cylinder and gold disk and gol
115. d disk and gold disk and gold disk Bare gold disk Chapter 8 153 8 4 8 4 8 4 8 4 AUTOLAB AUTOLAB AUTOLAB AUTOLAB Twingle Twingle Twingle Twingle configuration configuration configuration configuration The Autolab Twingle is configured as a flexible instrument controlled by a computer that can be configured to individual needs It is mainly composed of three parts Figure 8 5 Optics Surface Plasmon Resonance is generated by vibrating mirror optics Sensor One gold coated glass surface can be installed as sensor The cuvette separates two areas to monitor two macromolecular interactions at the same time Liquid handling The instrument is equipped with a continuously mixed cuvette Syringe pumps drain peristaltic pump cuvette Gold Hemi cylinder diode detector mirror laser polarisation filter scanner lens spindle 2 needles retaining screw Waste flask Buffer flask OPTICAL Channel 2 Channel 1 Figure Figure Figure Figure 8888 5 5 5 5 Schematic picture of the Schematic picture of the Schematic picture of the Schematic picture of the TWINGLE TWINGLE TWINGLE TWINGLE configuration configuration configuration configuration SPR Theory 154 8 4 1 8 4 1 8 4 1 8 4 1 Optics of the Optics of the Optics of the Optics of the Twingle Twingle Twingle Twingle system system system system The in
116. d to vibrations it will harm the mirror calibration and thus the measurements It is recommended to fill all tubes with milli Q or demineralized water to prevent bacterial growth and salt precipitation in the tubes when the autosampler is shut down for a period shorter than one week For longer periods it is recommended to fill the tubes with 0 05 sodium azide in milli Q or to remove the solution from all tubing to prevent bacterial growth Personal precautions The Autolab TWINGLE SPRINGLE instrument has a lift Samples can be aspirated and dispensed by the pipette tip Nothing should impede the movement of the pipette tip injury may result Never look directly into the laser beam or reflections of the laser failure to follow this instruction may seriously damage your eyes The peristaltic pump is an open system Do not put anything near the pump Keep the pump free from interference Do not allow untrained personnel to operate the instrument without supervision Electrical hazards There are no user serviceable parts inside Only factory qualified personnel should service the instrument Removal of front or back panels may expose potentially dangerous voltages Always disconnect the instrument from all power sources before removing protective panels Replace blown fuses only with new fuses in size and rating that are stipulated near the fuse panel holder and in the manual The power cord sh
117. de with help of an Autolab spincoater For example a thin film of polymer can be easily attached to the gold surface by the use of an Autolab spincoater An example of a commercially available modified gold disk is the dextran hydrogel modification The hydrogel covering the gold surface of the sensor chip is composed of non cross linked carboxymethylated dextran attached to the gold molecules via a thiol linker layer 8 Dextran is a linear polymer of glucose units which possesses very low non specific adsorption of biomolecules The dextran on the sensor chip is carboxymethylated with a composition of one carboxyl group per glucose residue Three purposes are achieved by the modification of dextran Incorporation of a functional group for immobilization procedures of biomolecules Negatively charged polymer at physiological pH values which allows positively charged biomolecules to adsorb electrostatically to the dextran layer under conditions of low ionic strength Enhancement of the hydrophilicity of the dextran layer by incorporation of carboxymethyl groups Biomolecules can be immobilized by reaction with activated carboxymethyl functional groups of the dextran layer Two functional groups of the ligand can be used for the immobilization Ligands are coupled by amine functional groups or by thiol functional groups 9 The dextran coating is very suitable to study a variety of macromolecular interactions It is especially useful in t
118. e Electrochemical 158 Hydrodynamic parameters 158 SPR configuration 152 Data acquisition 91 93 Temperature plot 98 Dielectric medium 149 disk 165 171 Disk 154 160 Cuvette 158 SPR situation 148 EDC 154 156 Electrical hazards 5 Electrical shock 4 Electrochemical
119. e Note Note Note some of the instructions have a corresponding command using the right click with the mouse TWINGLE TWINGLE TWINGLE TWINGLE menu menu menu menu shows all commands to control the instrument and the experiment Note Note Note Note some of the instructions have a corresponding button that is present in the toolbar Calibration only when the pipette tip is replaced Chapter 5 91 SPRINGLE SPRINGLE SPRINGLE SPRINGLE menu menu menu menu shows all commands to control the instrument and the experiment Note Note Note Note some of the instructions have a corresponding button that is present in the toolbar Calibration only when the pipette tip is replaced Option Option Option Optionssss menu menu menu menu used to choose personal settings execution of sequence or control of the scanner Communicati Communicati Communicati Communications ons ons ons menu menu menu menu used to select the right COM port or reconnect to the instrument User User User User menu menu menu menu used to control the accessibility of users Note Note Note Note This menu is only available after installing the security version Data Acquisition software 92 Help menu Help menu Help menu Help menu used to check the software version The The The The toolbar toolbar toolbar toolbar buttons buttons buttons buttons Clicking a spec
120. e Tw44 Curve a full kinetic plot_SA 35 ul sample Wait Baseline s Wait association s Wait dissociation s Wait regeneration s Wait interval 1 s Wait s 50 ul sample 50 ul sample 50 ul sample 50 ul sample Tw44 Curve a full kinetic plot_SA 50 ul sample Wait baseline s Wait association s Wait dissociation s Wait regeneration s Wait interval 1 s Wait s Flexible Flexible Flexible Flexible sample sample sample sample volume volume volume volume Flexible Flexible Flexible Flexible sample sample sample sample volume volume volume volume Flexible Flexible Flexible Flexible sample sample sample sample volume volume volume volume Flexible Flexible Flexible Flexible sample sample sample sample volume volume volume volume Flexible Flexible Flexible Flexible sample sample sample sample volume volume volume volume Flexible Flexible Flexible Flexible sample sample sample sample volume volume volume volume Tw44 Curve a full kinetic plot_SA adjustable volume Wait baseline s Wait association s Wait dissociation s Wait regeneration s Wait interval 1 s Wait s Chapter 6 131 To be able to fill out the time tab page in the automation control window the knowledge of the sequence to be used is necessary This knowledge can be gained by opening the sequence
121. e antigen by the second antibody Regeneration of the surface will usually break all non covalent interactions These methods have been used to study biomolecular interactions intensively Examples of biomolecular interactions are mentioned here Peptide antibody interaction 18 Peptide MHC interaction 19 Protein antibody interaction epitope mapping 20 Protein DNA interaction 21 Protein polysaccharide interaction 22 Protein virus interaction 23 Protein cell interaction 24 Protein T cell receptor interaction 25 Antibody antibody interaction capturing antibody 26 DNA DNA interaction 27 The activated NHS ester reacts with uncharged primary amino groups of biomolecules This means that the reaction rate is favoured by high pH values of the buffer The reaction can only occur if the ligand is available for reaction i e when it is inside the dextran layer This is achieved by pre concentration of the ligand Positively charged biomolecules adsorb electrostatically to the negatively charged dextran layer by pre concentration Consequently the ligand buffer should be lower than the isoelectric point pI of the ligand A compromise for pH values should be chosen for ligand solutions to fulfil the pre concentration condition and the reaction rate condition for ligands Chapter 8 157 Step 1 Step 2 Step 3 With R1 CH2CH3 R2 CH2CH2CH2N H CH3 2Cl R biomolecule Immobiliza
122. e SPR recording Update SPR recording Update SPR recording Records a SPR signal dip during the measurement Scope mode Scope mode Scope mode Scope mode Refreshing update SPR every 0 5 s Manual control Manual control Manual control Manual control Manual measurement Sampler position Sampler position Sampler position Sampler position To control the position of the lift Mix Mix Mix Mix Customize information window pops up Press the arrow and a selection of three different sequences can be made Inject Inject Inject Inject A selection of Press the arrow and a selection of three different sequences can be made Wash Wash Wash Wash de activates wash peristaltic pump Drain Drain Drain Drain de activates drain peristaltic pump Sequence editor Sequence editor Sequence editor Sequence editor The SPR sequence editor is used to automate measurements to create adjust and to execute sequences Auto Auto Auto Automation mation mation mation The Automation window handles the experimental setup parameters incubation times and the sequences to automate an experiment Data Acquisition software 94 5 3 5 3 5 3 5 3 File menu File menu File menu File menu New Opens a new data acquisition window for a new measurement Open Opens a data spr or ibo file window The spr file will have all data informati
123. e measurement with an incubation time Wait s 60 The inject sequence has the Wait s command in its sequence So the incubation time has to be changed in the sequence itself To change the time or any other item between brackets double click on the command and in the edit part of the window the command with its value to be changed is shown Whenever the Wait s has been changed in one of the other Wait xxxx s commands the inject sequence can also be executed via the Automation window The toolbar button inject icon will activate the Customize Inject window In this window a sequence can be chosen and the measurement time in the inject sequence can be specified The incubation time wait s as specified in the sequence will be overruled by this specified measurement time 6 5 6 4 6 5 6 4 6 5 6 4 6 5 6 4 The The The The stabilization stabilization stabilization stabilization sequences sequences sequences sequences These types of sequences have all the incubation command wait s to be filled out in the sequence editor These sequences are used to generate a stable baseline before the experiment starts The only difference between these sequences is the physical positions for the solutions used Figure Figure Figure Figure 6666 9999 The The The The inject sequence inject sequence inject sequence inject sequence Automation 134 6 5 6 5 6 5 6 5 6
124. e positions In the toolbar find the button lift positions Open the Manual Control window in the TWINGLE SPRINGLE Menu or with the button in the toolbar Figure Figure Figure Figure 3333 5555 Menu Menu Menu Menu TWINGLE TWINGLE TWINGLE TWINGLE to open Manual Control window to open Manual Control window to open Manual Control window to open Manual Control window Chapter 3 45 The lift position has to be calibrated every time you change the pipette tip The lift calibration sets the pipette position The cuvette position for the pipette is normally calibrated to 1 mm above the gold disk the middle position is calibrated with the pipette just about 2 mm inside the cuvette Procedure specified distances depend on the size of the used pipette tips 1 Press button Initialize lift 2 Enter a distance max 60 mm Start with e g 50 mm 3 Press the downward directed arrow button 4 Fine tune distance with 1 mm steps Figure Figure Figure Figure 3333 6666 Open the Lift calibration window Open the Lift calibration window Open the Lift calibration window Open the Lift calibration window Figure 3 Figure 3 Figure 3 Figure 3 7777 The lift calibration window The lift calibration window The lift calibration window The lift calibration window 46 Getting Started Autolab TWINGLE 5 Set new cuvette position
125. e slider Place the disk with a pair of tweezers in a storage box The sensor chip can be inserted in the sensor chip cover Place the sensor chip or disk in a storage box or tube and add some silica gel bags Close the box or tube and store it in the refrigerator for a longer period Always check the bioactivity after storing 9 4 9 4 9 4 9 4 Optics Optics Optics Optics Make it a routine to check the shape of the SPR dip with the update SPR command in order to verify the right quality of the sensor disk the matching of the disk with the hemi cylinder and the cleanness of the optics The possible errors in the SPR dip check that may occur are described in chapter 10 Chapter 9 167 9 5 9 5 9 5 9 5 Routine inspections Routine inspections Routine inspections Routine inspections Inspect all visible liquid connections pump syringes and valves needles drain and wash pump connections If any leaks are discovered clean and tighten the connections or replace tubing and seals if necessary Check the piston of the syringe pumps at least once a month Look for bacterial growth or salt crystals Check the tubing of the peristaltic pumps on signs of wear 9 6 9 6 9 6 9 6 Replacing syringe and piston Replacing syringe and piston Replacing syringe and piston Replacing syringe and piston The seal of each syringe should be changed with a minimum of once per year Seal lifetime varies a
126. eakage leakage leakage If there is leakage out of the cuvette onto the hemi cylinder the solution may come in contact with the detector The measurements will become very noisy A leakage with strong acids may detach the detector out of its calibrated position causing a hardware problem At this point the sensor disk can be reassembled 4 7 5 4 7 5 4 7 5 4 7 5 Fill tubing with buffer Fill tubing with buffer Fill tubing with buffer Fill tubing with buffer Exchange Exchange Exchange Exchange the buffer solution the buffer solution the buffer solution the buffer solution Why should there be liquid in the tubing Why should there be liquid in the tubing Why should there be liquid in the tubing Why should there be liquid in the tubing The solution in the tubing is used for washing the pipette tip Secondly fluid is not compressible like air which results in highly accurate sample volumes and flow rates Figure 4 14 Figure 4 14 Figure 4 14 Figure 4 14 Check for leakage outside of channel 1 Check for leakage outside of channel 1 Check for leakage outside of channel 1 Check for leakage outside of channel 1 Chapter 4 79 In a starting situation the tubing can be empty filled with air or filled with a solution Run the sequence initialization of instrument seq to fill the tubing with buffer or to change the running buffer as follows Select the menu bar item options Seq
127. eams passing through the hemi cylinder of glass cylinder of glass cylinder of glass cylinder of glass Chapter 8 151 In physics photons and electrons are described as waves and particles properties A plasmon is the particle name for the electron density waves When light hits the gold at a certain angle of incidence the energy of the photon can interact with the free fluctuating electron in the gold surface In general the electromagnetic wave phenomenon surface plasmon can be excited by the fields of charged particles and photons In our case the surface plasmon is excited by photons This is called surface plasmon resonance Therefore when in the total internal reflection situation the energy of the light is ideal SPR situation and the photons are converted to resonating plasmons there will be almost no reflected light to detect by the detector Plotting the light intensity versus angle of incidence will give a dip at the specific SPR angle Z Dielectric plasma metal Au Ag Cu m Dielectric ambient medium sample air a Z Evanescent field Ez 0 KSP EZ Figure Figure Figure Figure 8888 2 2 2 2 Electron fluctuation Electron fluctuation Electron fluctuation Electron fluctuation Electron fluctuations give rise to a surface plasmon wave Electron fluctuations give rise to a surface plasmon wave Electron flu
128. ecification Specificationssss Table 1 Specification of the Autolab TWINGLE system Technical Specification Technical Specification Technical Specification Technical Specification Definition Definition Definition Definition Measuring principle Surface plasmon resonance Transducer principle Scanning mirror Liquid handling Cuvette system Parallel channels Two Fixed wavelength 670 nm Sample loading and injection Manual or semi automatic Mixing Continuous wall jet Pumps 2 x Syringe pump 1 x peristaltic pump Flow rate range 0 8 l s 227 3 l s Syringe pump 30 l s 130 l s Peristaltic pump Sample volume 20 l 150 l Offset of SPR angle by spindle 62 78 Dynamic range 4000 m Angle resolution lt 0 02 m Minimum molecular weight 180 D Association constant range 10 3 10 7 M 1s 1 Dissociation constant range 10 5 10 1 s 1 Equilibrium affinity 10 4 10 10 M 1 Concentration range 10 11 10 3 M Refractive index 1 26 1 38 standard optional 1 32 1 44 or 1 40 1 52 Refractive index resolution lt 1 10 7 Measuring frequency 76 Hz Time interval range 0 1 s 300 s Baseline noise 0 1m during a measurement time interval of 1s Sensors Gold coated glass disk Standard supplied cuvette Combined Electrochemistry and SPR or SPR only no electrochemistry Extra cuvette slider Biacore sensor chip adaptor optional 14 Hardware Installation S
129. ed regularly because of inevitable spilling of immersion oil Unscrew the M3x3 screw on the slider Gently slide the hemi cylinder out of the slider Clean the hemi cylinder with ethanol Use only the lens tissue paper to clean and dry Alternatively the hemi cylinder and slider can be cleaned in an ultra sonic water bath After cleaning slide the hemi cylinder into the slider with the oil overflow hole to the oil overflow hole of the slider Reassemble the M3 screw One Gold disk provides 5 to 7 measuring positions Set a measuring position by gliding the disk over the hemi cylinder surface with a clean pipette tip Figure 4 10 Figure 4 10 Figure 4 10 Figure 4 10 Different positions on the gold disk Different positions on the gold disk Different positions on the gold disk Different positions on the gold disk 1 5 3 2 4 1 7 4 2 3 5 6 Chapter 4 77 4 7 3 4 7 3 4 7 3 4 7 3 Installation of the cuvette Installation of the cuvette Installation of the cuvette Installation of the cuvette There is only one way to position the cuvette in the cuvette holder Position the cuvette with the pin towards the slot in the cuvette holder The cuvette will slide in the cuvette holder tighten the cuvette with the ring Make sure that the ring is tightened firmly to prevent leakage outside the channel For extra tightening use the supplied SPR key Figure 4 11 Figure 4 11 Figure 4
130. end wearing gloves when handling the disk 9 10 Fig Fig Fig Fig 3 8 3 8 3 8 3 8 b The final steps of lift calibration b The final steps of lift calibration b The final steps of lift calibration b The final steps of lift calibration Figure Figure Figure Figure 3333 9999 A drop of immersion oil on top of the hemi A drop of immersion oil on top of the hemi A drop of immersion oil on top of the hemi A drop of immersion oil on top of the hemi cylinder cylinder cylinder cylinder 48 Getting Started Autolab TWINGLE o Touch only the frosted sides of the hemi cylinder to prevent scratches on the accurately polished round sides of the hemi cylinder o Manipulate the gold sensor disk with a fine tweezers especially when you take it out of the plastic case o The gold sensor disk in the plastic case is oriented with the gold surface facing down Cleaning the hemi Cleaning the hemi Cleaning the hemi Cleaning the hemi cylinder and slider cylinder and slider cylinder and slider cylinder and slider The slider and the hemi cylinder need to be cleaned regularly because of inevitable spilling of immersion oil Unscrew the M3x3 screw on the slider Gently slide the hemi cylinder out of the slider Clean the hemi cylinder with ethanol Use only the lens tissue paper to clean and dry Alternatively the hemi cylinder and slider can be cleaned in an ultra sonic water bath After cleanin
131. eplaced by PBS Only a stepwise change of the bulk refractive index should be measured The SPR dip is almost out of the dynamic range First adjust the spindle to 1500 mdegree set interval time on 0 5 seconds and then adjust spindle slowly to around zero mdegree Drift of the baseline signal Temperature in the laboratory is not constant There is a reciprocal correlation of temperature and angle shift The coating is not stable Place SPR in a climate chamber A spincoating of e g polystyrene should first be adapted to the new buffer Chapter 10 171 10 3 10 3 10 3 10 3 Troubleshoot list Troubleshoot list Troubleshoot list Troubleshoot list sample handling sample handling sample handling sample handling Problem Possible causes Suggested solutions The volume in the cuvette decreases during an experiment There is a leakage in one of the chambers of the cuvette The cuvette is damaged The cuvette is not properly mounted and is askew There is evaporation of sample during long experiments Try another cuvette Turn the mounting screw stepwise a little after each other in order to press down the cuvette as a whole perpendicular to the wafer Prevent evaporation with a cover The signal is not stable and noisy Air bubbles or solids are interfering with the mixture of the sample solution Mix with a decreased volume No mixing occurs or mixing falls out
132. eraction experi ist of interaction experiment sequences ment sequences ment sequences ment sequences Figure 6 Figure 6 Figure 6 Figure 6 8888 B B B B Springle l Springle l Springle l Springle list of interaction experiment sequences ist of interaction experiment sequences ist of interaction experiment sequences ist of interaction experiment sequences Automation 132 Ta Ta Ta Table ble ble ble 6666 2 The order of incubation times per sequence 2 The order of incubation times per sequence 2 The order of incubation times per sequence 2 The order of incubation times per sequence TW44 Curve Interaction plot 50ul sample seq Main sequence Tw44 Curve Baseline phase seq Tw44 Curve Association phase seq Tw44 Curve Regeneration phase seq Tw44 Curve Back to Baseline phase seq Include sequences inside the main sequence Tw44 Curve Inject 50 l Buffer Baseline seq Tw44 Curve_SA_Inject 50 l sample Association seq Tw44 Curve_SA_Inject 50 l sample Regeneration seq Tw44 Curve Inject 50 l Buffer Back to Baseline seq Second level of include sequences Include sequence Sequence SEQ Initialization Tw44 Curve Baseline Phase Tw44 Curve Association phase Tw44 Curve Regeneration phase Tw44 Curve Back to Baseline phase end Tw44 Conserv ation QA gold time time time time time 35 ul
133. eration cleaning Read include sequence Tw44 Immobilization_SA Regeneration cleaning step seq starts at command line 331 Within those include sequences commands for incubation times are written subsequently 1 Wait Baseline s line 86 2 Wait Associate s line 113 3 Wait Interval 2 s line 193 4 Wait Interval 3 s line 273 5 Wait Regenerate s line 354 Those times are linked with the number filled out in the automation window Define the analysis time settings Baseline 120s coupling buffer EDC NHS activation 300s EDC NHS activation time Ligand coupling 900s ligand coupling time Deactivation 600s deactivation time Regeneration 120s regeneration time The automation window enables to perform the immobilization semi automatically When the desired interval times are set see fig 3 24 the TAB sheet Parameters can be used to adjust system parameters for the experiment Figure 3 Figure 3 Figure 3 Figure 3 2 2 2 24444 The The The The automation automation automation automation window with three tab sheets to set up the window with three tab sheets to set up the window with three tab sheets to set up the window with three tab sheets to set up the experiment experiment experiment experiment 58 Getting Started Autolab TWINGLE With the button EDIT a new window System parameters pops up Within this window every item can be adjusted The setti
134. es shows the user configuration settings Keeps track of all actions in the Audit trail Figure Figure Figure Figure 5555 29 29 29 29 User User User User Figure 5 30 Figure 5 30 Figure 5 30 Figure 5 30 The Administration Control Panel The Administration Control Panel The Administration Control Panel The Administration Control Panel Chapter 5 115 5 11 5 11 5 11 5 11 Window menu Window menu Window menu Window menu Cascade Places several overlapping windows in a cascade Tile All binding plot curves windows are tiled within the data acquisition window next to each other Arrange icons Arrange data acquisition icons Close all Close all data presentation windows the data acquisition window cannot be closed 5 12 5 12 5 12 5 12 Help menu Help menu Help menu Help menu Graph action help Help on graph commands About SPR software Software version information Figure Figure Figure Figure 5 31 5 31 5 31 5 31 The Administration Control Panel The Administration Control Panel The Administration Control Panel The Administration Control Panel TTTThe access rules per he access rules per he access rules per he access rules per group can be limited Every menu group can be limited Every menu group can be limited Every menu group can be limited Every menu bar item with its commands can be bar item with its commands can
135. et up the experiment using EDIT 58 Figure 3 26 The sequence editor 60 Figure 3 27 The automation window with three tab sheets to set up the experiment 61 Figure 3 28 The automation window with Parameters tab sheets to set up the experiment using EDIT 62 Figure 3 28 An example of a binding experiment 63 Figure 4 1 Flow chart of the experimental setup 67 Figure 4 2 the back panel of the Autolab SPRINGLE 68 Figure 4 3 The draining tube from the drain peristaltic pump 71 Figure 4 4 Menu SPRINGLE to open Manual Control window 72 Figure 4 5 Open the Lift calibration window 72 Figure 4 6 The lift calibration window 73 Figure 4 7 a The lift calibration procedure 74 Fig 4 7 b The final steps of lift calibration 74 Figure 4 8 A drop of immersion oil on top of the hemi cylinder
136. ft Up command the needle pipette tip have direct access for a sample in a vial The Message Alert command the experiment stops the sequence and shows a message box window The message shown has been written in the sequence like With the continue button the sequence proceeds to aspirate the sample from the vial A new message alert will pop up and request to remove the vial Then after clicking the continue button the sequence will proceed with the experiment and start to measure Figure Figure Figure Figure 6666 12 12 12 12 Example of the Example of the Example of the Example of the sequence message alert box sequence message alert box sequence message alert box sequence message alert box Figure 6 Figure 6 Figure 6 Figure 6 13 13 13 13 An example An example An example An example of a sequence where the message alert has of a sequence where the message alert has of a sequence where the message alert has of a sequence where the message alert has been used been used been used been used Chapter 6 137 6 5 8 6 5 8 6 5 8 6 5 8 Writing a sequence Writing a sequence Writing a sequence Writing a sequence This section explains how to create a sequence to measure a binding curve For the explanation of the automation the Twingle has been used as an example The Springle has the same setup Basic routines of a binding curve 1 baseline buffer
137. g slide the hemi cylinder into the slider with the oil overflow hole to the oil overflow hole of the slider Reassemble the M3 screw One Gold disk provides 5 to 7 measuring positions Set a measuring position by gliding the disk over the hemi cylinder surface with a clean pipette tip Figure Figure Figure Figure 3333 1 1 1 11111 Different positions on the gold disk Different positions on the gold disk Different positions on the gold disk Different positions on the gold disk Figure Figure Figure Figure 3333 10 10 10 10 Assembly of a disk Assembly of a disk Assembly of a disk Assembly of a disk 1 5 3 2 4 1 7 4 2 3 5 6 1 2 1 2 1 2 1 2 3 3 3 3 4 5 6 4 5 6 4 5 6 4 5 6 Chapter 3 49 3 4 6 2 3 4 6 2 3 4 6 2 3 4 6 2 Installa Installa Installa Installation of the cuvette tion of the cuvette tion of the cuvette tion of the cuvette There is only one way to position the cuvette in the cuvette holder Position the cuvette with the pin towards the slot in the cuvette holder The cuvette will slide in the cuvette holder tighten the cuvette with the ring Make sure that the ring is tightened firmly to prevent leakage outside the channel For extra tightening use the supplied SPR key 3 4 7 3 4 7 3 4 7 3 4 7 Check for leakage between the two measurement channels Check for leakage between the two measurement cha
138. g of viral epitopes with conformationally specific monoclonal antibodies using biosensor technology J Chromatography 597 597 597 597 391 396 21 Bondeson K Frostell Bondeson K Frostell Bondeson K Frostell Bondeson K Frostell Karlsson L F gerstam and G Karlsson L F gerstam and G Karlsson L F gerstam and G Karlsson L F gerstam and G Magnusson Magnusson Magnusson Magnusson 1993 Lactose repressor operator DNA interactions SPR Theory 164 Kinetic analysis by a Surface Plasmon Resonance biosensor Anal Biochem 214 214 214 214 245 251 22 Mach H D B Volkin C J Burke C R Middaugh R J Linhardt Mach H D B Volkin C J Burke C R Middaugh R J Linhardt Mach H D B Volkin C J Burke C R Middaugh R J Linhardt Mach H D B Volkin C J Burke C R Middaugh R J Linhardt J R Fromm D Longanathan and L Mattson J R Fromm D Longanathan and L Mattson J R Fromm D Longanathan and L Mattson J R Fromm D Longanathan and L Mattson 1993 Nature of the interaction of heparin with acidic fibroblast growth factor Biochemistry 32 32 32 32 5480 5489 23 Dubs M Dubs M Dubs M Dubs M C D C D C D C D Altschuh and M H V van Regenmortel Altschuh and M H V van Regenmortel Altschuh and M H V van Regenmortel Altschuh and M H V van Regenmortel 1991 Interaction between viruses and monocl
139. he determination of kinetic parameters or antibody concentration Three methods are used to couple ligands Immobilization of the ligand to the dextran layer The ligand is coupled covalent either by amine functional groups or by thiol functional groups to the dextran layer Immobilization by amine functional groups of ligands is performed in three steps 1 reaction of carboxymethyl groups with a mixture of N hydroxysuccinimide NHS and N ethyl N dimethylaminopropyl carbodimide EDC to obtain an active NHS ester 2 reaction of the activated ester with primary amine functional groups of the ligand for a covalent ligand hydrogel bond 3 deactivation of excess activated ester groups with ethanolamine Non covalent binding of biotinylated ligand to a streptavidin immobilized gold disk Due to the severe biotin streptavidin interaction it is possible to regenerate the surface without disrupting SPR Theory 156 the non covalent biotin streptavidin bond This method is suitable to couple synthetic DNA molecules to the surface Immobilization of capturing antibodies Capturing antibodies are used when the activity of antibodies is reduced by the immobilization procedure Detection of antigens is achieved in three steps Firstly immobilization of the capturing antibody for example anti Rabbit Anti Mouse Fc Secondly binding of the second antibody a mouse antibody by the capturing antibody Thirdly specific binding of th
140. ific button in the toolbar can perform most of the manual handling for measuring SPR Figure 5 3 shows an overview of the DA toolbar and its buttons A The toolbar of the Springle B The toolbar of the Twingle Figure 5 Figure 5 Figure 5 Figure 5 3333 The Data Acquisition tool b The Data Acquisition tool b The Data Acquisition tool b The Data Acquisition tool bar ar ar ar The toolbar shows a number of buttons some of which might be grayed out in which case the attached instruction cannot be performed This section provides an overview of the toolbar buttons New New New New Opens a new data acquisition window for a new measurement Open Open Open Open SPR data files ibo old software versions or spr in a new window Save Save Save Save saves or saves as the currently measured SPR data Start measurement Start measurement Start measurement Start measurement starts the measurement Window Window Window Window menu menu menu menu used to organize multiple data acquisition windows Chapter 5 93 Pause measurement Pause measurement Pause measurement Pause measurement pauses the measurement Stop measurement Stop measurement Stop measurement Stop measurement stops recording the measurement Place Event Marker Place Event Marker Place Event Marker Place Event Marker Add a marker in a measurement plot Update SPR recording Updat
141. in the stored measurement Save Saves a data acquisition window as data file spr under the current name Previously saved files with the same name will be overwritten Remark The default directory to store the data needs to be specified in menu Options Customize settings User directories see Figure 5 26 Save as Opens a save as window to save recorded data as data file with a user created filename and directory Export Export data as a text file which can be imported in Excel for instance or export graphical plots as a BMP picture file Print Print plot Binding Curve Plot a data acquisition plot SPR angle vs time SPR Plots intensity of the reflected light vs angle Event Log event log file Print Setup Opens the print set up window for selection of printer and printer settings Exit Exits TWINGLE program and saves the current settings as default parameters Data Acquisition software 96 5 4 5 4 5 4 5 4 Edit menu Edit menu Edit menu Edit menu Cut Deletes the selected region of a text Copy Copies the selected object to the clipboard The copied object can be retrieved in Microsoft Word or Excel with the paste command Paste Copies the clipboard contents into the current selected object 5 5 5 5 5 5 5 5 View menu View menu View menu View menu The view menu contains commands for presentation of data acquisition plots Tool bar Option for opening or
142. in the sequence editor window and reading the sequence The table below shows sequences and their order of time commands used in measurements The SPRINGLE sequences use the same set of incubation time commands 6 5 6 2 6 5 6 2 6 5 6 2 6 5 6 2 The interaction plot sequence The interaction plot sequence The interaction plot sequence The interaction plot sequence The interaction plot sequence is an almost exact copy of the sequence called Tw44 Curve a full kinetic plot seq The only difference is the absence of the dissociation phase This sequence can be used if the dissociation constant is not of interest like for affinity constant or for qualitative results For the SPRINGLE the Sp44 Curve SA_Interaction plot 50 l sample seq sequence is the main sequence from which the other sequence is generated For the explanation of the automation the Twingle has been used as an example The Springle has the same setup To be able to fill out the time tab page in the automation control window the knowledge of the sequence to be used is necessary This knowledge can be gained by opening the sequence in the sequence editor window and reading the sequence The table below shows sequences and their order of time commands used in measurements Figure 6 Figure 6 Figure 6 Figure 6 8 A 8 A 8 A 8 A Twingle l Twingle l Twingle l Twingle list of interaction experi ist of interaction experi ist of int
143. incremental numbering the file names will start with interact001 and every next experiment with the same name will be up numbered up to interact999 Select the Start sequence from disk button at the bottom of the automation control window Figure 3 27 to select and to execute the specific experiment Have the samples ready for the experiment Sample Injection routine Vial 1 sample 1 Pipette tip 1 75 l analyte anti Insulin dilution1 in HEPES buffer Vial 2 sample 2 Pipette tip 2 75 l HEPES buffer Vial 3 sample 3 Pipette 1 and 2 1000 l Regeneration solution The HEPES Buffer is being used for all washing steps in the interaction experiment Therefore the buffer flask will be filled with this solution Figure 3 Figure 3 Figure 3 Figure 3 2 2 2 28888 The The The The automation automation automation automation window with Parameters t window with Parameters t window with Parameters t window with Parameters tab sheets to set ab sheets to set ab sheets to set ab sheets to set up the experiment using EDIT up the experiment using EDIT up the experiment using EDIT up the experiment using EDIT Chapter 3 63 Next experiments can be performed with the other prepared dilutions 2 and 3 3 7 3 7 3 7 3 7 The The The The Autolab Tw Autolab Tw Autolab Tw Autolab Twingle ingle ingle ingle data data data data 3 8 3 8 3 8 3 8 Cleaning of th Cleaning of th
144. ingle only Saves data Fill out a file name in the Sampler Window Sends out a DIO port trigger to the PGSTAT Receives a DIO port trigger from the PGSTAT Defines the drain left peristaltic pump pump 3 40rpm speed in 255 steps Drains the cuvette Stops draining the cuvette Inserts sequence file seq in opened sequence Opens KE software and creates a new overlay Add the channel 1 data to the KE overlay Add the channel 2 data to the KE overlay Add the differential data to the KE overlay Sends the needles pipette tips to the Inject positon 1mm above the gold Just within the cuvette position Home position Starts loop in sequence N defines number of cycles linked to Loop End command Stops loop linked to Loop Begin Saves the loop file by name counter e g protein001 Chapter 6 121 Stops measurement Holds measurement plot but the time is recorded Defines measurement interval time in seconds 0 1s 300s Clears the DA plot window and sets time to zero Saves measurement Starts measurement in channel 1 and channel 2 Starts measurement in channel 1 Starts measurement in channel 2 Shows message box with message and postpones sequence until message is confirmed by Continue or Abort button Sends out an email which is configured in Options_ Customize_ email Opens a window with all measurement paramete
145. interaction sensing with the Autolab Twingle Typical errors or misinterpretations are also presented For serious problems not found in this chapter please contact the local distributor 10 2 10 2 10 2 10 2 Troubleshoot list Troubleshoot list Troubleshoot list Troubleshoot list general general general general Problem Possible causes Suggested solution The entire instrument is not working Fuse defect No mains power Replace fuse if source is known Check for proper mains voltage The software is not working Wrong installation or combination of the SPR files Win98 2000 XP not properly installed Install with the most up to date version of the software First rename the SPR directory and delete the SPR icons See Chapter 2 for installation instructions The software is not working in combination with the Twingle The instrument is not connected to the host computer Serial ports are not correct The serial cable is defect First start the SPR program on the host computer then switch on the instrument Use preferably COM2 for the serial cable to connect the Twingle SPR starts but there is no initialisation sound of autosampler and syringe pumps Internal fuse defect Call the local distributor Status bar indicates not connected The link between the internal PC and the host computer is not working RS232 cable not connected properly or is defect
146. ion Adjustment of the baseline angle before immobilization Adjustment of the baseline angle before immobilization Adjustment of the baseline angle before immobilization Channel 1 Channel 2 Fix retaining screw Release retaining screw Turn spindle to adjust baseline Around 1500 Chapter 3 55 3 5 3 3 5 3 3 5 3 3 5 3 Stabilize rehydrate the dry 11 Stabilize rehydrate the dry 11 Stabilize rehydrate the dry 11 Stabilize rehydrate the dry 11 MUA di MUA di MUA di MUA disk sk sk sk Before the modified gold disk can be used for immobilization the baseline must be stabilized The Coupling Buffer will be used for all washing steps in the immobilization experiment Therefore the buffer flask will be filled with this solution Stabilize the surface of the gold disk using one of the sequences Stabilization with buffer from flask SEQ Stabilization with manually injected sample SEQ Stabilization with sample from vial SEQ Start the sequence and continue to wash until the baseline is sufficiently stable Eventually every solution should show the same SPR angle every time it is dispensed on the surface When the desired stability is reached the sequence can be stopped at any time by clicking the stop measurement button in the tool bar Figure 3 22 Figure 3 22 Figure 3 22 Figure 3 22 Stabilization cleaning Stabilization cleaning Stabilization cleaning Stabilization cleaning
147. ion of the front panel The LED in the power button lights up after a few seconds Start the Autolab SPR Data Acquisition software Wait until the instrument has finished initiating the TWINGLE SPRINGLE lift and the syringe pumps this will take about 20 seconds A warm up time of about 1 hour should be taken into account before measuring with the Autolab TWINGLE SPRINGLE 3 4 3 3 4 3 3 4 3 3 4 3 Liquid Handling set up Liquid Handling set up Liquid Handling set up Liquid Handling set up Autolab Autolab Autolab Autolab Before the system can be used all tubing needs to be filled with buffer Fill buffer flask with HEPES buffer and insert the inlet tubing of the syringe pump into the running buffer flask and the green outlet tubing of the peristaltic drain pump into the waste bottle Figure 3 Figure 3 Figure 3 Figure 3 3333 the back panel of the T the back panel of the T the back panel of the T the back panel of the TWINGLE WINGLE WINGLE WINGLE Left side the black power switch Left side the black power switch Left side the black power switch Left side the black power switch I O I O I O I O Figure Figure Figure Figure 3 3 3 3 4444 The draining tube from the drain peristaltic pump The draining tube from the drain peristaltic pump The draining tube from the drain peristaltic pump The draining tube from the drain peristaltic pump isisisis inserted into
148. ioned in between a Synchronized Mix Start with Measurement Start Both Channels and a Measurement End with Synchronized Mix Stop command In case of a Figure Figure Figure Figure 6 6 6 6 5555 E E E Example of a sequence with include xample of a sequence with include xample of a sequence with include xample of a sequence with include sequences sequences sequences sequences An An An An include sequence include sequence include sequence include sequence function function function function resembles a folder in window explorer resembles a folder in window explorer resembles a folder in window explorer resembles a folder in window explorer First include sequence Automation 126 xxxxx Mix Start before the Wait command also a xxxxx Mix Stop after the Wait command is necessary If a wait command is active without a Measurement Start command active the temperature registration in the software is not updated In this case the temperature is not updated in the software 6 5 4 6 5 4 6 5 4 6 5 4 Save data Save data Save data Save data There are three different commands to automatically save data while executing a sequence 6 5 4 1 6 5 4 1 6 5 4 1 6 5 4 1 Loop Save Loop Save Loop Save Loop Save xxxxxx00 xxxxxx00 xxxxxx00 xxxxxx00 Data measured during the sequential execution of a loop command will be stored under the same name with an increasi
149. isition software 100 SPR curve channel 1 Shows or hides a SPR plot of intensity versus of angle of channel 1 SPR curve channel 2 Shows or hides a SPR plot of intensity versus of angle of channel 2 Twingle only Position Shows or hides x and y positions of the mouse in the upper left corner of the data acquisition window Fixed position Connects the marker lines with channel 1 or channel 2 data points depending on the closest position of the mouse to a curve Marker lines Option to show or hide a flexible x and y axis as a mouse pointer this enables to read the interception values of a data point on the axes Event Markers Shows or hides the markers of channel 1 and or channel 2 in the data acquisition window 5 7 5 7 5 7 5 7 TWINGLE TWINGLE TWINGLE TWINGLE SPRINGLE SPRINGLE SPRINGLE SPRINGLE menu menu menu menu Figure Figure Figure Figure 5555 10 10 10 10 SPR curves of channel 1 an SPR curves of channel 1 an SPR curves of channel 1 an SPR curves of channel 1 and SPR curves of channel 2 d SPR curves of channel 2 d SPR curves of channel 2 d SPR curves of channel 2 With a right mouse click on the SPR plot window the user can select or deselect a recorded SPR curve In this example four SPR updates are still visible in the Channel 2 SPR plot Chapter 5 101 The TWINGLE SPRINGLE menu contains commands to control the hardware functions of the instrument 5 7 1 5 7 1 5
150. isk Storage of SPR disk Storage of SPR disk and sensor and sensor and sensor and sensor chip chip chip chip There are three recommended procedures to store a disk or sensor chip In the instrument during a relatively short period e g until the next day Store it in buffer with the lowest deactivating behaviour If the immobilized biomolecules can resist distilled water this is preferred Otherwise an ammonium carbonate buffer may be used because the salts of the buffer will evaporate In order to reduce the evaporation of the solution from the cuvette put some Parafilm on the cuvette In the slider during a relatively short period of maximal a week Wash the cuvette with distilled water Never wash with buffer because salts will dry and will destroy the coating Drain the cuvette and disconnect the cuvette Remove the slider from the instrument Place the slider in a plastic bag and store the slider in the bag in the refrigerator The plastic bag is necessary because otherwise moisture will condense on the hemi cylinder lens Let the slider equilibrate at room temperature before removing the plastic bag ca 30 minutes Place the slider with disk or chip in the holder and place the cuvette For storage of biomolecules on the disk or chip for a longer period first wash the coating with distilled water to remove the buffer containing salts Remove the cuvette and slider from the instrument and remove the disk or chip from th
151. istance Chemical Resistance Chemical Resistance Chemical Resistance The material of the pump the cuvette the disk chip and the Teflon tubing determine the chemical resistance of the instrument Aqueous buffer solution without organic compounds can be used without damaging the sample handling part of the instrument Table 1 7 1 and 1 7 2 Some organic solvents are not recommended for use in the TWINGEL SPRINGLE instrument they may damage the system Table 1 7 3 Table 1 7 1 Recommended buffer solutions Solution Solution Solution Solution Concentration Concentration Concentration Concentration pH pH pH pH Solution Solution Solution Solution Concentration Concentration Concentration Concentration pH pH pH pH ACES 50 mM 6 8 MES 50 mM 6 1 ADA 50 mM 6 6 MOPS 50 mM 7 2 BES 50 mM 7 1 MOPSO 50 mM 6 9 BICINE 50 mM 8 3 Phosphate 50 mM 7 5 BIS TRIS 50 mM 6 5 PIPES 50 mM 6 8 Borate 100 mM 8 8 POPSO 50 mM 7 8 CAPS 50 mM 10 4 TAPS 50 mM 8 4 CHES 50 mM 9 3 TED 50 mM 7 5 Citrate 50 mM 3 0 TRICINE 50 mM 8 1 EPPS 50 mM 8 0 TRIS HCl 75 mM 8 0 Glycine 50 mM 2 3 TRIZMA BASE 50 mM 8 1 HEPES 50 mM 7 5 22 Hardware Installation Table 1 7 2 Recommended regeneration solutions Solution Solution Solution Solution Concentration Concentration Concentration Concentration pH pH pH pH Acetonitrile 20 7 5 Hydrochloric acid 10 1000 mM E
152. lation repeat installation new disk chip Incorrect cuvette installation 1 cuvette not centered on laser spot 2 no buffer in cuvette 3 air bubbles between buffer and gold layer repeat cuvette installation add buffer in cuvette drain cuvette and inject buffer Optics 1 no laser light 2 laser spot is not centered on the hemi cylinder 3 other Contact the local distributor Chapter 10 177 Problem Possible cause Suggested solutions SPR dip shifted right Incorrect spindle position Adjust spindle to SPR signal Figure 10 4 and SPR signal out of range of approximately zero degrees Figure 9 1 SPR dip shifted left Incorrect spindle position Adjust spindle Figure 10 5 and SPR signal out of range Figure Figure Figure Figure 10 10 10 10 4 4 4 4 SPR dip SPR dip SPR dip SPR dip shifted right shifted right shifted right shifted right Figure Figure Figure Figure 10 10 10 10 5 5 5 5 SPR dip SPR dip SPR dip SPR dip shifted left shifted left shifted left shifted left Troubleshooting 178 Problem Possible cause Suggested solutions Unsymmetrical SPR dip fig 10 6 indicated by arrow 1 Particles in buffer sample 2 Dirt on hemi cylinder 3 Dirt in immersion oil 4 Dust in optics Filter buffer sample Clean half cylinder New immersion oil Clean optics please call the local distributor for advise Unsymmet
153. lder location Autolab Autolab Autolab Autolab TWINGL TWINGL TWINGL TWINGLEEEE Figure 2 8 Figure 2 8 Figure 2 8 Figure 2 8 Installation window 8 Installation window 8 Installation window 8 Installation window 8 Press Next to proceed with the installation procedure Chapter 2 31 A A A A Original folder location Autolab SPR Original folder location Autolab SPR Original folder location Autolab SPR Original folder location Autolab SPR B B B B New folder location New folder location New folder location New folder location Autolab Autolab Autolab Autolab TWINGL TWINGL TWINGL TWINGLEEEE Figure 2 9 Figure 2 9 Figure 2 9 Figure 2 9 Instal Instal Instal Installati lati lati lation window 9 on window 9 on window 9 on window 9 The program folder name can be changed in TWINGLE or SPRINGLE as well like in picture B Press Next to proceed with the installation procedure 32 Software Installation Figure 2 10 Figure 2 10 Figure 2 10 Figure 2 10 Install Install Install Installation window 10 ation window 10 ation window 10 ation window 10 Figure 2 1 Figure 2 1 Figure 2 1 Figure 2 11111 Installation window 11 Installation window 11 Installation window 11 Installation window 11 This screen pops up for just a very short moment Figure 2 1 Figure 2 1 Figure 2 1 Figure 2 12222 Installati
154. lled in the Good Laboratory Practice software version 2 4 2 2 4 2 2 4 2 2 4 2 Files in C Files in C Files in C Files in C Autolab SPR Autolab SPR Autolab SPR Autolab SPR Files in the root of the Autolab SPR folder are shown below The user folder will only be installed with the security software version Fig 2 1 Fig 2 1 Fig 2 1 Fig 2 15555 The desktop icons shown afte The desktop icons shown afte The desktop icons shown afte The desktop icons shown after the installation of the SPR r the installation of the SPR r the installation of the SPR r the installation of the SPR software software software software C C C C Figure 2 1 Figure 2 1 Figure 2 1 Figure 2 16666 Folder structure Folder structure Folder structure Folder structure Chapter 2 35 2 4 3 2 4 3 2 4 3 2 4 3 Examples of data files in Examples of data files in Examples of data files in Examples of data files in the sub the sub the sub the subfolder folder folder folder C C C C Autolab SPR Autolab SPR Autolab SPR Autolab SPR Data Data Data Data The shown files are original experimental data One measurement has four different saved files with different extensions IBO SPE SPO INI extension has all experimental data The current software will save measured SPR data in just one file with the extension SPR Figure 2 1 Figure 2 1
155. lot when printing Check this box to put the filename on the printouts Alert before executing a changed sequence Figure Figure Figure Figure 5 5 5 5 24 24 24 24 O O O Options menu ptions menu ptions menu ptions menu Data Acquisition software 112 Before the measurement will be executed an alert is shown to confirm the sequence change before starting the measurement Clear experimental title at new measurement Clear the experiment title before each start of a new measurement Try to connect SPR at start up If the instrument power is ON the software will automatically connect to the instrument at start up Initial temperature of water bath C This command field set the water temperature in the water bath The waterbath must be switched on before starting the Twingle software Figure 5 25 Figure 5 25 Figure 5 25 Figure 5 25 Customize Customize Customize Customize General tab page General tab page General tab page General tab page Figure 5 26 Figure 5 26 Figure 5 26 Figure 5 26 Customize Customize Customize Customize User directories tab page User directories tab page User directories tab page User directories tab page Chapter 5 113 Sequences See paragraph 5 7 3 figure 5 15 User directories tab page See Figure 5 26 The user can set default paths for a sequence and a data directory Email configuration ta
156. lution N Hydroxy Succinimide MW 217 13 Fluka 56485 Weigh 23 mg of NHS in a 3 ml vial and dissolve it in 2 ml demi water Ligand sample 200 l of ligand solution dissolved in coupling buffer in a 1 5 ml vial Preparation of 5 g ml insulin to immobilize on the sensor disk Insulin from bovine pancreas Mw 5733 49 Sigma I5500 Dissolve 1 mg insulin in 1 ml 1 M Acetic acid Dilute 50 l 200 x with 950 l 10 mM Acetate buffer Deactivation solution 200 l of 1 M ethanolamine pH 8 5 in a 1 5 ml vial Preparation of 1 M Ethanolamine solution Pipette 600 l of Ethanolamine in a 25 ml flask dilute it with 10 ml demi water and adjust the pH to 8 5 with 1 M HCl Regeneration solution Coupling buffer 0 1 SDS SDS Sodium Dodecyl Sulphate or Lauryl sulphate sodium salt 20 in H2O MW 288 38 Fluka 05030 When to prepare the solutions When to prepare the solutions When to prepare the solutions When to prepare the solutions _ EDC and NHS are not stable in solution Once prepared use it the same day and or store aliquots of 200 l at 20 C _ The pH of the acetate buffer can change in time so check the pH before use _ The ligand dissolved in acetate buffer should be freshly prepared The same preparation of acetate buffer should be used for all steps of the immobilization measurement Diluted antibodies can be stored at 4 C for a few days but be careful The experiment baseline buffer should be the same as the buffer fo
157. menu 96 Autolab SPR User manual 9 5 6 Plot menu 99 5 7 TWINGLE SPRINGLE menu 100 5 7 1 Manual Control of the Autolab 101 5 7 2 Lift position in the software 103 5 7 3 Inject 104 5 7 4 Wash 106 5 7 5 Drain 107 5 7 6 Place Event Marker 108 5 7 7 Update SPR recording 108 5 7 8 Start measurement 109 5 7 9 Pause measurement 109 5 7 10 Stop measurement 109 5 7 11 Set Baseline 109 5 7 12 Adjust to zero
158. mon Resonance is a physical process which occurs when light hits a metal under a special angle position during total internal reflection conditions If a light beam passes the glass of a hemi cylinder prism its path angle is changed when it leaves the prism into air beam 1 Figure 8 1 This change always occurs when light passes through a denser medium into a less dense medium or vice versa At a critical angle of incidence the light beam beam 2 Figure 7 1 does not leave the prism but will be reflected at the interface of the two media glass and air This is called total internal reflection In the SPR situation we have a replaceable glass disk coated with a thin layer of gold on the hemi cylinder Between the disk and the hemi cylinder is a thin layer of oil The refractive index of the hemi cylinder the oil and the disk is the same In this way the laser light will not bend passing the hemi cylinder the oil and the glass to reach the gold layer The photons hit the gold instead of air at the total internal reflection angle SPR Theory 150 There is a special situation for the photons when a dielectric medium is placed on top of the gold If the dielectric medium has an opposite or higher dielectric constant than gold the free electrons in the gold will fluctuate This electron fluctuation gives charge fluctuations in the metal The metal layer is very thin and therefore the charge fluctuations are only taking place at the su
159. n the How to open the How to open the Auto Auto Auto Automation mation mation mation Control Window Control Window Control Window Control Window The automation control window is activated by selecting the automation button at the tool bar or under menu lt options gt Figure 7 1 For the explanation of the automation the Twingle has been used as an example The Springle has the same setup The automation control window consists of a number of selection windows Parameters times and volumes tab sheets File name Execute button Figure Figure Figure Figure 7777 1 1 1 1 The autosampler control window selection The autosampler control window selection The autosampler control window selection The autosampler control window selection Figure 7 Figure 7 Figure 7 Figure 7 2 2 2 2 The The The The automation automation automation automation window with three tab sheets to set up the window with three tab sheets to set up the window with three tab sheets to set up the window with three tab sheets to set up the experiment experiment experiment experiment Automation 144 Although the name of the window suggests the experimental control is automatic measurements can only be performed semi automatic Whenever a sample needs to be introduced to the gold surface this action needs manually presenting the sample vial to the needles pipette tips 7 4 7 4 7 4 7 4
160. nces 133 6 5 6 5 The main immobilization sequence with all of its include sequences 134 6 5 7 The semi automatic sequences 135 6 5 8 Writing a sequence 137 Chapter 6 119 6 2 6 2 6 2 6 2 Introduction Introduction Introduction Introduction The sequence editor is a powerful tool to automate experiments Sequences can be used to describe experiment parameters i e flow speed mix volume sample volume etc sample positions measurement times and liquid handling In general sequences are used for automatic or semi automatic control of an experiment 6 3 6 3 6 3 6 3 Sequence editor window Sequence editor window Sequence editor window Sequence editor window Select from the toolbar or use menu Options Sequencer to open the Sequence Editor window The sequence editor window contains a list of simple commands in the left window and a list of commands in sequence to the right window The list of commands on the right forms a sequence By using the double click the selected command from the left will be added at the bottom of the assembled sequence The drag and drop functionality allows inserting a specific command at a selected
161. ng serial number A proper filename has to be specified before starting the sequence Within a sequence Loop Begin Repeat N N defines number of cycles Loop Save xxxxxx00 like sample00 or 3April00 Loop end The part of the sequence between the Loop Begin Repeat and Loop End will be repeated N times 6 5 4 2 6 5 4 2 6 5 4 2 6 5 4 2 Measurement Save Measurement Save Measurement Save Measurement Save filename filename filename filename At the end of an experiment the data are saved using the specified filename This command is useful at the end of a sequence to save one defined experiment 6 5 4 3 6 5 4 3 6 5 4 3 6 5 4 3 Automation Automation Automation Automation Save Save Save Save see automation see automation see automation see automation window window window window This is a command for a sequence that uses input from the Autosampler window Specify a filename in the Automation window The data are automatically saved using this filename All samples will have the same filename with an increasing serial numbers at the end Chapter 6 127 6 5 5 6 5 5 6 5 5 6 5 5 Commands with variables Commands with variables Commands with variables Commands with variables Command Command Command Command Valid entr Valid entr Valid entr Valid entry y y y Drain Speed 1 255 between 1 and 255 rpm 12
162. ng the installation of the software Manuals installed during the installation of the software Manuals installed during the installation of the software The Autolab SPR System Security manual will only be installed if the Security version has been installed C C C C Autolab SPR Autolab SPR Autolab SPR Autolab SPR Models Models Models Models Figure 2 Figure 2 Figure 2 Figure 2 20 20 20 20 Examples of kinetic evaluation Examples of kinetic evaluation Examples of kinetic evaluation Examples of kinetic evaluation models installed models installed models installed models installed with the with the with the with the software software software software C C C C Autolab SPR Autolab SPR Autolab SPR Autolab SPR Data Data Data Data Figure 2 Figure 2 Figure 2 Figure 2 22221111 Examples of kinetic evaluation Examples of kinetic evaluation Examples of kinetic evaluation Examples of kinetic evaluation projects projects projects projects installed installed installed installed with the with the with the with the software software software software Chapter 2 37 2 4 7 2 4 7 2 4 7 2 4 7 Autolab Autolab Autolab Autolab TWINGLE sequence TWINGLE sequence TWINGLE sequence TWINGLE sequence files in files in files in files in subdirectory subdirectory subdirectory subdirectory SEQUE SEQUE SEQUE SEQUENNNNCES CES CES CES
163. ng with the sample in the cuvette Lines 174 to 181 The fixed set of commands to perform the measurement Line 182 The include sequence has been written to be sure that the needle will have no sample left from previous actions SPECIALS lines 152 162 172 Line 136 Tw44 Curve Inject 50 l buffer dissociation The time involves about 10 seconds before measurement starts Replace this include sequence with a different file if this takes to long for the current application Automation 140 Lines 192 to 215 are similar to as lines 108 126 Line 192 The needle pipette tip is accessible to put a vial underneath Line 199 201 Stop the experiment to put the vial underneath the needle pipette tip Line 200 202 Aspirate the sample from the vial Line 203 Stops the experiment to give time to remove the vial from under the needle pipette tip Line 204 205 212 to drain away any solution on top of the gold before the next sample will be dispensed Lines 216 224 The fixed set of commands to perform the measurement SPECIALS lines 211 215 The syringe finishes its aspirate or dispense actions before going to the next step in the sequence experiment Line 182 The include sequence has been written to be sure that the needle will have no sample left from previous actions Lines 241 242 flush 450 l buffer to wash away the analyte Lines 245 246 the buffer sample to measure the dissocia
164. ngs System settings System settings Tab sheet pump2 for Twingle only Tab sheet pump2 for Twingle only Tab sheet pump2 for Twingle only Tab sheet pump2 for Twingle only Chapter 5 111 angle range So the number 2124 defines the dynamic scanning range of 4248 millidegrees The number 2124 needs to be written in the NOVA settings when performing ESPR measurements or should be checked in the C Windows ESPR ini file for the GPES software 5 8 5 8 5 8 5 8 Options menu Options menu Options menu Options menu 5 8 1 5 8 1 5 8 1 5 8 1 Sequencer Sequencer Sequencer Sequencer The SPR sequence editor is used to automate measurements See chapter 5 5 8 2 5 8 2 5 8 2 5 8 2 Auto Auto Auto Automation mation mation mation Opens the Automation window Here experimental parameters incubation times and storage file name can be addressed See chapter 6 5 8 3 5 8 3 5 8 3 5 8 3 Scope mode Scope mode Scope mode Scope mode Updates the SPR dip every 0 5 seconds Useful for manually adjusting the optical path 5 8 4 5 8 4 5 8 4 5 8 4 Scanner Scanner Scanner Scanner Stops and starts the scanner Only used for service items 5 8 5 5 8 5 5 8 5 5 8 5 Cust Cust Cust Customize omize omize omize Opens a window to specify software settings General settings tab page Include filename in p
165. ngs will be used for the experiment and loaded into the sequence with the command line 49 Automation Load Parameters Set 1 To finish the Automation window Give the experiment a name under which it will be stored filename example immobilization Because of automatic incremental numbering the file names will start with immob001 and every next experiment with the same name will be up numbered up to immob999 Select the Start sequence from disk button at the bottom of the automation control window Figure 3 25 to select and to execute the specific immobilization experiment Have the samples ready for the experiment Sample Injection routine Vial 1 sample 1 75 l EDC Vial 2 sample 2 75 l NHS When the EDC NHS sample is requested by the software mix both chemical components and present it to the pipette tip for incubation The chemicals are not alouwed to be mixed sooner Vial 3 sample 3 Pipette tip 1 75 l ligand Insulin in acetate buffer Vial 4 sample 4 Pipette tip 2 75 l acetate buffer Vial 5 sample 5 200 l 1M ethanolamine pH 8 5 Vial 6 sample 6 500 l regeneration buffer 0 1 M HCl Figure 3 Figure 3 Figure 3 Figure 3 2 2 2 25555 The The The The automation automation automation automation window with Parameters tab sheets to set window with Parameters tab sheets to set window with Parameters tab sheets to set window with Parameters tab sheets to set up the experiment using EDIT
166. nnel between channel between channel 1111 and and and and 2222 Figure 5 Figure 5 Figure 5 Figure 5 33 33 33 33 A zoom A zoom A zoom A zoom in on t in on t in on t in on the event log he event log he event log he event log from Fig from Fig from Fig from Fig 5555 32 32 32 32 A double A double A double A double click on a remark will result in an editable line below the event log click on a remark will result in an editable line below the event log click on a remark will result in an editable line below the event log click on a remark will result in an editable line below the event log window window window window Automation 118 Chapter Chapter Chapter Chapter 6666 6 6 6 6 Sequencer Sequencer Sequencer Sequencer 6 1 6 1 6 1 6 1 InInInIndex dex dex dex Chapter 6 118 6 Sequencer 118 6 1 Index 118 6 2 Introduction 119 6 3 Sequence editor window 119 6 4 Software Sequence editor
167. nnels Check for leakage between the two measurement channels Check for leakage between the two measurement channels To check for leakage pipette 125 l HEPES into channel 1 Be sure the fluid reaches the gold disk Check the SPR plot To monitor the dip continuously use the scope mode which is available under the Options menu and on the toolbar Deselect the scope mode by clicking the scope mode button again If there is no leakage from one channel to the other you will see a perfect dip Figure 3 Figure 3 Figure 3 Figure 3 1 1 1 12222 Installed SPR gold disk Installed SPR gold disk Installed SPR gold disk Installed SPR gold disk Figure Figure Figure Figure 3333 1 1 1 13333 LEFT LEFT LEFT LEFT An overview of the cuvette holder An overview of the cuvette holder An overview of the cuvette holder An overview of the cuvette holder The The The The SPRINGLE cuvette has only a channel ONE position SPRINGLE cuvette has only a channel ONE position SPRINGLE cuvette has only a channel ONE position SPRINGLE cuvette has only a channel ONE position Figure 3 Figure 3 Figure 3 Figure 3 1 1 1 14444 RIGHT RIGHT RIGHT RIGHT The positioning pin of a cuvette The positioning pin of a cuvette The positioning pin of a cuvette The positioning pin of a cuvette 50 Getting Started Autolab TWINGLE in channel 1 and a flat line around the abs
168. nts Change of roughness of the surface Filter the sample solution prior to addition Use other buffers or coating procedures Try another blocking agent Try another coating polymer Increase wash volumes to remove residual reagents more effectively Chapter 10 175 Figure Figure Figure Figure 10 10 10 10 1 1 1 1 Ideal dip Ideal dip Ideal dip Ideal dip Figure Figure Figure Figure 10 10 10 10 2 2 2 2 SPR dip SPR dip SPR dip SPR dip shift shift shift shift Ideal dips are smooth and symmetrical at the bottom of the dip At the beginning of a measurement the SPR dip is located at the x axis at zero degree Figure 9 1 and at the y axis zero degree The absolute intensity of an SPR dip is normally lower than 10 If it is above 10 air bubbles are present in most cases Upon binding the SPR dip will shift to the right as shown in Figure 9 2 Figure Figure Figure Figure 10 10 10 10 3 3 3 3 No SPR dip No SPR dip No SPR dip No SPR dip Troubleshooting 176 Problem Possible cause Suggested solutions No SPR dip Figure 10 3 Incorrect spindle position Adjust spindle position Incorrect scale settings Adjust scale Incorrect slider installation 1 hemi cylinder not clean 2 no immersion oil 3 disk reversed inserted gold layer not in buffer compartment 4 damaged gold layer clean cylinder repeat instal
169. of macromolecules to the sensor surface The response also depends on the refractive index of the bulk solution A change of 122 millidegrees represents a change in surface protein of approximately 1 ng mm 2 or in bulk refractive index of approximately 10 3 Table Table Table Table 8888 1 1 1 1 Correlation of SPR parameters The detection principle limits the size of the analyte which can be studied If the molecular weight of the compound is below 1000 Dalton then the change in refractive index upon binding to the sensor surface is too low to be detected directly The penetration depth of the evanescent wave of 300 400 nm also determines the size of macromolecules or particles that can be studied Particles larger than 400 nm cannot be measured totally As a result the signal is not linearly related to the amount of bound particles Under these circumstances it is possible to study the binding qualitatively but a quantitative or kinetic analysis cannot be performed SPR parameters Equivalent values SPR angle shift 122 millidegrees Change in protein surface concentration 1 ng mm 2 Change in bulk refractive index 0 001 Chapter 8 149 Background information history Background information history Background information history Background information history Augustin Fresnel presented in 1821 32 theories that in principle could have explained the SPR phenomenon James Clark Maxwell presented in 1873 all
170. of the go of the go of the go of the gold disk surface with ld disk surface with ld disk surface with ld disk surface with coupling buffer B4 coupling buffer B4 coupling buffer B4 coupling buffer B4 Why should the sodium acetate buffer SPR angle Why should the sodium acetate buffer SPR angle Why should the sodium acetate buffer SPR angle Why should the sodium acetate buffer SPR angle be set at be set at be set at be set at 1500 m 1500 m 1500 m 1500 m The solutions used in the immobilization differ significantly in refractive index and will change a number of times during the procedure The acetate buffer has the lowest refractive index and therefore the smallest SPR angle the Ethanolamine solution has the largest angle 56 Getting Started Autolab TWINGLE 3 5 4 3 5 4 3 5 4 3 5 4 Start the immobilization procedure Start the immobilization procedure Start the immobilization procedure Start the immobilization procedure The EDC NHS immobilization procedure is a standardized procedure that can be easily performed semi automatically Open the sequence editor window and select the file Tw44 immobilization_SA 50ul samples SEQ to have a glance on the procedure The experiment sequence set up 1 baseline Read include sequence Tw44 Immobilization Baseline with Coupling buffer seq starts at command line 50 2 EDC NHS activation Read include sequence Tw44
171. olute value of 90 in the empty channel 2 total reflection When in channel 2 the horizontal flat line goes down the cuvette is not correctly assembled Repeat the assembly of the cuvette until it is leakage free Drain the HEPES from channel 1 and check channel 2 for leakage Awareness Awareness Awareness Awareness of of of of possible possible possible possible leakage leakage leakage leakage If there is leakage out of the cuvette onto the hemi cylinder the solution may come in contact with the detector The measurements will become very noisy A leakage with strong acids may detach the detector out of its calibrated position causing a hardware problem Figure 3 1 Figure 3 1 Figure 3 1 Figure 3 15555 Check for leakage from channel 1 into channel 2 Check for leakage from channel 1 into channel 2 Check for leakage from channel 1 into channel 2 Check for leakage from channel 1 into channel 2 Figure 3 1 Figure 3 1 Figure 3 1 Figure 3 16666 Check for leakage from channel 1 into channel Check for leakage from channel 1 into channel Check for leakage from channel 1 into channel Check for leakage from channel 1 into channel 2 2 2 2 Check for leakage from channel 1 to channel 2 Leakage Compare with Fig 3 15 After a longer period of time a SPR minimum will appear Chapter 3 51 In case of leakage reassemble the sensor disk and check for leakage again 3 4 8 3 4 8 3 4 8 3 4 8
172. on in one file the SPR measurement plot max 50 000 data points the eventlog the SPR signal max 20 the experiment parameters and the sequence The ibo file an old software version used extension is linked to the ini spo and spe file extensions These file type are measurements performed with software version 4 1 and sooner Figure 5 Figure 5 Figure 5 Figure 5 4 4 4 4 File File File File menu menu menu menu The tooltip The tooltip The tooltip The tooltip Hovering your mouse pointer over one of the buttons will trigger a Tooltip to appear displaying some basic information on the functions of that button Chapter 5 95 Data file ibo for a data acquisition plot Procedure file ini for measurement settings SPR file spo for SPR curves of channel 1 12 maximum SPR file spe for SPR curves of channel 2 12 maximum Only the double channel will have the spe file Close Closes a data acquisition window without saving Permissions Allows the user to specify the level of visibility for their files This is explained in the Autolab SPR system security manual Electronic Signature Allows users to electronically sign files This is explained in the Autolab SPR system security manual Extract Parameters Opens the manual control window showing the used parameters of the stored measurement Extract sequences Opens the sequence editor showing the sequence used
173. on window 12 Installation window 12 Installation window 12 Installation window 12 This screen pops up for just a very short moment Chapter 2 33 Figure 2 1 Figure 2 1 Figure 2 1 Figure 2 13333 Installation window 13 Installation window 13 Installation window 13 Installation window 13 These two screens pop up at different positions a few times and disappear again very quickly 34 Software Installation A shortcut icon to the Data Acquisition program and the kinetic evaluation program will be installed on your desktop figure 2 15 2 4 2 4 2 4 2 4 Autolab SPR software setup on the hard disk after installation Autolab SPR software setup on the hard disk after installation Autolab SPR software setup on the hard disk after installation Autolab SPR software setup on the hard disk after installation 2 4 1 2 4 1 2 4 1 2 4 1 Folder structure Folder structure Folder structure Folder structure The Autolab SPR folder is created during the installation of the SPR software There are two folders for sequences in the Twingle software The main sequence folder filled with sequences to use and one sub folder for include sequences see chapter 6 The Autolab SPR root folder contains the executable for the Kinetic Evaluation software Fig 2 16 Shows the Autolab SPR folder structure C Autolab SPR with the TWINGLE SPRINGLE installation The subdirectory USER is only insta
174. onal antibodies studied by surface plasmon resonance Immunol Let 31 31 31 31 59 64 24 Watts H J and C R Lowe Watts H J and C R Lowe Watts H J and C R Lowe Watts H J and C R Lowe 1994 Optical biosensors for monitoring microbial cells Anal Chem 66 66 66 66 2465 2470 25 Brigham Brigham Brigham Brigham Burke M J R Edwards and D J O Shannessy Burke M J R Edwards and D J O Shannessy Burke M J R Edwards and D J O Shannessy Burke M J R Edwards and D J O Shannessy 1992 Detection of receptor ligand interactions using surface plasmon resonance model studies employing the HIV 1 gp 120 CD4 interaction Anal Biochem 205 205 205 205 125 131 26 Johne B M Johne B M Johne B M Johne B M Gadnell and K Hansen Gadnell and K Hansen Gadnell and K Hansen Gadnell and K Hansen 1993 Epitope mapping and binding kinetics of monoclonal antibodies studied by real time biospecific interaction analysis using surface plasmon resonance J Immunol Methods 160 160 160 160 191 198 27 Wood S J Wood S J Wood S J Wood S J 1993 DNA DNA hybridisation in real time using BIAcore Microbiochem J 47 47 47 47 330 337 Chapter 9 165 Chapter Chapter Chapter Chapter 9999 9 9 9 9 Maintenance Maintenance Maintenance Maintenance 9 1 9 1 9 1 9 1 Index Index Index Index Chapter 9
175. ontents Table of contents Table of contents Welcome 2 Safety rules 4 Chapter 1 11 1 Hardware Installation 11 1 1 Index 11 1 2 Introduction 12 1 3 Computer requirements 12 1 4 Autolab TWINGLE SPRINGLE Hardware 12 1 5 Specifications 13 1 6 Hardware Installation 17 1 7 SPR and ESPR setup 18 1 8 Chemical Resistance 21 1 9 Materials 24 Chapter 2
176. opic techniques 146 include sequence subroutine sequence 123 Instrument Precautions 4 Interaction Peptide MHC 155 Protein antibody 155 Protein cell 155 Protein DNA 155 Protein polysaccharide 155 Protein T cell receptor 155 Protein virus 155 Interactions 153 154 155 KEL F 24 Kinetic 116 154 Kretschmann configuration
177. orts of SPR and PGSTAT 5 Connect cell cables to electrodes in the cuvette A1 ESPR cuvette A2 SPR cuvette 20 Hardware Installation Figure 1 3 Figure 1 3 Figure 1 3 Figure 1 3 AAAA The Electrochemical cuvette The Electrochemical cuvette The Electrochemical cuvette The Electrochemical cuvette and the normal SPR cuvette and the normal SPR cuvette and the normal SPR cuvette and the normal SPR cuvette Figure 1 3 Figure 1 3 Figure 1 3 Figure 1 3 BBBB The Electrochemi The Electrochemi The Electrochemi The Electrochemical cuvette cal cuvette cal cuvette cal cuvette B A 1 The electrochemical SPR TWINGLE cuvette with the three electrode connections WE RE and CE 2 The normal SPR cuvette for the Twingle Compared with the SPRINGLE cuvettes the SPRINGLE will have ONE channel less and ONE drain less B Connecting the cuvette electrodes to the potentiostat a Counter electrode wire for the platinum bar CE black b Reference electrode wire for the Ag AgCl RE blue c Working electrode wire for the gold contact WE red d Connecting the CE of the cuvette with the CE of the potentiostat e Connecting the RE of the cuvette with the RE of the potentiostat f Connecting the WE of the cuvette with the WE of the potentiostat g Connecting the ground of the electrode wires with the ground of the potentiostat Chapter 1 21 1 8 1 8 1 8 1 8 Chemical Res
178. ould be in command of Figure 2 Figure 2 Figure 2 Figure 2 5555 Installation window 5 Installation window 5 Installation window 5 Installation window 5 Press Next to proceed with the installation procedure A Autolab A Autolab A Autolab A Autolab SPR SPR SPR SPR or rename or rename or rename or rename B Autolab B Autolab B Autolab B Autolab TWINGLE TWINGLE TWINGLE TWINGLE Figure 2 Figure 2 Figure 2 Figure 2 6666 Installation window 6 Installation window 6 Installation window 6 Installation window 6 Twingle Twingle Twingle Twingle Not necessary but the name of the destination folder can be changed So for the SPRINGLE installation the name can be changed in SPRINGLE Again it is not necessary Press OK to confirm the new folder name and location 30 Software Installation Figure 2 7 Figure 2 7 Figure 2 7 Figure 2 7 Installation window 7 Installation window 7 Installation window 7 Installation window 7 When the folder does not exist press Yes to create the folder If the folder name has been changed into Autolab SPRINGLE this box would ask for confirmation as well A A A A Original folder location Original folder location Original folder location Original folder location Autolab SPR Autolab SPR Autolab SPR Autolab SPR B B B B New folder location New folder location New folder location New fo
179. ould be placed so that it cannot be damaged The main cable should not be bent laid over sharp edges walked over or exposed to any chemicals If the insulation on the main cable has been damaged this may cause electric shocks and or fire Replace or repair faulty or frayed insulation on power cords and control cables Autolab SPR User manual Replace control cables only with original spare parts When replacing a power cord use only approved type consistent the regulations Check all connected equipment for proper grounding Do not attempt to move the instrument with power cords connected This instrument may only be connected to a power supply that has the voltage and frequency stated on the type plate It may only be connected to the power supply using the power cable provided Incorrect voltages may damage the instrument Disconnect the power cord during thunderstorms Voltage surges from lightning strikes or other causes may damage the instrument through the main power supply Customer service Customer service Customer service Customer service Metrohm Autolab B V and its worldwide network of distributors provide you with instrument service and help with technical questions If you need assistance please contact your local representative On our web page www Metrohm Autolab com we maintain an up to date list with address details of our distributors Autolab SPR User manual 7 Table of contents Table of c
180. pincoater To spincoat standard gold disks 100 10 000 rpm optional Weight 23 kg Dimensions H x W x D 330mm x 400mm x 360mm Interface RS 232 Power requirements 170 W 100 240 V 50 60 Hz Figure 1 1 Figure 1 1 Figure 1 1 Figure 1 1 A A A A The Autolab The Autolab The Autolab The Autolab TWINGLE TWINGLE TWINGLE TWINGLE Chapter 1 15 Table 2 Specification of the Autolab SPRINGLE system TTTTechnical Specification echnical Specification echnical Specification echnical Specification Definition Definition Definition Definition Measuring principle Surface plasmon resonance Transducer principle Scanning mirror Liquid handling Cuvette system Parallel channels One Fixed wavelength 670 nm Sample loading and injection Manual or semi automatic Mixing Continuous wall jet Pumps 1 x Syringe pump 1 x peristaltic pump Flow rate range 0 8 l s 227 3 l s Syringe pump 30 l s 130 l s Peristaltic pump Sample volume 20 l 150 l Offset of SPR angle by spindle 62 78 Dynamic range 4000 m Angle resolution lt 0 02 m Minimum molecular weight 180 D Association constant range 10 3 10 7 M 1s 1 Dissociation constant range 10 5 10 1 s 1 Equilibrium affinity 10 4 10 10 M 1 Concentration range 10 11 10 3 M Refractive index 1 26 1 38 standard optional 1 32 1 44 or 1 40 1 52 Refractive index resolution lt 1 10 7 Measuring
181. position on the right in the assembled sequence A user defined sequence can be stored or retrieved from disk it will have the extension seq A sequence can execute other include sequences this makes it possible to develop a standard set of sequences for general purposes These subroutine sequences are called include sequences During installation of the software a list of sequences and include sequences are stored in the Autolab SPR software directory All sequence commands and their functions are listed below Figure Figure Figure Figure 6666 1 1 1 1 Two ways to activate the Sequencer Two ways to activate the Sequencer Two ways to activate the Sequencer Two ways to activate the Sequencer via the Menu via the Menu via the Menu via the Menu Options or the Tool Options or the Tool Options or the Tool Options or the Toolbar button bar button bar button bar button Automation 120 Loads the parameters settings determined in the automation control window parameters tab Aspirate the sample volume filled out in the volume tab in pump 1 for channel 1 Dispense the sample volume filled out in the volume tab in pump 1 for channel 1 Aspirate the sample volume filled out in the volume tab in pump2 for channel 2 Aspirate the sample volume filled out in the volume tab in pump 2 for channel 2 Pump2 commands are available in Tw
182. procedure starts with using the Automation window First get some inside knowledge of the procedure itself Open the Sequence editor and browse for the Tw44 Curve a full kinetic plot_SA 50ul sample SEQ The experiment sequence set up 1 Baseline Read include sequence Tw44 Curve Baseline Phase seq starts at command line 60 2 Association Figure 3 Figure 3 Figure 3 Figure 3 2 2 2 26666 The sequence editor The sequence editor The sequence editor The sequence editor showing the sequence showing the sequence showing the sequence showing the sequence Tw44 Tw44 Tw44 Tw44 Curve Curve Curve Curve a full kinetic plot_SA 50ul sample SEQ a full kinetic plot_SA 50ul sample SEQ a full kinetic plot_SA 50ul sample SEQ a full kinetic plot_SA 50ul sample SEQ Chapter 3 61 Read include sequence Tw44 Curve_SA Association Phase 50ul sample seq starts at command line 109 3 Dissociation Read include sequence Tw44 Curve Dissociation Phase long seq starts at command line 135 4 Regeneration Read include sequence Tw44 Curve_SA Regeneration Phase seq starts at command line 192 5 Back to Baseline Read include sequence Tw44 Curve Back to Baseline Phase seq starts at command line 226 Within those include sequences commands for incubation times are written subsequently 1 Wait Baseline s line 94 2
183. programs Insert the Data acquisition installation software CD in the CD drive Wait until the automatic setup screen appears IF the automatic Setup screen does not appear after inserting the CD Select Run from the windows Start menu Browse to the Autolab SPR CD ROM Open SETUP EXE Select OK in the RUN screen Figure 2 1 Figure 2 1 Figure 2 1 Figure 2 1 Start of the Setup procedure Start of the Setup procedure Start of the Setup procedure Start of the Setup procedure 28 Software Installation Figure 2 Figure 2 Figure 2 Figure 2 2222 Installation window 2 Installation window 2 Installation window 2 Installation window 2 This window appears for a very short time and disappears automatically Figure 2 Figure 2 Figure 2 Figure 2 3333 Installation window Installation window Installation window Installation window 3 Welcome 3 Welcome 3 Welcome 3 Welcome Press Next to proceed with the installation procedure Figure 2 Figure 2 Figure 2 Figure 2 4444 Installation window 4 for Autolab Installation window 4 for Autolab Installation window 4 for Autolab Installation window 4 for Autolab TWINGLE TWINGLE TWINGLE TWINGLE Press Next to proceed with the installation procedure Chapter 2 29 Select the Autolab SPR instrument type TWINGLE which the software sh
184. pter 4 73 The lift calibration sets the pipette position The cuvette position for the pipette is normally calibrated to 1 mm above the gold disk the middle position is calibrated with the pipette just about 2 mm inside the cuvette Procedure specified distances depend on the size of the used pipette tips 11 Press button Initialize lift 12 Enter a distance max 60 mm Start with e g 50 mm 13 Press the downward directed arrow button 14 Fine tune distance with 1 mm steps 15 Set new cuvette position 16 Move lift to top 17 Enter distance of 38 mm and fine tune with 1 mm steps 18 Set new middle position 19 Move lift to top 20 Close See Figure 4 7 a and b step 1 to 10 Figure 4 6 Figure 4 6 Figure 4 6 Figure 4 6 The lift calibration window The lift calibration window The lift calibration window The lift calibration window 74 Getting Started Autolab SPRINGLE 1 2 and 3 and 4 5 6 7 8 Figure 4 7 Figure 4 7 Figure 4 7 Figure 4 7 a The lift calibration procedure a The lift calibration procedure a The lift calibration procedure a The lift calibration procedure 9 10 Fig 4 7 Fig 4 7 Fig 4 7 Fig 4 7 b The final steps of lift calibration b The final steps of lift calibration b The final steps of lift calibration b The final steps of lift calibration Chapter 4 75 4 7 4 7 4 7 4 7
185. quences SPRINGLE has equal functionality SPRINGLE has equal functionality SPRINGLE has equal functionality SPRINGLE has equal functionality Chapter 5 107 5 7 5 5 7 5 5 7 5 5 7 5 Drain Drain Drain Drain The DRAIN button in the toolbar will switch ON or OFF the peristaltic drain pump The speed is defined in the manual control window Figure 5 18 Figure 5 18 Figure 5 18 Figure 5 18 Example of the Customize window to change a Example of the Customize window to change a Example of the Customize window to change a Example of the Customize window to change a linked linked linked linked sequence shown in the Menu_Twingle Wash sequence shown in the Menu_Twingle Wash sequence shown in the Menu_Twingle Wash sequence shown in the Menu_Twingle Wash SPRINGLE has equal functionality SPRINGLE has equal functionality SPRINGLE has equal functionality SPRINGLE has equal functionality Figure 5 Figure 5 Figure 5 Figure 5 19 19 19 19 Direct access to Direct access to Direct access to Direct access to start and stop the drain pump start and stop the drain pump start and stop the drain pump start and stop the drain pump Data Acquisition software 108 5 7 6 5 7 6 5 7 6 5 7 6 Place Event Marker Place Event Marker Place Event Marker Place Event Marker To mark a position on the data line during a measurement select the
186. quences for the Autolab equences for the Autolab equences for the Autolab equences for the Autolab SPRINGLE instrument SPRINGLE instrument SPRINGLE instrument SPRINGLE instrument Chapter 3 39 Chapter 3 Chapter 3 Chapter 3 Chapter 3 3 3 3 3 Getting started Getting started Getting started Getting started Autolab Autolab Autolab Autolab TWINGLE TWINGLE TWINGLE TWINGLE 3 1 3 1 3 1 3 1 Index Index Index Index Chapter 3 39 3 Getting started Autolab TWINGLE 39 3 1 Index 39 3 2 Introduction 40 3 3 Two days before the experiment 42 3 3 1 Gold surface modification with a 11 MUA layer 42 3 4 One day before the experiment 42 3 4 1 Washing the 11 MUA modified gold surface 42 3 4 2 Startup of the Autolab instrument 42 3 4 3 Liquid Handling set up Autolab 43 3 4 4
187. r the dilution of the samples 54 Getting Started Autolab TWINGLE 3 5 2 3 5 2 3 5 2 3 5 2 Set angle position of the sodium acet Set angle position of the sodium acet Set angle position of the sodium acet Set angle position of the sodium acetate buffer ate buffer ate buffer ate buffer Put 50 l of acetate buffer on the gold disk and check the dip by selecting the Scope Mode button To deactivate the scope mode press this button again The scope mode will update the SPR angle every 0 5 seconds Every TWINGLE instrument is calibrated with water on a bare gold disk The lowest value of the SPR dip is between 0 and 10 percent absolute reflection To change the position of the dip release the retaining screw of the optical path Adjusting the position of the dip is done by turning the micrometer spindle Click on start measurement in the tool bar and follow the change of the angle in real time Set the baseline around 1500 millidegrees m After adjusting the baseline fasten the retaining screw again Figure Figure Figure Figure 3 19 3 19 3 19 3 19 SPR dip SPR dip SPR dip SPR dip Figure Figure Figure Figure 3 20 3 20 3 20 3 20 The optical path cover The optical path cover The optical path cover The optical path cover Figure Figure Figure Figure 3 21 3 21 3 21 3 21 Adjustment of the baseline angle before immobilizat
188. rate wait period Defined in the Automation Window Wait periods Defined in the Automation Window Wait periods Defined in the Automation Window Wait periods Defined in the Automation Window Wait periods Defined in the Automation Window Chapter 6 123 6 4 6 4 6 4 6 4 Software Sequence editor Software Sequence editor Software Sequence editor Software Sequence editor The Sequence Editor toolbar see Figure 5 3 contains the shortcuts to the menu items new sequence open sequence save sequence print numbering and execute respectively The functions for the menu and toolbar items will be explained in the next section 6 4 1 6 4 1 6 4 1 6 4 1 The sequence editor menu and toolbar The sequence editor menu and toolbar The sequence editor menu and toolbar The sequence editor menu and toolbar New Sequence Clears the sequence editor window Set temperature of the waterbath Julabo or Lauda Set temperature of the waterbath and wait until the temperature is reached before continuing with the next sequence step Figure Figure Figure Figure 6666 3333 The menu The menu The menu The menu bar and tool bar and tool bar and tool bar and tool bar buttons bar buttons bar buttons bar buttons Figure 6 Figure 6 Figure 6 Figure 6 4 4 4 4 The sequence menu The sequence menu The sequence menu The sequence menu Automation
189. rface and cause an electromagnetic surface wave called surface plasma oscillations Ritchie 1957 Remarks Definition of plasma plasma is a medium with equal concentration of positive and negative charges of which at least one charge type e g electron is mobile e g metal Metals are conductors of electricity and insulators like plastics glass are called dielectrics All kind of components liquid gasses metals and salts have a dielectric constant Electron charge fluctuations are possible in the volume of a plasma and in its boundary with a dielectric The SPR situation has only the boundary electron charge fluctuation because the metal layer is very thin Legend Beam 1 is a refracted beam Beam 2 is a reflected beam O intensity incoming light R intensity reflected light T intensity refracted light i angle of incident light r angle of reflected light t angle of refracted light i beam 2 r beam 2 air refractive index of air 1 glass refractive index of glass 2 in general air lt glass hemi cylinder Beam 1 Beam 2 Beam 1 T Beam 2 R air glass 0 0 t r i air glass Figure Figure Figure Figure 8888 1 1 1 1 An overview of light beams passing through the hemi An overview of light beams passing through the hemi An overview of light beams passing through the hemi An overview of light b
190. rical SPR dip consequences fig 10 7 Noisy signals when a new SPR dip is located at the disturbance of the first dip see above see above SPR dip shifted up intensity minimum gt 10 SPR curve also broader 1 Particles on surface 2 Air bubbles in cuvette hydrophobic surfaces Wash cuvette thoroughly Wash cuvette thoroughly avoid to drain the cuvette completely for hydrophobic surfaces like bare gold and polystyrene coatings Figure Figure Figure Figure 10 10 10 10 7 7 7 7 Consequences of an Consequences of an Consequences of an Consequences of an unsymmetri unsymmetri unsymmetri unsymmetrical SPR cal SPR cal SPR cal SPR dip dip dip dip Figure Figure Figure Figure 10 10 10 10 8 8 8 8 SPR dip SPR dip SPR dip SPR dip shifted up shifted up shifted up shifted up Figure Figure Figure Figure 10 10 10 10 6 6 6 6 Unsymmetrical SPR Unsymmetrical SPR Unsymmetrical SPR Unsymmetrical SPR dip dip dip dip Chapter 11 179 Chapter 1 Chapter 1 Chapter 1 Chapter 11111 11 11 11 11 Figures Figures Figures Figures Figures 180 Table of figures Table of figures Table of figures Table of figures Figure 1 1 A The Autolab TWINGLE 14 Figure 1 1 B The Autolab SPRINGLE
191. roduction Two approaches are available to study macromolecular interactions with the biosensor The interactions can be studied directly or indirectly Direct measurements monitor the binding of analytes with immobilized or coated ligands A direct measurement can be performed in multiple steps i e sequential binding of two or more components If the molecular weight of an analyte is too low for detection direct measurements are not possible In that case binding of the analyte can be determined by an indirect measurement Before a high molecular weight analyte is added to the immobilized or coated ligand the binding sites are blocked by pre incubation with a low molecular weight compound The blocked analyte is not able to bind the ligand anymore and therefore the binding curve will disappear partially or totally With indirect measurements the response is related to the amount of unblocked analyte and thereby to the amount of blocker added during pre incubation In an indirect measurement two components compete with each other in a parallel process and not in a serial process as in multiple direct measurements The serial and parallel interaction processes can be combined freely Direct and indirect measurements can be performed with the SPR disk and the sensor chip An important possibility of SPR is the determination of kinetic parameters of biomolecular interactions and determination of analyte concentrations It is possible to sep
192. rom the stock solution into a 1 5 ml vial and add 900 l HEPES buffer 2 Dilution 2 1 16000 Pipette 200 l from dilution 1 into a 1 5 ml vial and add 200 l HEPES buffer 3 Dilution 3 1 32000 Pipette 200 l from dilution 2 into a 1 5 ml vial and add 200 l HEPES buffer 60 Getting Started Autolab TWINGLE 3 6 2 3 6 2 3 6 2 3 6 2 Stabilize the surface Stabilize the surface Stabilize the surface Stabilize the surface Change the coupling buffer in the flask with the HEPES buffer use the initialization of instrument seq Stabilize the surface as described in section 3 5 3 of the immobilization paragraph Thiol layers Dextran layers or surfaces with immobilized ligands have to be stabilized to minimize matrix effects that are caused by differences in pH or ionic strength high low salt concentrations of the different buffers used throughout the experiment The matrix effects influence the SPR signal Due to exposure of the layer with the different buffers of the experiment the layer will respond in a more predictive way and will continuously give a SPR signal at the same angle When the desired stability is reached the sequence can be stopped at any time by clicking the Stop measurement button in the tool bar 3 6 3 3 6 3 3 6 3 3 6 3 Start the association procedure Start the association procedure Start the association procedure Start the association procedure The association
193. rs Prints a hardcopy of the binding plot Prints a hardcopy of the SPR signal Defines aspirate volume in l for channel 1 Defines dispense volume in l for channel 1 Defines pump 1 flow in l s for channel 1 Starts mixing pump 1 advice always in Lift Down position Stops mixing pump 1 Defines mix volume in l for channel 1 Switches pump valve position to needle pipette tip for pump 1 Switches pump valve position to buffer for pump 1 Automation 122 Defines aspirate volume in l for channel 2 Defines dispense volume in l for channel 2 Defines pump flow in l s for channel 2 Starts mixing pump 2 advice always in Lift Down position Stops mixing pump 2 Defines pump 2 mix volume in l for channel 2 Switches pump valve position to needle pipette tip for pump 2 Twingle only Switches pump valve position to buffer for pump 2 Defines pump 1 amp 2 flow in l s for channel 1 and 2 Starts mixing pump 1 amp 2 Stops mixing pump 1 amp 2 Twingle only Defines pump 1 amp 2 mix volume in l for channel 1 and 2 Records an SPR plot for channel 1 and 2 Adds event with text to event recorder Set Relative Response to zero Wait period in seconds Baseline wait period Defined in the Automation Window Associate wait period I Defined in the Automation Window Dissociate wait period Defined in the Automation Window Regene
194. s with thiol containing compounds Gold interacts with sulfur 14 By applying this property for peptides self assembled receptor layers were developed 15 Direct measurement of low molecular weight compounds by response enhancement with latex particles Low molecular weight compounds can be attached to carboxy modified latex by a carbodiimide coupling reaction 16 17 Direct binding of low molecular weight compounds coupled to latex particles can be determined using a coated ligand at the sensor surface Polymer coated surfaces A thin film of 20 30 nm of the polymer also used for ELISA microtiter plates can be attached to the gold surface of the SPR disk by spincoating ELISA methods can be used to study biomolecular interactions with the polymer coated gold surface Latex coating Latex particles can be immobilized with ligand by a carbodiimide coupling reaction 16 17 The latex particles can be coated on the SPR disk resulting in a biospecific latex layer covering the sensor surface An advantage of this layer is that total regeneration of the gold surface is possible with a sodium dodecyl sulphate SDS buffer solution 8 6 8 6 8 6 8 6 References References References References 1 Kooyman R P H H Kolkman J van Gent and J Greve Kooyman R P H H Kolkman J van Gent and J Greve Kooyman R P H H Kolkman J van Gent and J Greve Kooyman R P H H Kolkman J van Gent and J Greve
195. s modified with an offset in contrast to the previous option The adjust to zero option can be used to adjust both channels to zero 5 7 13 5 7 13 5 7 13 5 7 13 Lift Calibration Lift Calibration Lift Calibration Lift Calibration Figure Figure Figure Figure 5555 2 2 2 22222 Lift calibration window Lift calibration window Lift calibration window Lift calibration window Data Acquisition software 110 The lift calibration calibrates the pipette position The cuvette position for the pipette is normally calibrated to 1 mm above the gold disk the middle position is calibrated with the pipette just about 2 mm inside the cuvette Procedure specified distances depend on the size of the used pipette tips Initialize lift Enter a distance max 60 mm Start with e g 54 mm and fine tune with 1 mm steps Set new cuvette position Move lift to top Enter distance of 42 mm Set new middle position Move lift to top Close 5 7 14 5 7 14 5 7 14 5 7 14 System Parameters System Parameters System Parameters System Parameters System settings All hardware setup settings are defined in the System Settings These numbers should not be changed One important number to read is the Mirror Amp which refers to a specific instrument unique dynamic scanning Figure Figure Figure Figure 5555 23 23 23 23 System settings System setti
196. sampler is controlled by the Data Acquisition software The liquid handling circuit is shown in Figure 8 8 Figure Figure Figure Figure 8888 8 8 8 8 Schematic picture of the instrument Schematic picture of the instrument Schematic picture of the instrument Schematic picture of the instrument Two three Two three Two three Two three way valves the pump 1 valve channel 1 and the pump 2 way valves the pump 1 valve channel 1 and the pump 2 way valves the pump 1 valve channel 1 and the pump 2 way valves the pump 1 valve channel 1 and the pump 2 valve valve valve valve channel 2 de channel 2 de channel 2 de channel 2 determine the liq termine the liq termine the liq termine the liquid handling One uid handling One uid handling One uid handling One peristaltic peristaltic peristaltic peristaltic pump pump pump pump isisisis used used used used for draining and washing for draining and washing for draining and washing for draining and washing drain peristaltic pump cuvette gold layer prism half cylinder diode detector Waste flask Buffer flask Needle Channel 2 Needle Channel 1 To needle To buffer Syringe pump 2 Syringe Pump 1 Chapter 8 161 8 5 8 5 8 5 8 5 SPR methods SPR methods SPR methods SPR methods 8 5 1 8 5 1 8 5 1 8 5 1 Introduction Introduction Introduction Int
197. se proximity to or integrated with the signal transducer to give a reagentless sensing system specific to a target compound analyte Transducers are the physical components of the sensor that react to a signal due to the interaction between the biological sensing element and the target analyte Biosensing occurs only when the analyte is recognized specifically by the biological element The biological recognition elements can be divided into two distinct groups catalytic and non catalytic The catalytic group includes enzymes micro organisms and plant or mammalian tissue while the non catalytic or affinity class includes antibodies receptors and nucleic acids The interaction output can be amplified stored or displayed The advantage of a biosensor is the label free detection of the interaction The label free form in most cases is achieved by immobilizing the biological recognition element In general the immobilization matrix may function purely as a support or else it may also be concerned with mediation of the signal transduction mechanism associated with the analyte Immobilization techniques include physical entrapment by an inert membrane physical chemical adsorption binding to a functionalised support and entrapment in an active membrane Surface Plasmon Resonance is a specific biosensor a special case of the interaction of light with matter SPR signals are related to the refractive index close to the sensor surface
198. self or as a sequence variable that is defined in the automation window Below an example of the sequence Tw44 Curve a full kinetic plot_SA 50 l sample seq is explained Automation 138 Syringe starts in default middle position Lines 4 5 67 set syringe pumps to middle Line 9 Cleans the DA window Line 11 fills wash needle pipette tip and peristaltic tubing Lines 19 20 max aspiration volume to fill up the 500ul syringe barrel Lines 26 27 Dispenses volume to clean the needle pipette tip and cuvette Lines 31 32 dispense 50 l on the gold surface Line 36 will put the syringe to middle position Lines 38 39 together with 42 and 45 aspirate 50 l air into the needle pipette tip SPECIALS lines 7 28 37 46 The command to the valve of the syringe is used to let the syringe finish its aspirate or dispense actions before going to the next step in the sequence experiment The basic measurement routine is defined in a loop line 42 till line 274 which is repeated 1 times Lines 48 58 define all parameters for the measurements which is also done by Line 59 which is linked to the automation window parameters The baseline include sequence Lines 76 77 flush 125 l buffer over the gold Lines 80 81 define the baseline sample volume Lines 84 85 take up 50 l of air Lines 88 97 A measurement has a fixed set of commands start mixing line 88 start the measurement line 90
199. seline angle before immobilization Adjustment of the baseline angle before immobilization Adjustment of the baseline angle before immobilization Adjustment of the baseline angle before immobilization Why should the sodium acetate buffer SPR angle be set at Why should the sodium acetate buffer SPR angle be set at Why should the sodium acetate buffer SPR angle be set at Why should the sodium acetate buffer SPR angle be set at 1500 m 1500 m 1500 m 1500 m The solutions used in the immobilization differ significantly in refractive index and will change a number of times during the procedure The acetate buffer has the lowest refractive index and therefore the smallest SPR angle the Ethanolamine solution has the largest angle Channel 1 Fix retaining screw Release retaining screw Turn spindle to adjust baseline Around 1500 m Time s 82 Getting Started Autolab SPRINGLE Start the sequence and continue to wash until the baseline is sufficiently stable The sequence can be stopped at any time by clicking the end measurement button in the tool bar Eventually every solution should show the same SPR angle every time is it dispensed on the surface 4 8 4 4 8 4 4 8 4 4 8 4 Start the immobilization procedure Start the immobilization procedure Start the immobilization procedure Start the immobilization procedure The EDC NHS immobilization procedure is a standardized procedure that can
200. sociation phase is performed by washing the sample away with association buffer The association buffer is the same buffer as the dilution buffer for the anti BSA The dissociation phase is used for determining the strength of the interaction After the dissociation phase all bound anti Insulin is removed from the Insulin coated gold disk by diluted SDS Sodium dodecyl sulphate regeneration buffer Then the baseline of the modified disk will be restored by washing the regeneration solution replacing it with the baseline HEPES buffer BSA BSA complex K association K dissociation Chapter 4 67 Figure 4 Figure 4 Figure 4 Figure 4 1 1 1 1 Flow chart of Flow chart of Flow chart of Flow chart of the experimental setup the experimental setup the experimental setup the experimental setup After one initial immobilization numerous SPR experiments can be performed The surface plasmon resonance SPR measures angle versus time There is a linear relationship between the amount of bound material and shift in SPR angle The SPR angle shift in millidegrees m is used as a response unit to quantify the binding of macromolecules to the sensor surface A change of 122 m represents a change in surface protein mass of approximately 1 ng mm 2 MUA coating of the gold disk Stabilization BSA layer Assembling hemi cylinder gold disk Start up instrument Stabilization of the MUA layer
201. software 104 5 7 3 5 7 3 5 7 3 5 7 3 Inject Inject Inject Inject Figure 5 13 Figure 5 13 Figure 5 13 Figure 5 13 Example of t Example of t Example of t Example of two wo wo wo TWINGLE TWINGLE TWINGLE TWINGLE DA screens with Lift Positions DA screens with Lift Positions DA screens with Lift Positions DA screens with Lift Positions choices choices choices choices via Menu bar or Toolbar via Menu bar or Toolbar via Menu bar or Toolbar via Menu bar or Toolbar SPRINGLE has e SPRINGLE has e SPRINGLE has e SPRINGLE has equal functionality qual functionality qual functionality qual functionality Figure 5 14 Figure 5 14 Figure 5 14 Figure 5 14 Two Two Two Two TWINGLE TWINGLE TWINGLE TWINGLE DA scr DA scr DA scr DA screens showing two ways eens showing two ways eens showing two ways eens showing two ways to get to get to get to get quick quick quick quick access to access to access to access to customize and to customize and to customize and to customize and to sequences sequences sequences sequences SPRINGLE has equal functionality SPRINGLE has equal functionality SPRINGLE has equal functionality SPRINGLE has equal functionality Chapter 5 105 Press the toolbar inject picture and the Customize information window pops up The Customize window is needed for selection of a sequence that will be linked Select Customize and the customi
202. ssociation procedure Start the association procedure Start the association procedure Start the association procedure The experimental association procedure is a standardized procedure that can be easily performed semi automatically Open the sequence editor Figure 4 21 Figure 4 21 Figure 4 21 Figure 4 21 The sequence editor The sequence editor The sequence editor The sequence editor showing the sequence showing the sequence showing the sequence showing the sequence immobilization SEQ immobilization SEQ immobilization SEQ immobilization SEQ 84 Getting Started Autolab SPRINGLE window and select the file Curve SA a full kinetic plot SEQ The lines 67 120s 86 3600s 117 600s 146 300s and 178 120s are the incubation times for each different step in the full kinetic plot procedure With a double click on the command line the settings can be edited Select the green button with the arrow to start the experiment Whenever an action of the user is required a message alert will pop up with information to act upon 4 10 4 10 4 10 4 10 The SPRINGLE The SPRINGLE The SPRINGLE The SPRINGLE Data Data Data Data Figure 4 22 Figure 4 22 Figure 4 22 Figure 4 22 Sequence Editor Window Sequence Editor Window Sequence Editor Window Sequence Editor Window showing the curve showing the curve showing the curve showing the curve
203. taltic pump tubing Peristaltic pump tubing Peristaltic pump tubing Figure 1 7 Figure 1 7 Figure 1 7 Figure 1 7 Syringe pump Syringe pump Syringe pump Syringe pump Item Item Item Item Material Material Material Material 1 Cuvette standard KEL F special PVDF 2 Fitting Tefzel ETFE 3 Tubing Teflon 4 Connector peristaltic tubing PVDF 5 Peristaltic pump tubing Pharmed 6 Valve Kel F 7 Syringe barrel Borosilicate glass 8 Syringe seal Teflon 9 Syringe plunger Stainless steel RVS Chapter 1 25 PVDF Polyvinyllidene Fluoride Excellent chemical resistance Ideal for the cuvette and tubing connections KEL F PCTFE polychloro trifluoroethylene Excellent chemical resistance ideal for fittings and sealing surfaces THF tetrahydrofuran and a few halogenated solvents will react with it PEEK Polyetheretherketone Excellent chemical resistance although not recommended with nitric acid sulphuric acid halogenated acids and pure halogenated gases Also a swelling effect occurs with methylene chloride THF and DMSO Teflon FEP and PFA Fluorinated ethylene propylene and perfluoroalkoxy alkane Inert to virtually all chemicals Tefzel ETFE Ethylene tetrafluoroethylene Excellent solvent resistance Kel Kel Kel Kel FFFF PEE PEE PEE PEEkkkk PVDF PVDF PVDF PVDF FEP PFA FEP PFA FEP PFA FEP PFA Tefzel Tefzel Tefzel Tefzel Solvent Sol
204. tensity of the reflected light p polarized with a wave length of 670 nanometer is measured over a range of 4000 millidegrees A scanning mirror with a frequency of 76 Hertz is used to obtain an angle scan of 4000 millidegrees in approximately 13 milliseconds The SPR angle of a buffer solution can be fixed manually by a spindle with an offset SPR angle of 62 78 degrees which corresponds to a refractive index range of 1 33 1 43 of the sensor surface The SPR angle scan is performed around the manually fixed SPR position The optical reflectance of incident light at different angles around the fixed SPR angle for a buffer solution is measured while the laser beam is kept at one spot of the sensor surface of approximately 2 mm 2 This is accomplished by applying cylindrical optics A half cylinder is used as a prism for the optical contact with the sensor surface The optics are designed in such a way that a parallel light beam will be inside the half cylinder while scanning The function of the cylindrical lens is two fold It projects the rotating axis of the vibrating mirror at the centre of the hemi cylinder and compensates its converging effect The advantage of the Twingle optical configuration is that unwanted defects in the ligand specific layer will be averaged and artefacts due to spatial inhomogeneties are eliminated The optical configuration results in an accurate reproducible and sensitive detection In the vibrating mirror set up 7
205. thanol 10 100 Formic acid 1 20 Glycine 0 01 2 M 2 5 3 5 Phosphoric acid 0 1000 mM SDS 5 20 Sodium carbonate 200 mM 11 5 Sodium chloride 1000 mM Sodium hydroxide 10 1000 mM Table 1 7 3 Organic solvents NOT NOT NOT NOT recommended for the Autolab TWINGLE instrument Chapter 1 23 Solution Solution Solution Solution Concentration Concentration Concentration Concentration Solution Solution Solution Solution Concentration Concentration Concentration Concentration Acetone 100 Ethyl chromide 100 Amyl acetate 100 Methyl Ethyl Ketone 100 Benzene 100 Methylene Chloride 100 Butyl alcohol 100 Nitric Acid 100 Carbon tetrachloride 100 Pyridine 100 Chlorine 100 Sulphuric Acid 100 Chloroform 100 Toluene 100 Chromic acid 100 Trichloroethylene 100 Cyclohexane 100 Xylene 100 Ethyl acetate 100 24 Hardware Installation 1 9 1 9 1 9 1 9 Materials Materials Materials Materials Figure Figure Figure Figure 1 5 1 5 1 5 1 5 Peristaltic pump Peristaltic pump Peristaltic pump Peristaltic pump Figure 1 4 Figure 1 4 Figure 1 4 Figure 1 4 Cuvette tubing and fitting Cuvette tubing and fitting Cuvette tubing and fitting Cuvette tubing and fitting Figure 1 6 Figure 1 6 Figure 1 6 Figure 1 6 Peristaltic pump tubing Peris
206. the interaction of the sample anti insulin antibody with the BSA protein molecule 3 5 3 5 3 5 3 5 DDDD day day day day The immobilization The immobilization The immobilization The immobilization 3 5 1 3 5 1 3 5 1 3 5 1 Sample preparation Sample preparation Sample preparation Sample preparation For the immobilization procedure prepare the following samples and solutions Coupling buffer for baseline and wash steps Preparation of coupling buffer 10 mM Acetate buffer pH 4 5 Dissolve 68 4 mg NaAc in 45 ml demi water Adjust the pH to 4 5 with acetic acid Adjust volume to 50 ml with demi water The pH of the coupling buffer depends on the pI of the ligand protein The general rule for the pH of coupling buffer is pHbuffer pIligand 0 5 Figure 3 18 Figure 3 18 Figure 3 18 Figure 3 18 TWINGLE TWINGLE TWINGLE TWINGLE The sequence Initialization of The sequence Initialization of The sequence Initialization of The sequence Initialization of IIIInstrument SEQ nstrument SEQ nstrument SEQ nstrument SEQ Chapter 3 53 EDC NHS activation solution 60 l of 1 1 freshly mixed 0 4 M EDC and 0 1 M NHS in a 1 5ml vial Preparation of 400 mM EDC solution Dimethylaminopropyl N Ethylcarbodiimide N 3 hydrochloride MW 191 70 Fluka 03449 Weigh 153 4 mg of EDC in a 3 ml vial and dissolve it in 2 ml demi water Preparation of 100mM NHS so
207. the waste bottle green tubing inserted into the waste bottle green tubing inserted into the waste bottle green tubing inserted into the waste bottle green tubing in reality in reality in reality in reality Both Both Both Both syringe syringe syringe syringe tube tube tube tubessss are are are are inserted into the buffer flask inserted into the buffer flask inserted into the buffer flask inserted into the buffer flask SPRINGLE has only ONE SPRINGLE has only ONE SPRINGLE has only ONE SPRINGLE has only ONE syringe tubing syringe tubing syringe tubing syringe tubing Com port Main switch 44 Getting Started Autolab TWINGLE 3 4 4 3 4 4 3 4 4 3 4 4 Initialization o Initialization o Initialization o Initialization of the instrument f the instrument f the instrument f the instrument Before using the instrument Calibrate lift positions Assemble a new sensor disk Assemble the cuvette Fill the tubing with running buffer solution 3 4 5 3 4 5 3 4 5 3 4 5 Autolab lift Autolab lift Autolab lift Autolab lift position calibration position calibration position calibration position calibration There are three ways to find out if the lift is calibrated In the menu TWINGLE SPRINGLE find the lift position item to check the positions up middle down In the menu TWINGLE SPRINGLE find the Manual control item to check the thre
208. theories necessary to model SPR He introduced the displacement current and wrote down the relations between the electric and magnetic fields now known as the Maxwell s Equations Loss of light incident onto a grating was first observed by R W Wood in 1902 while he was studying diffracted spectra of metallic gratings Woods anomalies In 1941 Frano suggested an excitation of electromagnetic surface waves Pines and Bohm assumed that the observed energy losses were due to the excitation of plasma oscillations or plasmons of the conducting electrons 1951 This was in 1957 theoretically explained as Surface Plasmons SP by Ritchie This theory was confirmed experimentally in reflection studies by Powell and Swan 1959 Later Stern and Ferrell observed significant effect on the angle of incidence at which energy losses occurred 1960 Turbadar presented experimental results of the SPR phenomena and showed that it could be predicted by the thin film theory 1968 Otto invented the attenuated total reflection ATR method to excite a surface plasmon Between the prism and metal layer is a layer with air In 1971 the method was improved by Kretschmann by applying a thin metal film directly onto an ATR prism denoted the Kretschmann configuration The Kretschmann configuration is the most used configuration which is also the basic configuration of the Autolab Twingle instrument Figure 8 3 Theory Theory Theory Theory Surface Plas
209. tion Lines 247 248 250 to put the syringe in the middle position Lines 252 253 259 260 aspirate 50 l air This to prevent contamination of the buffer in the tubing with the sample in the cuvette Lines 263 to 271 The fixed set of commands to perform the measurement SPECIALS lines 243 251 261 The syringe finishes its aspirate or dispense actions before going to the next step in the sequence experiment Lines 225 272 Define a loop 2x for measuring the baseline twice Lines 231 249 Define a loop 2x to wash the gold twice Line 227 Tw44 Curve Inject 50 l buffer back to baseline Aspirates 500ul buffer from flask to wash the needles with 500ul and subsequently 50 l is used for the incubation The loop will repeat this procedure 2x Chapter 6 141 Line 273 saves the experiment with a file name defined in the automation window Line 274 finish the experiment Line 275 to be sure all actions are stopped Lines 285 335 Preserve the quality of the molecules attached to the gold surface Lines 295 296 flush 150 l buffer to wash the surface Lines 299 305 drain away the solution from the gold Lines 307 308 the buffer sample to measure the baseline Lines 311 aspirate 50 l air This to prevent contamination of the buffer in the tubing with the sample in the cuvette Every hour the solution will be replace by a new fresh solution Line 329 1 hour of incubation Line 326 333 w
210. tion by thiol functional groups of ligands is performed by a thiol coupling reagent 1 activation of carboxymethyl groups to an NHS ester by EDC NHS chemistry 2 reaction with 2 2 pyridinyldithio ethaneamine PDEA to introduce reactive disulfide bridges 3 reaction of disulfide bridges with thiol ligand groups 4 deactivation of excess disulfides with cysteine O O HO N C O O O O N C O OH C N N R1 R2 C O O C N N R1 R2 H C N N R1 R2 H H O H2N R H C O N R C O O O O N H2NCH2CH2OH H C O N CH2CH2O HO O O N O O HO N SPR Theory 158 Step 1 Step 2 Step 3 Step 4 CH2CH2S S H C O N R N S H HS R CH2CH2S S H C O N N CH2CH2SS H C O N CH2CCO2H NH2 H NH2 H HSCH2CCO2H CH2CH2S S H C O N N C O OH C N N R1 R2 C O O C N N R1 R2 H C O O O O N C N N R1 R2 H H O O O HO N C O O O O N N SSCH2NH2 CH2CH2S S H C O N N O O HO N Chapter 8 159 8 4 3 8 4 3 8 4 3 8 4 3 Cuvette Cuvette Cuvette Cuvette The cuvette limits the physical parameters for the reaction volume and position of the reaction on the gold layer The disk Eco Chemie B V
211. to the gold surface To get a reproducible quality thiol layer filter the solution prior to the incubation and perform the incubation overnight 3 4 3 4 3 4 3 4 One day before the experiment One day before the experiment One day before the experiment One day before the experiment 3 4 1 3 4 1 3 4 1 3 4 1 Washing Washing Washing Washing the the the the 11 11 11 11 MUA MUA MUA MUA modified modified modified modified gold surface gold surface gold surface gold surface Wash the disk three times with alcohol to remove the excess thiol To remove the alcohol rinse three times with demineralized water Blow the disk dry with compressed air or nitrogen gas The thiol covered gold disk can be stored dry up to 2 months in the original container 3 4 2 3 4 2 3 4 2 3 4 2 Startup of the Autolab i Startup of the Autolab i Startup of the Autolab i Startup of the Autolab instrument nstrument nstrument nstrument Install the power supply cable and connect the com port of the instrument to the computer with the RS232 connector cable Switch on the Autolab instrument Use the main switch on the back panel Figure 1 1 and the power button situated at the right top Figure 3 2 Figure 3 2 Figure 3 2 Figure 3 2 6 Well Microtiter culture plate 6 Well Microtiter culture plate 6 Well Microtiter culture plate 6 Well Microtiter culture plate Chapter 3 43 posit
212. tories The study and characterisation of molecular interactions is essential to explore the structure function relationships of biomolecules and to aid our understanding of biological systems in life sciences like antibody antigen ligand receptor protein nucleic acid cell adhesion and drug development With the option of electrochemistry an extra tool is available for the study of enzymes ion channels membrane proteins polymer layers polymerisation and polymer biomolecular interactions SPR provides quantitative information which can be used to determine reaction kinetics and affinity constants for molecular interactions as well as the active concentration of biomolecules in solution It also provides qualitative information and allows small molecule screening to epitope mapping and complex assembly studies The SPR technique is related to the nature of the surface plasmon Methods of optical excitation and basic properties of surface plasmon resonance that are important in the sensor application are summarized in this manual The optical detection principle of the Autolab SPR instrument has been derived from technology developed at the University of Twente The Netherlands which was supported financially by the Dutch Foundation of Technical Sciences STW The hardware and software of the Autolab instrument was developed by Metrohm Autolab BV Another SPR system is the Autolab ESPRIT a fully automated double channel SPR system The user
213. uence then the remark is already specified between the brackets in the sequence See for example the event text lines like 1 association and 2 association in Figure 4 32 and Figure 4 33 Remarks The event log remarks can be edited with a double click on the line Deleting event log lines is also possible If a marker is deleted and a new marker is put onto the DA plot the event log will continue to increment the number If the position of the new marker is coincidently the same position as the deleted marker the old marker number will be used A SPR plot can handle up to twelve updates of recorded dips Chapter 5 117 Figure 5 Figure 5 Figure 5 Figure 5 32 32 32 32 An example of a kinetic An example of a kinetic An example of a kinetic An example of a kinetic experiment experiment experiment experiment with event log data with event log data with event log data with event log data The upper The upper The upper The upper line is c line is c line is c line is channel hannel hannel hannel 1111 t t t the lower line is channel he lower line is channel he lower line is channel he lower line is channel 2222 data The data The data The data The middle middle middle middle curve is the differential angle curve curve is the differential angle curve curve is the differential angle curve curve is the differential angle curve between channel between cha
214. uencer 5 Click sequence in the menu bar and select Open sequence 6 Select from the list of sequences the file Initialization of Instrument SEQ 7 Press the green arrow toolbar button to run the procedure to fill exchange the tubing solution with the buffer from the buffer flask 8 The procedure will flush the liquid handling system which will give the opportunity to check for leaks at tubing connectors At this point the sensor disk can be exchanged Do not forget to check for leakage after disk exchange The instrument is now ready for use the 11 MUA modified gold disk and the cuvette are assembled correctly the tubing is filled with the correct buffer The measurement can start The experiments consist of two parts The first part is to physically attach the BSA protein molecule to the 11 MUA surface This chemical binding step is called immobilization The second part is the interaction of the sample anti BSA antibody with the BSA protein molecule Figure 4 15 Figure 4 15 Figure 4 15 Figure 4 15 Two ways to activate the Sequencer Two ways to activate the Sequencer Two ways to activate the Sequencer Two ways to activate the Sequencer via the Menu via the Menu via the Menu via the Menu Options or Options or Options or Options or the Toolbar button the Toolbar button the Toolbar button the Toolbar button Figure 4 16 Figure 4 16 Figure 4 16 Figure 4 16 S
215. uffer solution B 4 Dilute 100 l 10x with 900 l 10mM Acetate buffer B 4 B 6 Preparation of 100mM NHS solution Weigh 23 mg of NHS Mw 115 09 A 8 in a 3 ml vial and dissolve it in 2 ml demi water B 7 Preparation of 400mM EDC solution 70 Getting Started Autolab SPRINGLE Weigh 153 4 mg of EDC Mw 191 70 A 9 in a 3 ml vial and dissolve it in 2 ml demi water B 8 Preparation of 1M Ethanolamine solution Pipette 600 l of Ethanolamine in a 25 ml flask dilute it with 10 ml demi water and adjust the pH to 8 5 with 1 M HCl Preparation of anti BSA dilutions B 9 Dilution 1 1 100 Pipette 5 l of the concentrated stock solution anti BSA A 6 in a vial of 1 5 ml dilute this with 500 l HEPES buffer Mix by pipetting several times up and down B 10 Dilution 2 1 300 Pipette 100 l from dilution 1 into a 1 5 ml vial and add 200 l HEPES buffer B 11 Dilution 3 1 900 Pipette 100 l from dilution 2 into a 1 5 ml vial and add 200 l HEPES buffer When to prepare the solutions When to prepare the solutions When to prepare the solutions When to prepare the solutions _ EDC and NHS are not stable in solution Once prepared use it the same day and or store aliquots of 200 l at 20 C _ The pH of the acetate buffer can change in time so check the pH before use _ The ligand dissolved in acetate buffer should be freshly prepared The same preparation of acetate buffer should be used for all steps of the immobiliz
216. up see Figure 5 18 Via the Menu TWINGLE Wash a user defined sequence can be defined to be executed or choose to open the Customize window Figure Figure Figure Figure 5555 16 16 16 16 An example of a TWINGLE An example of a TWINGLE An example of a TWINGLE An example of a TWINGLE inject sequence inject sequence inject sequence inject sequence However However However However Line Line Line Line 70 72 70 72 70 72 70 72 has a defined incubation time which will be overwritten has a defined incubation time which will be overwritten has a defined incubation time which will be overwritten has a defined incubation time which will be overwritten by the time shown in the Customi by the time shown in the Customi by the time shown in the Customi by the time shown in the Customize ze ze ze Sequence Sequence Sequence Sequence tab sheet tab sheet tab sheet tab sheet Fig Fig Fig Fig 5555 15 15 15 15 Figure Figure Figure Figure 5 17 5 17 5 17 5 17 Two Two Two Two TWINGLE TWINGLE TWINGLE TWINGLE DA screens showing two ways to DA screens showing two ways to DA screens showing two ways to DA screens showing two ways to be be be be able to get quick access to able to get quick access to able to get quick access to able to get quick access to customize and to customize and to customize and to customize and to sequences sequences sequences se
217. ve their intensity maximum in the surface and exponentially decaying field perpendicular to it The surface plasmon is a p plane polarized surface bound electromagnetic wave propagating at the interface between a metal and a dielectric Our SPR system measurement principle Our SPR system measurement principle Our SPR system measurement principle Our SPR system measurement principle Surface plasmon resonance 1 4 occurs under certain conditions when a thin film of metal gold or silver is placed inside the laser beam When the incoming light is monochromatic and p polarized i e the electric vector component is parallel to the plane of incidence the free electrons of the metal will oscillate and absorb energy at a certain angle of incident light The angle of incidence at which SPR occurs is called the SPR angle SPR is detected by measurement of the intensity of the reflected light At the SPR angle a sharp decrease or dip in intensity is measured The position of the SPR angle depends on the refractive index in the substance with a low refractive index i e the sensing surface The refractive index of the sensor surface changes upon binding of macromolecules to the surface As a result the SPR wave will change and therefore the angle will change accordingly There is a linear relationship between the amount of bound material and shift in SPR angle 5 The SPR angle shift in millidegrees is used as a response unit to quantify the binding
218. vent Solvent Solvent Aromatics R R R R R Chlorinated M M R R R Ketones R R R R R Aldehydes R R R R R Ethers M M R R R Amines R R R R M Aliphatic sol R R R R R Organic Acids R M R R R Inorganic Acids R M R R M Bases R R R R R Sulfonated Compounds R M R R R Thread strength Thread strength Thread strength Thread strength Good Excellent Excellent Good Good R Recommended NR Not recommended M Moderate resistance 26 Software Installation Chapter 2 Chapter 2 Chapter 2 Chapter 2 2 2 2 2 Software Installation Software Installation Software Installation Software Installation 2 1 2 1 2 1 2 1 InInInIndex dex dex dex Chapter 2 26 2 Software Installation 26 2 1 Index 26 2 2 Introduction 27 2 3 Installation of the Autolab SPR software 27 2 4 Autolab SPR software setup on the hard disk after installation 34 2 4 1 Folder structure
219. with buffer or to change the running buffer as follows Select the menu bar item options Sequencer 1 Click sequence in the menu bar and select Open sequence 2 Select from the list of sequences the file Initialization of Instrument SEQ Figure 3 17 Figure 3 17 Figure 3 17 Figure 3 17 Two ways to activate the Sequencer Two ways to activate the Sequencer Two ways to activate the Sequencer Two ways to activate the Sequencer via the Menu via the Menu via the Menu via the Menu Options or the Toolbar button Options or the Toolbar button Options or the Toolbar button Options or the Toolbar button 52 Getting Started Autolab TWINGLE 3 Press the green arrow toolbar button to run the procedure to fill exchange the tubing solution with the buffer from the buffer flask 4 The procedure will flush the liquid handling system which will give the opportunity to check for leaks at tubing connectors At this point the sensor disk can be exchanged Do not forget to check for leakage after disk exchange The instrument is now ready for use the 11 MUA modified gold disk and the cuvette are assembled correctly the tubing is filled with the correct buffer The measurement can start The experiments consist of two parts The first part is to physically attach the Insulin protein molecule to the 11 MUA surface This chemical binding step is called immobilization The second part is
220. ze window will pop up On the tab page Sequences a link can be established to the inject sequence icon in the Menu_Twingle Inject see Figure 5 15 Using the menu TWINGLE Inject the name of the sequence which could be linked would be replacing the words not verified Inject Sequence not verified Using a measurement sequence the Measurement time in inject sequences s incubation time shown in the customize window will overwrite the incubation time command wait in the sequence see Figure 5 16 The flexibility of the customize window is the ability to link all other kinds of sequences besides inject sequences to the inject toolbar arrow Figure Figure Figure Figure 5555 15 15 15 15 Example of a Example of a Example of a Example of a Customize window to change a linked Customize window to change a linked Customize window to change a linked Customize window to change a linked sequence shown in the Menu_Twingle Inject sequence shown in the Menu_Twingle Inject sequence shown in the Menu_Twingle Inject sequence shown in the Menu_Twingle Inject SPRINGLE has equal functionality SPRINGLE has equal functionality SPRINGLE has equal functionality SPRINGLE has equal functionality Data Acquisition software 106 5 7 4 5 7 4 5 7 4 5 7 4 Wash Wash Wash Wash Press the toolbar WASH icon and the Customize information window pops

Autolab Twingle-Springle SPR user manual 4.4.0-6

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