Chapter 5 on Microrheology
Contents
1. Therefore to avoid this interaction the amount of sample added with respect to the tracer must be controlled This is done by measuring the sample zeta potential measurement against the tracer zeta potential The resultant measurement and comparison must show that the sample measurement is within a set percentage of the tracer measurement This comparison is termed the acceptable zeta ratio Zetasizer Nano accessories guide Page 5 11 Chapter 5 Micrarhealagy Page 5 12 For the microrheology measurement to measure reliably the acceptable zeta ratio is 25 or less Note gt It is important to note that stable dispersion of the tracer particles in the sample may take some time It has been noticed that with certain systems gentle mixing for example using a sample roller will help to disperse the tracer particles but that surface interactions can take several hours to man ifest themselves During the measurement this ratio is checked If the ratio is acceptable the measurement will continue if not and the zeta potential ratio is more than 25 different the software will advise There are two possible causes for the zeta potential shows significant differences between the tracer and the sample tracer systems m The tracer concentration is not high enough in the sample IF the tracer concentration is too low the zeta potential result will be domi nated by the sample not the tracer adding more tracer pa2. Microrheology Introduction This chapter gives an overview of the mierorheology option in the Zetasizer software It can be used with either a Zetasizer Nano ZS or ZSP The microrheology option allows the measurement of the viscoelastic modulus of samples within the linear viscoelastic region ZENS600 _Zetasizer Nano ZSP instrument ZEN3600 Zetasizer Nano ZS instrument It describes how to connect control and operate the microrheology optioned instrument to ensure reliable and consistent measurements DLS Microrheology Th called DLS Microrheology as it is a cross over between DLS and rheology and allows rheological measurements of low viscosity and weakly structured samples to be made casurement protocol for the Zetasizer Nano ZS and ZSP has bet Advantages of DLS Microrheology are m Rheological characterization of low viscosity weakly structured samples Via access to very high frequency short time dynamics which is highly relevant for dilute samples or weak structures Very small sample volumes can be used approx 124 This is particularly suitable for precious protein samples m Provides rheological parameters G G rt Data can be verified using the same sample measured on a rotational rheometer where measurement ranges overlap Data can be exported and used in the Malvern rSpace software Zetasizer Nano accessories guide Page 5 1 Chapter 5 Micrarhealagy m Extends viscoelastic
3. cP or mPas The Correlogram plot against Lag times _ Plot against angular frequency G Prime Prime Pa G Prime Pa Viscous component plot againstangu Elastic component plot against lar frequency angular frequency These can be imported into Space software using an appropriate sequence Note gt It is important to note that for the first release of microrheology the time is exported from the Zetasizer software in ts and will be imported into the Space software in s Zetasizer Nano accessories quide Page 5 5 Chapter 5 Micrarhealagy Controlling the Microrheology measurement via an SOP A microrheology measurement follows the same SOP format as performed when doing a normal size measurement with a few exceptions When a microrheology measurement is chosen some extra dialogues Optimization and tracer will be included in the SOP selections The SOP Editor and setup is described in fall in the Zetasizer Nano user manual Most of the SOP sections are common to Measurement types and these are described in the above manual The other SOP sections are specific to the microrheology measurement being performed these are described below Also note that some of the other dialogue pages will alter slightly to accommodate extra parameters necessary to perform the microrheology measurement Creating or editing an SOP Measurement type selection m To create a new SOP select File New SOP This will open u
4. main Zetasizer Nano user manual Page 5 16 man 0487 Mierorheolagy Chapter 5 Running only the microrheology test The microrheology test can be run on it s own as long as the user is confident that the other steps have been fulfilled to their satisfaction For example if a range of concentrations of the same sample are being tested the zeta potential testing can be run once on just a single concentration If the same tracer is being used the tracer size can be measured once and the same value can be set in the SOP by checking the edit box next to the nominal tracer size and changing the nominal tracer size to the size that has been measured Fer IT DEDE Since Sim een Term FSR Displaying the microrheology measurement report To display a micr rheology report select a Microrheology group type measurement record and then select the appropriate report tab The report will show all appropriate measurement information for that record Standard report microrheology measurements Once a microrheology measurement is completed there are a number of standard reports available for reviewing the measurement results Zetasizer Nano accessories quide Page 5 17 Chapter 5 Micrarhealagy Page 5 18 These are m Microrheology Correlogram and MSD mean squared displacement m Rheological properties Eta Complex viscosity G Moduli Creep compliance Microrheology MSD mean squared d
5. measurements into ranges inaccessible by mechanical rheometry techniques Fast measurements are possible with all frequencies effectively sampled simultaneously m Applications for viscosity of protein solutions and onset of protein aggregation DLS Microrheology basic theory DLS Microrheology uses tracer probe particles to measure the relationship between stress and deformation Both DLS and ELS measurements are done to ensure robust measurement methodology A measurement consists of the 3 main measurement steps m Ensure suitable tracer particle surface chemistry Zeta potential measurement step to show no significant interaction between the tracer particles and measurement sample The tracer is initially measured in the sample buffer dispersant or solvent alone with no sample added sample is then added and a second measurement performed The two zeta potential results are then compared a small relative change in zeta potential in the presence of the sample indicates minimal inter action between the sample and tracer particles m Ensure suitable concentration and dispersion of tracer particles Size measurement step for evaluation of appropriate scattering signal and PSD to mect this condition A size measurement is run to see that tracer scattering dominates signal and has a narrow monomodal Intensity PSD peak ie tracers are dispersed prop erly m Measure Correlation function of tracer particles P
6. ace software Exporting the microrheology data csv using the File option To export the measurement data as a csv file m Selecta microrheology measurement record The export will not work if this is not done Select File Export ed requesting the export destination Enter the destination rs and settings as required m A window is display and alter the paramet m To export the data as a esv file select the Browse button and alter the file Note if Ok is pressed the data will be exported and saved as a text txt file Note 9 Refer also to the Exporting results section in the main user manual Exporting the microrheology data xml csv using the Microrhe ology utilities To export the measurement data as either a xml file or a csv file w Selecta microrheology measurement record The export will not work if this is not dor m Select Tools Utilities Microrheology utilities m The Microrheology data processing window will be displayed Select Save and input the export destination Alter the file extension to xml file or csv as required Page 5 4 man 0487 Micrarhealagy Chapter 5 w On selecti OK the data will be exported The parame rs that are exported Sample Name Date File Name Tag times is Times 35 Angular Frequency rad s Greep Compliance Mean Square Displacement Plot against Times Plot against Times Channel Values Complex Viscosity
7. d measurement sample are mixed A measurement of the zeta potential of the tracer in the measurement sample is then performed Once this measurement is complete the software compares the two measured zeta potential results and tests to sce if sample measurement is within a set percentage of the tracer measurement this is the acceptable zeta ratio Ifthe ratio is less than 25 the measurement process will continue to the next step otherwise with a dialogue appearing indicating the next step to perform Ifthe ratio is more than 25 the software will give sample preparation advice and advise on repeating steps 1 and 2 Refer to the zeta potential accepta ble zeta ratio section man 0487 Mierorheology Chapter 5 tips mad Daponaie zung arvete DTSCH Themen ptr nth pesare of tarota en bom me og via te heresy 0 Thee men ha sea BSS anal ise charge na part ne preoer d wre nals nema tion betwee Pe agin ana urn Ws m ee Gren torre meray da ek Metta paced wth as messen D Cineni to en mesovnnert Size measurement to monitor tracer and sample scattering Measure size of measurement sample Use a folded capillary cell or disposable sizing cell I Prepare a fresh measurement sample and perform a size measurement m When finished a dialogue will appear indicating the next step to perform Measure size of measurement sample and tracer particles Use a folded capillary cell or disposable sizing cell Add a quan
8. ements for the measurement is important to ensure that reliable and repeatable results are achieved The concentration of tracer samples used will depend to a great extent on the concentration of yonr sample in the dispersant Following are some important considerations for the preparation Zeta potential measurements For the initial tests of the zeta potential the following starting concentrations are suggested m Tracer in dispersant buffer or solvent only 0 5pl tracer particles to 10ml sample 0 05pd ml m Tracer in dispersant buffer or solvent including sample Add 10 100 of the tracer in dispersant to 1ml of the sample in dispersant Start at the low end and increase the concentration if required Size measurements to hide sample scattering Size measurements are performed to check that the tracer particle s scattering completely hides the sample scattering therefore the size of the tracer particle should be larger than the sample man 0487 Mierorheolaogy Chapter 5 The following starting concentrations are suggested m Tracer particles in measurement sample Start with a few p up to about Syd of neat tracer to 1ml of sample If more tracer is required to hide the sample scattering add yl at a time dilute 1004 in 1ml of buffer filter using an appropriately sized filter and add Spd at a time if more probes are required to hide the sample scat tering m Filtering It is suggested that to ge
9. erform a microrheology measurement and extract microrheology data from Mean Square Displacement MSD plot Page 5 2 man 0487 Mierorheolaogy Chapter 5 Microrheology utilities The Zetasizer software has a utilities section for the Microrheology suite which can be accessed via either the Tools menu Tools Utilities Microrheology utilities or from right clicking on a microrheology record The following tab selections are available m Mean square displacement m Viscoelastic Moduli m Complex viscosity Each tab page contains controls that allow different models to be fitted to the microrheology data graph It is important to note however that not all models will be applicable to all the datasets Due to the measurement inconsistencies at very short timescales there will be areas of the data that will not be appropriate to use with the models In order to change the area used and displayed in the Fit results area left click and drag the range pointers the red triangles on the x axis to an appropriate point on the chart To export the microrheology data refer to the next section Zetasizer Nano accessories guide Page 5 3 Chapter 5 Micrarhealagy Exporting the Microrheology data On completion of the measurement the rheology data can be exported from the Zetasizer Nano software saved as an xml or a esv file Note The csy file should be used if the results are to be imported into the Mal vern rSp
10. isplacement The mean squared displacement is a representation of the movement of the tracer particles within the sample A purely viscous Newtonian sample will show a straight line Correlogram The correlogram shows the base data from which the microrheology result is calculated The correlation function displayed is a measurement of the amount of movement the probe particles make within the test sample over a range of timescales Rheological properties Eta Complex viscosity This report shows the relationship of the complex viscosity to the shear viscosity using the Cox Merz rule This rule is generally only fully applicable for simple systems and that the differences between complex viscosity and shear viscosity increases as the sample structure becomes more complex G Moduli The Moduli tab shows the viscoelasticity of the sample it shows two sets of data on the chart G the elastic storage component and G the viscous loss component Ina microrheology measurement it is expected that the viscous component will be dominant for at least most of the measured frequency range This is because the technique relies on the tracer particles being able to move and therefore produce a correlogram For example a gel system where the elastic component G is dominant for all of the frequency range will hold the tracer particles still within the gel matrix which means that the correlogram will be close to a flat li
11. l Selector deselect the zeta potential and size tick boxes as SOP Size SOP required Refer to the schematic description below Toad Load a saved SOP file The SOP loaded will be shown in the above entry box Edit Edit a new zeta potential or size SOP Schematic The schematic shows the optimization process that will be conducted If either or both of the zeta potential or size SOP tick boxes are deselected the schematic will grey out that part of the schematic and that part of the measurement stage will be ignored Zetasizer Nano accessories quide Page 5 7 Chapter 5 Micrarhealagy Tracer The Tracer SOP window shows details about the tracer used in the measurement Press Select to open the tracer manager window where the tracer to be used can be selected On 0 aun ew ze a me Settings Description Select Highlight the tracer required and press OK The Tracer SOP will now be populated with the selected tracer Edit Enables the editing of the nominal tracer size Notes Add any additional information Measurement Advanced This window is the same as the standard size Measurement Advanced except for the addition of the Acceptable zeta ratio setting Settings Description Acceptable zeta This is the acceptable ratio between the zeta potential meas ratio red for the tracer particles in dispersant alone and of that measured in dispersant and sample ma
12. luted suspension of the tracer particles is added An initial measurement of the zeta potential and size of the tracer is then performed Cate eared Deporte sung curse T5012 The min ptr pr mb Far as ben naan REGS Tenet sep menu ecw pats ne Ok Moto proceed th a ase messen BE Conte aaa th mesnsmnere Zetasizer Nano accessories guide Page 5 13 Chapter 5 Micrarhealagy Page 5 14 The tracer size measurement is used later in the microrheology measurement step The tracer size needs to be measured to ensure the microrheology result is as accurate as possible When finished a dialogue will appear indicating the next step to perform Calia auei Zas dp ol ZENO The vce pace ma hat been menmam as 100m Sane The race patie ma paaria has boen meanaed me S0 T AV aol avert of vacer panes zes sanie 1o a coromrtrton Aria aa n es belio napero EE e na e nen Kader cel ard men nu he ee ee Fox mia moster data De vacar patna on ro ony wove sere name Tt tes ty mamma nce pe ae Taaroa ne patens On sarna m onesna co TE PE DATS E oeron Ck hiato proceed wth a zta sare meaner er Measure tracer zeta potential in dispersant buffer or solvent including sample This is termed the sample measurement Use a zeta potential cell Prepare the sample to be used for the measurement then add small amount of tracer particles do this at a concentration similar to that used in the previous step Ensure the tracer an
13. n 0487 Mierorheolaogy Chapter 5 om o Bun om For a description of the other measurement parameters refer to the Measurement Advanced description in the size SOPs section of the main user manual w For all other microrheology SOP windows refer to the size SOP section in the main user manual Microrheology measurement process With the cell loaded into the instrument and the SOP configured a measurement can be performed Overall to make a microrheology measurement only one measurement is technically needed this isa size measurement that the software uses to calculate the relaxation times and therefore the viscosity and moduli values However in order to get to this point and in order to make reliable measurements the full microthcology measurement process consists of several basic steps these are described in the following paragraphs below As the measurement progresses through the measurement steps the software will prompt instructions for the user to perform where relevant Each instruction will involve a manual operation and the user must open the cell area lid perform the specified action then close the lid again before continuing The process a microrheology measurement performs is shown in the optimization stage of the SOP Zetasizer Nano accessories quide Page 5 9 Chapter 5 Micrarhealagy Sample preparation advice The correct preparation of the tracer and sample el
14. ne and the MSD will be effectively 0 Creep compliance Creep compliance is another way of viewing the viscoelasticity of a sample man 0487 Mierorheolagy Chapter 5 A standard report MSD The standard microrheology report MSD mean squared displacement M gives similar information as seen in a standard size and zeta potential report plus additional information relating to the microrheology measurement itself len sora Measurement Dut and Tine 00 December 2011 11 51 44 oral Probe Sue 515 Probe surtace Chen Catania eased probe size cm 2828 aime viscose 951 Temperature Pek 25 0 Dura una a 50 Countrate denen 989 Measurement Poston mm 055 Ccenveserotor Disposable siang amts Ansmann amp ee Saure Dsaceren pel Zetasizer Nano accessories quide Page 5 19 Chapter 5 Micrarhealogy Page 5 20 man 0487
15. p the SOP Edi tor The SOP Editor consists of several dialogues that can be stepped through by using the Next arrow button To edit an existing SOP choose Open SOP instead m Complete the SOP Editor as described in the Zetasizer Nano user manual m Once the SOP has been created press Finish and save the new SOP The various SOP dialogues are described below Measurement type options Select a microrheology measurement type then complete the SOP creation as required Optimization The optimization SOP window is required for preparation optimization of the sample and measurements before the microrheology measurement proper Optimization is the process of performing zeta potential and size measurements on the tracer particle and then subsequently on the measurement sample to ensure all requirements are met prior to performing the microrheology measurement The zeta potential and size optimization measurements are configured by clicking on the Edit buttons this will open the standard zeta potential and size SOP windows as described in the main user manual This stage is not always necessary Page 5 6 man 0487 Mierorheolaogy Chapter 5 depending upon the measurement setup and data available both the zeta potential and size optimization measurements can be ignored with the user going directly to the microrheology stage ow oan ou Fee iy Sar re Bes Settings Description Zeta potentia
16. rticles and re testing can show if the difference is due to concentration rather than interaction m The tracer and sample are interacting Ifthe tracer and sample are interacting adding more tracer won t improve the ai ce in zeta potential unless so much is added that it completely domi s the zeta potential measurement Measurement process A fall microrheology measurement consists of several steps As the measurement progresses through the measurement steps the software will display instructions examples are shown in the following text On pressing the Start button a dialogue will appear indicating the first step to perform Pressing Next in the measurement dialognes will then move the measurement onto the next step man 0487 Mierorheology Chapter 5 Calpe ured Zus dp cot ENO Pre lund bene cer ac m ao iter soen er we Kippe cng port Su d rest racer patil napengn Ton er a tearan Fred aed Pace BAGTE rsa par abe ab dnote io ranet Taten ip memes the zn para rds ct e vacar materi n tho ber Een enema ct Motta proceed nt zeta poterti nenn net ek Cancalf wen meresnzenen Zeta potential measurement to study sample tracer interaction Measure tracer zeta potential in dispersant buffer or solvent alone This is termed the tracer measurement Use a zeta potential cell m A zeta potential cell is filled with the dispersant buffer that is to be used in the later mierorheology measurement and a di
17. t the most reproducible result the tracers are filtered with appropriately sized i e filter size larger than the tracers filter Size sample measurement to check sample scattering visibility During the measurement the software makes a measurement of the sample size then asks for tracer particles to be added The size is re measured and checked against the original sample size measurement If the sample scattering is still visible The software will encourage the user to add more tracer IF there is too much tracer in the sample it will become turbid and measurement will be difficult It is therefore important to use just enough tracer to mask the sample but not put in so much that the sample becomes turbid Note S The measured size of the tracer may not be correct This is becanse of the viscosity of the sample and does not matter The important point is that the scattering from the tracer should hide the scattering from the sample Zeta potential acceptable zeta ratio Testing the difference in the zeta potential between the tracer and measurement sample is important because large changes would indicate thatthe tracer particle and the sample are interacting With proteins that could mean that the proteins are adhering to the tracer s surface and therefore not only changing the tracer s zeta potential but also the size of the tracer The tracer then becomes part of the system and directly influences the rheology
18. tity oftracer particles to the measurement sample Once done performa size measurement Zetasizer Nano accessories guide Page 5 15 Chapter 5 Mierorhealogy With the measurement is complete an intensity plot should also be displayed showing one peak The software compares the two measured size results and tests for the following m The size of the tracer particle should be larger than the sample particles m The tracer particle s scattering should completely hide that of the sample scattering Ifthe sample scattering is still visible the software will prompt for more tracer to be added to the sample The tracer scattering must obscure the sample scattering by more than 90 but not so much that the sample becomes turbid therefore making any subsequent measurement difficult When the software is satisfied with the comparison between the two size measurements the microrheology measurement can be started m A dialogue will appear indicating the next step the microrheology measurement can be performed Press next to start the microrheology measurement automatically Microrheology measurement This measurement is a single size measurement from which the MSD and rheological parameters are calculated The microrheology measurement display The microrheology measurement displays are virtually identical to those shown when performing a standard zeta potential or size measurement These standard tabs are explained in the
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