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njit-etd2005-112 - New Jersey Institute of Technology

1. eem 3 1 Description of Spotting Experiment ik ss 3 2 Control Methodology used for Spotting sese TETRA AT Spo Fom MERE EE EE ER EEE 4 2 pot Dee Fensexeda dua duos tesa bare cue DRE 4 3 Production of Uniform Size MicroarmraySs waemnamama 4 4 Experiments with Molecular Beacon csse 5 XONCLUSION AND FUTURE WO Rusas illa vil Page TABLE OF CONTENTS Continued Chapter Page APPENDIX A HARDWARE SPECIFICATIONS oooccccccccnncnccccanannnancnnnnnnonnoo 45 APPENDIX B SOURCE CODE uta i 54 REFERENCES PE 82 viii Table 2 1 22 2 3 3 1 4 1 LIST OF TABLES Page Product Specifications of Zaber Stage 16 Instruction Format of Zaber Stage 17 Command Reference of Zaber Stage 17 Look up Table for the Controller 25 Sample Solutions of Molecular Beacons 40 Figure 1 1 1 2 1 3 24 2 2 2 3 2 4 2 9 2 6 PANI 2 8 2 9 2 10 2 11 3 3 3 3 LIST OF FIGURES Page Microarrayer with impact printing technology left and contact DEHIUDS DID Hl i ecose cenis debxwu do sequ e REO NE I DEREN e BRI M exe 2 Image of a section of a Microarray Experiment using the Center for Applied Genomics CAG Human 19K DNA oligo array The chip was hybri
2. ResetEvent NCommEndEvnt 0 _RobJogXYZFunc 3 WaitForSingleObject NCommEndEvnt 0 INFINITE Get the index of end time isensDAQPosDataldx2 SensDAQDataldx iWhenToPickRef if WhenToPickRef gt 4 iCurvRefldx SensDAQPosDataldx1 j else break j Elongation i e move robot up ResetEvent NCommEndEvnt 0 RobJogXYZFunc 9 WaitForSingleObject RCommEndEvnt 0 INFINITE SetCtrlVal iPnlHdl PANEL CURRENTROW iCurrRow SetCtrlVal iPnlHdl PANEL CURRENTCOL iCurrCol iSpotNum iCurrRow 1 iMaxCol iCurrCol SetCtriVal iPnlHdl PANEL SPOTNUM iSpotNum Stop sensor data recording iSensDAQStartRec 0 Save data to file sprintf strFileName row dcol d dat iCurrRow iCurrCol iFileHdl OpenFile strFileName VAL WRITE ONLY VAL OPEN AS IS VAL BINARY WriteFile iFileHdl char dSensDAQData sizeof dSensDAQData 0 iSensDAQDataldx CloseFile iFileHdl if iSpotNum 96 6 0 Push more droplet ResetEvent NCommEndEvnt 1 _ZabJogZFunc 5 0 WaitForSingleObject NCommEndEvnt INFINITE Move robot to next Column ResetEvent NCommEndEvnt 0 RobJogXYZFunc 5 WaitForSingleObject hCommEndEvnt 0 INFINITE Move robot to next Row ResetEvent NCommEndEvnt 0 RobJogXYZFunc 7 WaitForSingleObject hCommEndEvnt 0 INFINITE 80 ResetEvent hExpStartEvnt 1 AI EE A LES ES o ee I Te
3. 31 Graph depicting the intensity change for 50 glycerol T T T Y T j Sakae 0 12 Ec a d te arme amp o o 3 j 01 A 2 0 08 o 7 4 gt j E 0 06 w t o e E 0 04 0 02 o 0 10 20 30 40 50 50 70 80 90 100 110 Air gap distance from the droplet um Figure 4 3 Intensity plot for 50 glycerol spot formation 1 y i t 043 675 1000 000 1200 000 1400 000 1600 000 1866 Figure 4 4 Graph depicting the intensity change during 190um droplet formation for 50 glycerol 0 065 gt fot L 19200 0 06 o gt e gt 3 fot L 19150 gt 0 055 t o o a m E ES o e E gt o gt 3 gt gt E E gt 0 05 hay by aad fort 19100 e v gt gt y o a 0045 j o 3 e 9 E LA 4 gt 5 gt E 0 04 e gt o a gt a gt o D 0 035 j Eg H 0 03 x J a ct 20 40 60 80 100 120 Air gap in um Figure 4 5 Intensity plot for water Cy3 spot formation 32 1000 000 Figure 4 6 Graph depicting the intensity change during 210um droplet formation for water Cy3 In addition to the optical lever the SmartPin also utilizes the total internal reflection principle due to the difference in refractive indices of air glass pin sample material and the shape of t
4. MessagePopup CreateThread Message strErrMsg return 1 break return 0 j A RN RR RR Thread for the 2nd part of the experiment i RR OPE EOM DWORD WINAPI AutoExpMonProc2 LPVOID IpParam while 1 Trigger the process WaitForSingleObject hExpStartEvnt 1 INFINITE Initialize variables CurrRow 1 CurrCol 1 iSpotNum 0 SetCtriVal iPnIHdl PANEL CURRENTROW iCurrRow SetCtriVal iPnIHdl PANEL_CURRENTCOL iCurrCol SetCtriVal iPniHdl PANEL SPOTNUM iSpotNum Go to the first spot ResetEvent NCommEndEvnt 0 _RobGo21stSpotFunc WaitForSingleObject hCommEndEvnt 0 INFINITE GetCtrlVal iPnlHdl PANEL MAXROW amp iMaxRow GetCtrlVal iPnlHdl PANEL MAXCOL amp iMaxCol for iCurrRow 1 iCurrRow lt iMaxRow iCurrRow for iCurrCol 1 iCurrCol lt iMaxCol iCurrCol Push drop ResetEvent NCommEndEvnt 1 _ZabJogZFunc 5 0 WaitForSingleObject hCommEndEvnt INFINITE Wait for stable condition of sensor Sleep 2000 Trigger sensor data recording iSensDAQDataldx 0 SensDAQStartRec 1 iSensDAQPosDataldx1 0 isensDAQPosDataldx2 0 IWhenToPickRef 0 CurvRefldx 0 Robot touches the Z limit 79 while dRobCurr 2 gt dRobLim 2 Spot formed if TestSpotForm 1 Remember the index of start time iSensDAQPosDataldx1 SensDAQDataldx Move robot one step down
5. inti bttn stat iSensDAQStartRec 0 GetCtrlVal iPniHdl PANEL EMERGENCYSWITCH amp i bttn stat 0 released 1 pressed switch i bttn stat case 0 break case 1 break default break Callback function to handle the receive of DAQ data int CVICALLBACK _SensDAQReadData int iPnlHdl int iCtriHdl int iEvntHal void pvCallBackData int iEvntData1 int iEvntData2 switch iEvntHdl case EVENT DAQ NUMERIC DATA READY if SensDAQStartRec amp amp SensDAQDataldx lt SNESDAQNUMIDXLIM 1 Get the raw data dSensDAQRaw iSensDAQDataldx double iEvntData1 Use Butterworth lowpass filter Bw LPF dSensDAQRaw SensDAQDataldx 50 00 1 00 2 dSensDAQData iSensDAQDataldx dSensDAQTime iSensDAQDataldx dSensDAQTime iSensDAQDataldx 1 SENSDAQNUMINT DeleteGraphPlot iPniHdl PANEL SENSGRAPH 1 VAL DELAYED DRAW PlotXY iPnlHdl PANEL SENSGRAPH dSensDAQTime dSensDAQData iSensDAQDataldx 1 VAL DOUBLE VAL DOUBLE VAL THIN LINE VAL EMPTY SQUARE VAL SOLID 1 VAL RED 76 break return 0 7 DARE AE POPE PEPTIDE MI TRUE IA ARS IRA Callback function to handle the change of Robot limit ARA MA A AAN int CVICALLBACK RobLimZChng int iPnlHdl int iCtriHdl int iEvntHdl void pvCallBackData int _iEvntData1 int iEvntData2 switch _iEvntHdl case EVENT VAL CHANGED GetCtrlVal iPnlHdl PANEL ROBLIMITZ amp dR
6. 0 0 dRobTarg 1 0 0 dRobTarg 2 0 0 SetCtriVal iPniHdl PANEL ROBTARGX dRobTarg 0 SetCtrlVal iPnlHdl PANEL ROBTARGY dRobTarg 1 SetCtriVal iPniHdl PANEL ROBTARGZ dRobTarg 2 SetCtriVal iPnlHdl PANEL EXPMSG1 ROBOT GOING HOME SetCtriVal iPniHdl PANEL EXPMSG2 SetCtriVal iPniHdl PANEL ROBMSG COMMI strRobCmdq 0 0 ResetEvent hCommsStartEvnt 0 ResetEvent hCommEOLEvnt 0 _SendRobCmd 1 0 0 0 0 0 0 HOME SetEvent hCommsStartEvnt 0 A RUD EPI WEE ay Callback functions to move the Robot to liquid well il o ts SOUT OE Nae a e e ASAP RENE S RHEIN int CVICALLBACK GoToWell int iPniHdl int _iCtriHdl int iEvntHdl void pvCallBackData int iEvntData1 int iEvntData2 switch iEvntHdl case EVENT COMMIT _RobGo2WellFunc break return 0 void RobGo2WellFunc void SetCtriVal iPnIHdl PANEL EXPMSG1 ROBOT GOING TO WELL SetCtriVal iPnlHdl PANEL EXPMSG2 SetCtriVal iPniHdl PANEL ROBMSG COMM GetCtrlVal iPnlHdl PANEL WELLX amp dRobWell 0 GetCtrlVal iPnIHdl PANEL WELLY amp dRobWell 1 GetCtrlVal iPnlHdl PANEL ROBLIMITZ amp dRobLim 2 dRobWell 2 dRobLim 2 ROBLIMGAP SetCtriVal iPnIHdl PANEL WELLZ dRobWell 2 strRobCmd 0 0 ResetEvent hCommsStartEvnt 0 ResetEvent NCommEOLEvnt 0 SendRobCmd 2 dRobWell 0 dRobWell 1 dRobCurr 2 _SendRobCmd 2 d
7. Callback functions to move to next column f Bat EA E C AA wa Tran int CVICALLBACK _GoNextCol int iPnlHdl int iCtriHdl int iEvntHadl void pvCallBackData int iEvntData1 int iEvntData2 switch _iEvntHdl case EVENT COMMIT _RobJogXYZFunc 5 break return 0 nr GM Gem SA Aa A A ee da ee LES J Callback functions to move to next row Dr BG Eg tn aa ae e e J int CVICALLBACK _GoNextRow int iPnlHdl int iCtriHdl int iEvntHdl void pvCallBackData int iEvntData1 int iEvntData2 switch iEvntHdl I case EVENT COMMIT RobJogXYZFunc 7 break return 0 r ACI EEE Se IAEA A ANI RETA Callback functions to jog the Robot p Along X axis je nn Le OLE SE EA NA E G ae ENE E int CVICALLBACK RobJogMinuX int iPniHdl int iCtriHdl int iEvntHdl void pvCallBackData int iEvntData1 int iEvntData2 switch iEvntHdl case EVENT COMMIT _RobJogXYZFunc 1 break return 0 68 int CVICALLBACK _RobJogPlusX int iPnlHdl int iCtriHdl int iEvntHdl void pvCallBackData int iEvntData1 int iEvntData2 switch _iEvntHdl case EVENT COMMIT _RobJogXYZFunc 4 break return 0 a LAKEN AIR ARE IO RS A AN MERA rm Callback functions to jog the Robot Y Along Y axis d p AREA o Et Ser int CVICALLBACK _RobJogMinuY int _iPniHdl int _iCtriHdl int iEvntHadl void pvCallBackData int iEvntDatat1 int iEvntData2
8. strcat strRobCmd strTempRobWrt break default break p INE A NN EUN A A er m nr VEG a A Thread for controlling communication with Robot ji hCommStartEvnt O control the start of communication hCommEOLEvnt 0 control the end of communication fr ENE A PR A RENTE DWORD WINAPI RobCommMonProc LPVOID IpParam char pstr_temp char pstr temp1 char str temp 50 int i idx while iPortOpen 0 1 WaitForSingleObject hCommsStartEvnt 0 INFINITE pstr temp strRobCmd while pstr temp 0 FlushinQ iCommpPort 0 FlushOutQ iCommPort 0 ResetEvent hCommEOLEvnt 0 i idx strcspn pstr temp n 1 str temp 0 0 strncat str temp pstr temp i idx 64 pstr temp i idx pstr temp1 strstr str temp T1 if pstr temp1 NULL pstr temp1 3 GetCoorVal pstr temp1 amp dRobTarg 0 amp dRobTarg 1 amp dRobTarg 2 SetCtriVal iPniHdl PANEL ROBTARGX dRobTarg 0 SetCtriVal iPniHdl PANEL ROBTARGY dRobTarg 1 SetCtriVal iPnlIHdl PANEL ROBTARGZ dRobTarg 2 iStrSize 0 StringLength str temp iBytsSent 0 ComWrt iCommPort 0 str temp iStrSize 0 WaitForSingleObject RCommEOLEvnt 0 INFINITE SetCtriVal iPniHd l PANEL EXPMSG2 DONE SetCtriVal iPniHdl PANEL_ROBMSG OK ResetEvent hCommsStartEvnt 0 SetEvent hCommEndEvnt 0 return 1 A A i LAE SS Callback function to deal with
9. 2 dRobTarg 0 dRobTarg 1 dRobTarg 2 SetEvent NCommsStartEvnt 0 A A O O Prepare the command string to Zaber y f iCmdType 20 move absolutely gi po 21 move relatively PF 60 read absolute position PF e da AA ida void SendZabCmd unsigned char cCmdType long int plZ unsigned char pc temp strZabCmd iCmdldx 1 0 ZABUNIT strZabCmd iCmdldx 1 1 cCmdType switch cCmdType case 20 pc temp unsigned char plZ strZabCmd iCmdldx 1 2 pc temp 71 strZabCmd iCmdldx 1 3 pc_temp 1 strZabCmd iCmdldx 1 4 pc temp 2 strZabCmd iCmdldx 1 5 pc_temp 3 break case 60 strZabCmd iCmdldx 1 2 0 strZabCmd iCmdldx 1 3 0 strZabCmd iCmdldx 1 4 0 strZabCmd iCmdldx 1 5 0 break default break iCmdldx 1 ZABCNT JA Wa IE EE REMORE RR EZ TI E O E ee A E Thread for controlling communication with Zaber ss p hCommsStartEvnt 1 control the start of communication hCommEOLEvnt 2 control the end of communication qe A si A A IA ui DWORD WINAPI ZabCommMonProc LPVOID IpParam char pstr temp char str_temp 50 int i idx while iPortOpen 1 1 l WaitForSingleObject hCommsStartEvnt 1 INFINITE pstr temp strZ2abCma i idx 0 while i idx iCmdldx 1 FlushinQ iCommPort 1 FlushOutQ iCommPort 1 ResetEvent NCommEOLEvnt 1 iStrSize 1 ZABCNT if pst
10. 4 2 Graph depicting the intensity deed 160um T formation for 100 glycerol ere Tm M RT REESE 30 4 3 Intensity plot for 50 glycerol spot formation v v c 31 4 4 Graph depicting the intensity change during 190um droplet formation or 309 NENNE e DNE YA 31 4 5 Intensity plot for water Cy3 spot formation 31 4 6 Graph depicting the intensity change during 210um droplet formation A NE EN 32 4 7 Ray Tracing result for SmartPin before Up and after Down droplet EDEN NR 33 4 8 Raster scam DI since veste Ebo Peur cpu ta ibldetietat ida A E 34 4 9 Graph depicting the 3D view of a 220micron diameter droplet and a cross sectional measurement of the spot which is about 18 microns high at the tme of SCAN asco ees iesu mu odia wien tar Desin Ee N 36 4 10 Graph depicting the 3D view of a 400micron diameter droplet of 100 eo EEE ES piste uad EAD RM MU Due E 36 4 11 Spot matrix formed out of 100 glycerol each spot measures 160microns left and 50 glycerol each spot measuring 190 microns isle ERE 37 4 12 Microarray with 100 glycerol and 80 microns diameter dcc M RE 37 4 13 Microarray with spotting solution 3XSSC with 160 microns diameter Sij m 38 xi LIST OF FIGURES Continued Figure Page 4 14 Microarray with spotting solution 3XSSC with 160 microns diameter spots and 130 mi
11. The second byte is the command number Bytes 3 4 5 and 6 are data in long integer 2 s complement format with the least significant byte transmitted first How the data bytes are interpreted depends on the command The two move commands are tabulated in Table 2 3 17 Table 2 2 Instruction Format of Zaber Stage Unit Command Data Data Data Data number number least significant most significant byte byte Table 2 3 Command Reference of Zaber Stage 17 Data bytes Reply data Move absolute The device Absolute position in Absolute position If moves to the position given by micro steps the data is out of the data bytes The position range the device will must be within the acceptable not move but will range for the device return 255 in byte 2 as well as the absolute position Absolute position If the data is out of range the device will not move but will return 255 in byte 2 as well as the absolute position Move relative The device moves to the position given by its position before the command plus the value in the data bytes The final position must be within the acceptable range for the device Relative position can be negative in Micro steps Each unit replies with its device ID after it finishes renumbering Renumber This command must Ignored always be issued with a 0 in Byte
12. data 1s stored as the highest point The forthcoming data values lying in the lower zone can be subtracted from highest point and this difference when divided by the gap value gives the slope in voltage If this slope in voltage is less than the threshold mentioned in the panel then the spot formation has occurred and its time for the needle to back up and once again the main routine takes over the task Thus spotting is controlled by the repeated voltage pattern observed CHAPTER 4 TEST RESULTS 4 1 Spot Formation Three types of solutions are considered 100 glycerol 50 glycerol and water In each case a 10 Cy3 dye 1s added to the solution for fluorescence Results of spotting 100 glycerol are shown in Figure 4 1 where the top traces depict sensor intensity as the pin approaches the slide The droplet on the pin opening comes into contact with the slide at 20 microns air gap distance at which the sensor intensity drops off by 83 due to the loss of total internal reflection The disengagement of pin is characterized by elongation of the spot due to the viscosity of glycerol and the binding to the slide surface which has been coated with poly L lysine The total elongation is about 70 microns after which the spot and the pin separate The results are highly repeatable Graph depicting the intensity change during droplet disengagement for 100 Glycerol 0 4 om ee ap EEE ET inte
13. expression consist of 1 microarray fabrication 2 hybridization and 3 detection DNA chips also known as microarrays present an efficient way to manipulate the huge numbers of reagents that would be required to probe for a large collection of genes and this technology has become a central platform for functional genomics 1 These bio devices are dense grids of DNA bound to a solid matrix that can be probed with a complex mixture of labeled DNA or cDNAs The major advantages of microarrays over other technology are the increase in the number of genes being analyzed the substantial reduction in sample size requirements and the use of fluorescence detection schemes for high signal to noise ratios Among the many applications for DNA microarrays are e Expression profiling of human cancers e Identification of diagnostic signature transcriptional profiles for host responses to infectious agents e Gene expression profiling following injury e Expression profiling following drug treatment Currently there are two major approaches for the creating DNA microarrays photochemical process and printing process The photochemical process pioneered by Affymetrix 19 has a high inherent degree of reproducibility due to the spatial specificity of the manufacturing process However this approach tends to be inflexible and expensive thus excluding most small medium scale laboratories from conducting innovative research The conventional printing t
14. int iPortldx switch iCommerr iPortldx default if iGommerr iPortldx lt 0 Fmt strErrMsg s lt RS232 error number 96i iCommErr iPortldx MessagePopup RS232 Message strErrMsg break case O MessagePopup RS232 Message No errors break case 2 Fmt strErrMsg Yos Invalid port number must be in the range 1 to 8 MessagePopup RS232 Message strErrMsg break case 3 Fmt strErrMsg Yos No port is open n Check COM Port setting in Configure MessagePopup RS232 Message strErrMsg break case 99 Fmt strErrMsg Yos Timeout error nn Either increase timeout value in check COM Port setting orn i check device MessagePopup RS232 Message strErrMsg break f NR A 3 NS et SL Re ui Prepare the command string to Robot 63 iCmdType 1 HOME iCmdType 2 MOVE iCmdType 3 DISP ja rate See AA AA A AA EN SEU NUNC RENE af void SendRobCmd int iCmdType double dX double dY double dZ switch _ CmdType case 1 strcpy strTempRobWrt HOMEWAnDISPIANQUIT An strcat strRobCmd strTempRobWrt break case 2 strcpy strTempRobWrt DO CLEARWWM strcat strRobCmd strTempRobWrt sprintf strTempRobWrt DO T1 2f 2f 2f 2f r n dX dY dZ 0 0 strcat strRobCmd strTempRobWrt strcat strRobCmd DO MOVE T1WMnNDISPIANQUITIAN break case 3 strcpy strTempRobWrt DISPWnQUIT Wn
15. switch iEvntHdl case EVENT COMMIT RobJogXYZFunc 2 break return 0 int CVICALLBACK _RobJogPlusY int iPnlHdl int iCtriHdl int iEvntHdl void pvCallBackData int iEvntData1 int iEvntData2 switch iEvntHdl case EVENT COMMIT RobJogXYZFunc 5 break return 0 p EE EE ND a Callback functions to jog the Robot Along Z axis er ee Mee M DEEE NS NE TE int CVICALLBACK RobJogMinuZ int iPnlHdl int iCtriHdl int iEvntHdl void pvCallBackData int iEvntData1 int iEvntData2 switch iEvntHdl case EVENT COMMIT RobJogXYZ Func 3 break return 0 69 int CVICALLBACK _RobJogPlusZ int iPnlHdl int iCtriHdl int iEvntHdl void pvCallBackData int iEvntData1 int iEvntData2 switch iEvntHdl case EVENT COMMIT RobJogXYZFunc 6 break return 0 tn A RO NN e Et at EN RC ai Type 1 Minus X 4 Plus X 2 Minus Y 5 Plus Y Next Column jr 3 Minus Z 6 PlusZ je 7 Next Row 8 Get up ERO ERE A St OE i void RobJogXYZFunc int iType GetCtrlVal iPniHdl PANEL_ROBSTEPX amp dRobStep 0 GetCtrlVal iPniHdl PANEL ROBSTEPY amp dRobStep 1 GetCtrlVal iPniHdl PANEL ROBSTEPZ amp dRobStep 2 GetCtrlVal iPnIHdl PANEL 1STSPOTY amp dRob1stSpot 1 SetCtrlVal iPnIHdl PANEL ROBMSG COMMI switch iType case 1 Minus X dRobTarg 0 dRobCurr 0 d
16. void _ MylnitApp void void _ MyExitApp void int MylnitExp void void _DispRS232Err int iPortldx DWORD WINAPI SendRobCmd int iCmdType double dX double dY double dZ RobCommMonProc LPVOID _lpParam void CVICALLBACK RobRplyEOLFunc int iPortNum int iEvntMask void void void void void DWORD WINAPI void pvCallBackData _RobGoHomeFunc void _RobGo2WellFunc void _RobGo21stSpotFunc void RobJogXYZFunc int iType SendZabCmd unsigned char cCmdType long int plZ ZabCommMonProc LPVOID IpParam void CVICALLBACK ZabRplyEOLFunc int iPortNum int iEvntMask void void void DWORD WINAPI DWORD WINAPI void vpCallBackData _ZabJogZFunc int iType long int IStep _ZabGoHomeFunc void GetCoorVal char strVal double pdX double _pdY double pdZ AutoExpMonProc1 LPVOID IpParam _AutoExpMonProc2 LPVOID IpParam int _TestSpotForm void void _EmgyProc void f AAN SOI SEWER IRA AN IEA SER J File Static Variables f ee de ganen EE o e REN MERE I static int PniHdl JA RITE IANS AT Se ee KA Ua ERT LP Te RCT TENE BRE OT B E g Volatile Variables I ANDREE THREE A A ENS ARS PERDERE Senn double dRobWell 3 double dRob1stSpot 3 double dRobLim 3 double dRobStep 3 double dRobTarg 3 double dRobCurr 3 unsigned char strRobCmd ROBCMDLEN unsigned char unsigned char unsigned char long int long int
17. 1 i e it must be issued to all units simultaneously As shown in the front panel of Figure 2 10 the instruction 1s being transmitted to the Zaber stage The format of the instruction is a group of 6 bytes as explained before Command reference is issued as per the required Zaber movement it can either be relative or absolute The data field specifies how far the leadscrew driven by the stepper motors should move For the experiments carried out the Zaber is moved down to the maximum limit of 20 000um to fit the needle and then to draw the sample the Zaber stage is moved upwards to about 1000um relative from maximum limit making the absolute position as 19 000um to make room for the sample to enter the pin The reply data in um indicates the present position of the Zaber stage Figure 2 10 Front Panel and LabView program for Zaber stage The mechanical assembly in the practical model is a sliding bar with springs attached that roles up and connects the Zaber stage Seiko robot and the smart pin stage together as shown in Figure 2 11 Figure 2 11 Mechanical integration of Zaber CHAPTER 3 METHODOLOGY AND CONTROLLER DESCRIPTION The smart pin consists of a glass tube pointed at the end resembling a pin The Fotonic sensor of diameter 504um driven by the Zaber stage is placed inside the glass tubing such that 1t can move freely but sampai enough The robotic arm holds the above arrangement The robot is commanded t
18. 4 3 2 4 o Figure A 5 MTI 1000 Fotonic Sensor characteristics APPENDIX B SOURCE CODE The automatic spotting sequence method explained in the thesis is implemented using Measurement Studio Lab Windows CVI of National Instruments The following is the source code for the entire process A A A E ORE PEE A fn anten mai FILE spotting c PURPOSE Automate the SPOTTING experiment n NOTES Make sure all serial cables are connected properly a F SER NER RR TS A ARO ASES ARA AA J O TR el E ae ARRANCAR Include Files A Rr EE ENTENDER E EN HE II II O A CORR NE O DRE J include lt windows h gt include daq num h include lt analysis h gt include daqchart h include lt ansi_c h gt include lt formatio h gt include lt rs232 h gt include lt utility h gt include lt cvirte h gt include lt userint h gt include spotting h a a a A A a E mn Constants g p EC DESEE EN Tr e as Zdefine ROBEOL gt Zdefine ROBLIMGAP 0 2 define ROBCMDLEN 512 define ZABUNIT unsigned char 1 define ZABCNT define ZABCONVFACT 0 09921875 define ZABCMDLEN 512 define SENSRNGGAP 0 2 define SNESDAQNUMIDXLIM 2000 define SENSDAQNUMRATE 50 00 Zdefine SENSDAQNUMINT 1 0 SENSDAQNUMRATE define SENSSPOTTESTTH 5 54 55 P EREE AE A o E ELEA E RASES Local Function Prototypes JA ber Ar Sue E NER ae ds E RE RI ER
19. 5 Further decrease in gap distance actually results in reduced intensity measurement corresponding to the front slope region as shown in Figure 2 5 At contact or zero gap most of the light exiting the transmitting fibers is reflected directly back into those fibers No light is provided to the receiving fibers and the output signal is zero and the primary characteristics are given in the Appendix A section 4 11 Figure2 4 MTI 1000 Fotonic Sensor with a MTI 3802 sensor module According to the manufacturer s specifications 14 a major advantage of the Fotonic sensor is its ability to operate directly with a large variety of surfaces from specular to diffuse and materials from conductors to insulators As shown in Figure 2 5 the gap and displacement range over which the initial rise in signal takes place and at which the maximum occurs is primarily determined by the diameter and the numerical aperture of the fibers and the intensity distribution within the operating field of the fibers Most commercial devices of this type use multiple transmit and receive fibers in order to obtain the higher levels of intensity at the photo detectors needed to insure acceptable levels of performance OPTICAL PEAK E 2 S yA ae A x Sy 16 KA OPTICAL yA FIBRE Lo fe PROBE m KN eo O Oy e EET a GAP Figure 2 5 Receiver illumination and instrument output 12 Fiber Distribution Goncentric CTh R
20. SetEvent hCommsStartEvnt 1 j REFERENCES 1 Dangond F Chips around the world Physiological Genomics Online Volume 2 Issue 2 March 13 2000 Pages 53 58 2 Cheung V G Morley M Aguilar F Massimi A Kucherlapati R and Childs G Making and reading microarrays Nature Genetics Volume 21 Issue 1 Supplement January 1999 Pages 15 19 3 Duggan D J Bittner M Chen Y Meltzer P and Trent J M Expression profiling using cDNA microarrays Nature Genetics Volume 21 Issue 1 Supplement January 1999 Pages 10 14 4 Chang T N Hou E and Godbole K Optimal Input Shaper Design For High Speed Robotic Workcells to appear in the Journal of Vibrational and Control Sage Science Press 5 Chang T N Kwadzogah R and Caudill R Vibration Control On Linear Robots With Digital Servocompensator IEEE ASME Transactions on Mechatronics Volume 8 Issue 4 December 2003 Pages 439 445 6 Chang T N Dani B Ji Z and Caudill R Contactless Magnetic Leadscrew Vibration control and resonance compensation IEEE ASME Transactions on Mechatronics Volume 9 Issue 2 June 2004 Pages 458 461 7 Chang T N and Sun X Control of hysteresis in a monolithic nanoactuator Proceeding to the 2001 American Control Conference Arlington VA June 2001 8 Chang T N and Sun X Analysis and Control of Monolithic Piezoelectric Nano actuator IEEE Transa
21. can be the interior of the pin cavity or a pressurized fluid chamber connected to the cavity 2 A fluid delivery plunger made with an optical fiber probe and a driver The fiber driver is a precision lead screw assembly along the Z axis as shown in Figure 2 2 In the present version the optical fiber bundle has a diameter of 150 microns When the fiber is moved downwards a metered amount will be dispensed onto the tip of the pin Similarly aspiration can be accommodated with upward movement of the probe the holding volume is about 0 2 L nanoliter per micron where L is the vertical displacement in microns For a 2cm vertical cavity the liquid storage volume is about 4 microliters 3 The fiber probe also determines the distance between the fiber tip and the slide The optical sensing scheme utilizes the fiber itself as an optical lever There are two ways the optical sensors can be realized active and semi active In the active method a controlled light source generates a collimated beam directed towards the fiber tip and slide The reflected light is collected through a photodetector The position information is inferred ratiometrically from the intensity of the reflected light by a digital signal processor For the semi active method the glass slide is uniformly illuminated from below The optical fiber picks up the light beam and transmits it to a photodetector In addition to the optical lever the SmartPin also utilized the total inter
22. control setting Turn the intensity coarse control to the next higher switch settings Then turn the intensity fine control clockwise to bring the indicator to the set cal zone 14 9 Move the robot downwards to decrease the gap and move into the front slope operating range Observe the indicator it will move down scale of the set cal zone as the target moves towards the sensor probe Continue to move the robot until the indicator is positioned in the operate zone on the scale The gap is now calibrated to the front side slope on the calibration curve The plot of the gap distance from the mirror to the intensity measure read out on the panel gives the calibration curve for the Fotonic sensor as shown in Figure 2 8 10 If the back slope area is to be used to obtain more range or standoff distance then the robot must be moved upwards This will increase the gap and move the probe into the back side operating area 11 While calibrating the plug in module MTI 3802 using instrument MTI 1000 the settings used were 2K maximum intensity and datum reference 10 The target being referred to is a polished plane mirror The front side slope sensitivity of the Fotonic sensor obtained from the slope curve as shown in Figure 2 8 is 0 05785um mv and the back side slope is 0 1034 um mv Calibration of Fotonic Sensar Gap vs Intensity Curve intensity in volts o 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160
23. dud i 1 t 1 Ala P U Po Rate 3 05500 a CA P ES la AS T 0 e qna Fi Be 7 ity few i vi I i as 3 A Pano 3 io fe 3 An C da e A i f TM ii Fest Led Medes na ner lard Ce TENT GN ho opcode a hreg Figure 4 19 Alternate columns of buffer with 10 Cy3 and distilled water spotting showing no cross contamination of material handled by the pin CHAPTER 5 CONCLUSION AND FUTURE WORK In this work a novel liquid dispensing system termed SmartPin is described It is based on integrated active sensing and control to produce precise and uniform liquid spots in a contactless manner Due to active fluid displacement the pin is capable of handling highly viscous materials such as glycerol Primary application of this system includes DNA and protein microarray fabrication and other situations where aspiration of small liquid quantity is required The current laboratory prototype is capable of spotting arrays of circular dots with uniform dimensions and controlled diameters from 80 200 microns The features of the pin include not only spotting and spot detection but 1t also can locate the missing spots based on the optical sensor intensity information and can redo the spotting In the future the piezoelectric actuator can be incorporated to the model to ensure precise positioning of the slide and to break the elongation of the droplet sooner to get smaller spots The sensing capability of the smart pin can be exploi
24. is shown in Figure 4 9 where a 3 D view of the spot is imaged left Cross sectional view depicting the height of the spot at the time of scanning 1s shown in the right Figure 4 10 shows a 3 D view of a 400 micron droplet formed by 100 glycerol Raster scan is done to find the geometry of the droplet The 3 D view of the spot can be captured The pin opening affects the intensity pick up A pin with a bigger diameter opening admits more light and can therefore detect smaller spots For this experiment the pin is placed 60 microns above the slide having the sample The robot Z axis distance is thus fixed and now the robot s X and Y coordinates have to be changed to get the raster scan as shown in Figure 4 8 Figure 4 8 Raster scan procedure 35 Raster scan procedure l First a column wise scan can be performed by fixing the Y ax s value as constant as the Smartpin has to keep moving in the same vertical line path and the X axis 1s programmed to move by 10um increment The intensity picked by the sensor is recorded when it moves along a column The position of the robot is noted as points 1 and 2 as shown in Figure 4 8 when the change in intensity begins and drops during the column scan The difference in the position value of the start point of intensity change and end point of intensity change will help us fix the midpoint 5 as in Figure 4 8 along the X axis Similarly by fixing the Z coordinate and fixing X coordinate at po
25. long int long int long int long int long int char strRobRply ROBCMDLEN strTempRobWrt ROBCMDLEN strTempRobRd ROBCMDLEN IZabLowLim ZabUppLim IZabStep IZabTarg IZabTargTemp IZabTargTemp1 IZabCurr IZabCurrTemp strZabCmd ZABCMDLEN char char char strZabRply ZABCMDLEN strTempZabWrt ZABCMDLEN strTempZabRd ZABCMDLEN unsigned short int iCurrRow iCurrCol unsigned short int iMaxRow iMaxCol unsigned short int iSpotNum double dCurrV char bEmgSwch int iCmdldx 2 int iPortOpen 2 int iCommErr 2 int iDevErr 2 int iCommPort 2 int iBaudRate 2 int iPortldx 2 int iParity 2 int iDataBits 2 int iStopBits 2 int ilnptQ 2 int iOuptQ 2 int iXMode 2 int iCTSMode 2 int iStrSize 2 int BytsSent 2 int iBytsRead 2 int iBrkStat 2 int CommsStat 2 int ilnQLen 2 int iOutQLen 2 double dTimeOut 2 HANDLE hCommsStartEvnt 2 HANDLE hCommEOLEvnt 2 HANDLE hCommeEndEvnt 2 HANDLE hCommThra 2 DWORD dwCommThrdID 2 char strRobPortName 30 char strZabPortName 30 char strErrMsg 200 int iSensDAQNumID int iSensDAQStartRec int isensDAQDataldx double dSensDAQRaw SNESDAQNUMIDXLIM double dSensDAQData SNESDAQNUMIDXLIM double dSensDAQTime SNESDAQNUMIDXLIM int SensDAQPosDataldx1 isensDAQPosDataldx2 int isensDAQPosDataldx 56 57 double dSensDAQPosData SNESDAQNUMIDXLIM double dSensDAQPos SNESDAQNUM
26. of Sensor Voltage volt 0 000 Graph of Sensor Voltage volt 0 100 Threshhold 100 0 030 J 70 90 0 080 Me 80 nozo Rob Dist mm E 70 0 060 A 60 E end 0 12 50 2 0 040 40 0 030 w 0020 0 010 30 20 10 Sensor Voltage volt 0 000 0 1 0 0 1 2 1394 5 6 7 8 9 10 Gap between pin amp shde mm Figure 3 2 Front Panel of spotting experiment When the spotting process begins the pin is directed to the well and the Zaber is pulled back 1000um to fill the needle with the sample liquid Then the robot is taken to the home position and the tip and sides of the needle are cleaned Now the needle goes to the first spotting location as specified in the panel by the user which is about 120um from the surface of the slide A step increment of 20um is given in the Zaber to get a metered amount of liquid at the tip of the needle The present row number is checked with the maximum row number and if the last row has been reached then the present row is at maximum and spotting is stopped else the present column number is checked with the 23 maximum column number and 1f the last column has been reached then the robot positions to the next row and gets a drop again else the robot checks if the robot Z limit has been reached or not If the robot Z limit has been reached then it means the robot has to be pulled back and spotting at the next column has to be execute
27. well ResetEvent NCommEndEvnt 0 RobGo2WellFunc WaitForSingleObject hCommEndEvnt 0 INFINITE Wait for the capillary attraction Sleep 3000 Suct liquid into needle GetCtriVal iPnlHdl PANEL ZABCURRZ amp lZabCurr GetCtriVal iPniHdl PANEL ZABLOWZLIM amp lZabLowLim while IZabCurr gt IZabLowLim ResetEvent NCommEndEvnt 1 _ZabJogZFunc 1 0 Get liquid WaitForSingleObject hCommEndEvnt 1 INFINITE Move Robot up ResetEvent hCommEndEvnt 0 _RobJogXYZFunc 8 WaitForSingleObject hCommEndEvnt 0 INFINITE ResetEvent hExpStartEvnt 0 J EAE PE dk OENE o NEE J Callback function to automate the 2nd part of the experiment gi JA A a e a es do le e dd int CVICALLBACK AutoExpStep2 int iPnlHdl int iCtriHdl int iEvntHdl void pvCallBackData int iEvntDatai int iEvntData2 switch _ EvntHal case EVENT COMMIT if RExpStartEvnt 1 NULL hExpStartEvnt 1 CreateEvent NULL TRUE TRUE NULL if hexpStartEvnt 1 NULL I Fmt strErrMsg s lt CreateEvent failed i n GetLastError MessagePopup CreateEvent Message strErrMsg return 1 ResetEvent hExpStartEvnt 1 78 SetEvent hExpStartEvnt 1 if hexpFuncThra 1 NULL hExpFuncThrd 1 CreateThread 0 0 AutoExpMonProc2 0 0 amp dwExpFuncThrdID 1 if RExpFuncThrd 1 NULL Fmt strErrMsg s lt CreateThread failed i n GetLastError
28. 170 18D 190 200 210 220 230 240 250 260 270 280 290 300 gap in um e intensity in volts e intensity in volts Linear intensity in volts Front Slope Chart Linear intensity in volts Back slope chart 10 10 B s 2 8 O o gt gt 6 E 6 D 2 w 4 o 4 3 3 E 2 E 2 0 0 0 50 100 150 0 100 200 300 400 gap in um gap in um Figure 2 8 Calibration of Fotonic sensor up and front side and back side slope charts of Fotonic sensor down 15 To implement this system as a part of the hardware construction light intensity is taken as a reference the output data will be used to measure gap distances between the sample liquid and the sensor s probe In turn this data will allow the user to determine the spot formation and uniformity of the spots 2 4 Description of Zaber Stage Zaber Technologies Inc series of computer controlled positioning products as shown in Figure 2 9 use stepper motors and leadscrews to achieve open loop position control The product specifications are given in Table 2 1 The stepper motor turns by a constant angle called a step for every electrical impulse sent to it This allows a system to be built without feedback reducing total system cost Zaber positioning devices are driven by stepper motors using a micro stepping drive with 64 microsteps per step All position data sent to or received from Zaber devices must be in units of microsteps the software converts position data enter
29. 32 communication package Host can have the ability to start and stop the robot Host could be a Cathode Ray Tube CRT Personal or Business Computer Vision Systems It uses ASCII for communication and any computer or peripheral device compatible to a UART Conditions to Match the Protocol e Several Port characteristics must be matched for 2 devices to communicate using EIA s RS 232 It 1s Important to have devices to be set as DTE Data Terminal Equipment or DCE Data Communication Equipment It is not important that which device is DTE or DCE it 1s only important that both are not set as same Speed at which the communication is done should also be same for both the communicating devices The Robot controller has a UART Universal Asynchronous Receiver Transmitter which is Intel 8251A e Note that the Baud Rate factor B1 BO is never to be changed from 1 0 for current version of communication package as per the manual It s according to the clock setting of the processor Also a Null Modem can be used incase both terminals are either DTE DCE The integer in its binary form determines the characteristics of the port Figure A l demonstrates how the desired characteristics of RS 232 port are reduced to the integer in DARL statement 49 EVEN PARITY PARITY ENABLE STOP BITS CHARACHTER LENGTH BAUD RATE FACTOR 0 NO PARI ODD ENABLE SYNC MODE 1X 16X 645 0 0 INVALID 0 0 SBITS B 4 UBT 0 6BITS BES 0 7
30. AQ in the flowchart stands for Data Acquisition 27 CONTROLLER DECISION SENSOR READ OUT FROM DAQ INTERRUPT FOR EVERY 0 02 SEC TO SAVE THE DATE STORE THE DATA IN THE FORM OF AN ARRAY GET THE NEXT DATA FROM ARRAY TABLE AND ASSIGN AS PRESENT DATA AND MAKE THE CURRENT PRESENT AS PREVIOUS YES PRESENT DATA VOLTAGE WITHIN HIGH VOLTAGE RANGE PRESENT DATA VOLTAGE gt PREVIOUS VOLTAGE NO NO SLOPE PREVIOUS PRESENT 1000 GAP DISTANCE NO PRESENT DATA VOLTAGE WITHIN LOW VOLTAGE RANGE Y YES SLOPE gt THRESHOLD STORE PREVIOUS DATA AS HIGHEST LOWER ROBOT SLOPE Z BY 30 UM HIGHEST PRESENT 1000 GAP DISTANCE YES SPOT FORMED SLOPE THRESHOLD NO BACK ROBOTZ BY 30 UM Figure 3 5 Controller action flow chart for spotting experiment 28 The main routine 1s interrupted by a subroutine every 0 02 sec 1e sampled at 50 Hz The data from the sensor is stored in the form of an array and the present data is compared with the previous value and is checked for the range in which it lies If the value is higher than previous and as well lies in the higher voltage range then the robot has to jog down by another 10um and again the same check 1s repeated until the present data is leaser than the previous and lies in the lower zone At this juncture the previous
31. BITS 3BITS di Figure A 1 Reduction of RS 232 characteristics into integers in DARL statement Connecting the Robot to the Computer In the Robot side first of all the physical connection is done using a 25 9 pin cable build using connectors The wires are shielded to avoid picking stray signals Also the length of the cable should be approximately 1 5 meter for errorless transmission The default input and output unit of the robot s controller is the Teach Terminal At the robot end the important 8 pins used out of 25 pins of RS 232 Port Robot Controller are listed below 1 Ground 5 Clear To Send 2 Transmit Data 6 Data Send Ready 3 Receive Data 7 Signal Ground 4 Request to Send 20 Data Terminal Ready 50 DARL statement OUNIT n assigns the output unit of the controller and n is the unit which will act as the output unit DARL statement IUNIT n assigns the input unit of the controller Values of n 0 LCD Key Board on Teach Terminal RS 232 Port 1 2 RS 232 Port 22 there are 2 Ports on the back panel 3 Printers Example of MONITOR Mode DO OUNITI press enter DO IUNITI press enter The Z statement holds the key in communication Its syntax is Z integer Now this integer value is the DECIMAL equivalent of the 8 Bit Binary number from the UART Example if Z 78 DEC ie 0 1 0 0 1 1 1 0 BIN it implies that the controller is set to following parameters for communication 8 Characters NO Parity Stop Bit Exa
32. Copyright Warning amp Restrictions The copyright law of the United States Title 17 United States Code governs the mahing of photocopies or other reproductions of copyrighted material Under certain conditions specified in the law libraries and archives are authorized to furnish a photocopy or other reproduction One of these specified conditions is that the photocopy or reproduction is not to be used for any purpose other than private study scholarship or research If a user makes a request for or later uses a photocopy or reproduction for purposes in excess of fair use that user may be liable for copyright infringement This institution reserves the right to refuse to accept a copying order if in its judgment fulfillment of the order would involve violation of copyright law Please Note The author retains the copyright while the New Jersey Institute of Technology reserves the right to distribute this thesis or dissertation Printing note If you do not wish to print this page then select Pages from first page to last page on the print dialog screen The Van Houten library has removed some of the personal information and all signatures from the approval page and biographical sketches of theses and dissertations in order to protect the identity of NJIT graduates and faculty ABSTRACT AUTOMATED LIQUID DISPENSING PIN FOR DNA MICROARRAY APPLICATIONS Suganya A This thesis describes the research and deve
33. EE an IE E EEEE TO TE AR RD ID ERR NER NR A A Push a drop out of the needle ig AA EARNE A AE APR AI E NEPE EN II IEA int CVICALLBACK ZabPushDrop int iPnlHdl int iCtriHdl int iEvntHdl void pvCallBackData int iEvntDatai int iEvntData2 switch iEvntHdl case EVENT COMMIT _ZabJogZFunc 2 0 break return 0 73 IN A AE RS A A PEDES IAE Callback functions to jog the Zaber Along Z axis ja NT Aa amme n IIS O A RON int CVICALLBACK _ZabJogMinuZ int _iPnlHdl int iCtriHdl int iEvntHdl void pvCallBackData int _iEvntData1 int iEvntData2 switch iEvntHdl case EVENT COMMIT _ZabJogZFunc 1 0 break return 0 int CVICALLBACK _ZabJogPlusZ int iPnlHdl int iCtriHdl int iEvntHdl void pvCallBackData int iEvntData1 int iEvntData2 switch _iEvntHdl case EVENT COMMIT _ZabJogZFunc 2 0 break return O ig E EEE ee EE das nies aa Se EM EE lo ee x Type 1 Minus Z Fr 2 Plus Z yi JA 3 Ready for getting liquid i p 4 Minus Z big step Ei hai 5 Plus Z big step PF RUPEE RS PI A RI EA A void ZabJogZFunc int iType long int IStep GetCtrlVal iPniHdI PANEL_ZABSTEP amp lZabStep GetCtriVal iPniHdi PANEL ZABCURRZ amp lZabCurr GetCtriVal iPnlHdl PANEL ZABLOWZLIM amp IZabLowLim GetCtriVal iPnIHdl PANEL ZABUPPZLIM amp lZabUppLim switch _iType case 1 IZabTargTemp IZabCurr
34. IDXLIM int iSsensDAQFiltMaxldx iSensDAQFiltMinldx double dSensDAQFiltMax dSensDAQFiltMin int iSensDAQFiltldx double dSensDAQFiltData double dSensDAQFilt 10 HANDLE hExpStartEvnt 2 HANDLE hExpFuncThrd 2 DWORD dwExpFuncThrdiD 2 int iFileHdl int iFileStat char strFileName 64 int iWhenToPickRef int iCurvRefldx F PARAR II AAA TOS AR A A ISS NS CET ORDRE UE Main procedure i A MV EN EE STE ELELEE E ta Ae OR E A Se S E int main int argc char argv Initialize variables MylnitApp if InitCVIRTE 0 argv 0 0 return 1 out of memory if iPnIHdl LoadPanel 0 spotting uir PANEL 0 return 1 DisplayPanel iPnIHdl RunUserlnterface DiscardPanel iPniHdl Release occupied resources _MyExitApp return O A eer RUE DECR ASEE LA EEEE ES EA EAE Initialize Variables p A A A a IDONEUS O O void MylnitApp void int idx for i idx 20 i idx lt 2 i idx iPortOpen i idx 0 hCommStartEvnt i_idx NULL hCommEOLEvnt i idx NULL hCommEndEvnt i idx NULL hCommThrd i_idx NULL hExpStartEvnt i_idx NULL hExpFuncThrd i idx NULL dwExpFuncThrdID i idx 0 58 iSensDAQNumID 0 isensDAQDataldx Q isensDAQStartRec 0 dSensDAQTime 0 0 0 iSensDAQFiltlIdx 0 F AE AA AA IE i Uninitialize Variables el A REESE AEE AA E E NUN el SO YR ee int CVICALLBACK _Q
35. IZE l ROBOTZ Z ZSTEP 10 UM IN THE SPOTTING PANEL INPUT THE MAXIMUM NUMBER Y AXIS OF ROWS AND COLUMNS COLUMN 1 COLUMN 2 COLUMN 3 X AXIS GO TO WELL AND PULL UP ZABER TO ROW 1 C C C 19000 UM TO FILL NEEDLE ROW2 O O O BACK UP ROBOT TO ROW 3 HOME AND CLEAN C O O NEEDLE TIP AND SIDES MODEL OF THE C SLIDE WITH SPOTS IN 3 COLUMNS AND 3 ROWS Figure 3 3 Flow chart for the entire automatic spotting sequence 25 3 2 Control Methodology used for Spotting The controller s decision is the most crucial part in the formation of spots The controller used here is simple look up table rules based The observation from the initial experimental results for spot formation indicated that each time the spot is formed a particular pattern of graphical display is generated in the plot for Gap vs Sensor voltage for each sample substance used These graphs and spot formations are observed to be repeatable It is observed that when the pin with the sample at the end approaches the slide the sensor voltage keeps increasing as the gap distance becomes closer to the slide But when the non contact spotting is done meaning the droplet contacts the slide then there is a drastic decrease in the voltage readout from the sensor At this point the needle can be pulled back and during this the droplet spotted on the slide tries to cling to the needle tip and elongates until the gap distance increase to a poin
36. IZabStep break case 2 IZabTargTemp IZabCurr IZabStep break case 3 IZabTargTemp IZabUppLim 4 IZabStep break case 4 74 IZabTargTemp IZabCurr 4 IZabStep break case 5 IZabTargTemp IZabCurr 4 IZabStep break j if IZabTargTemp lt IZabLowLim IZabTargTemp IZabLowLim if IZabTargTemp gt IZabUppLim IZabTargTemp IZabUppLim IZabTarg ceil double lZabTargTemp ZABCONVFACT SetCtriVal iPnIHdl PANEL ZABMSG COMMA iCmdldx 1 0 ResetEvent hCommsStartEvnt 1 ResetEvent RCommEOLEvnt 1 _SendZabCmd 20 amp lZabTarg SetEvent hCommsStartEvnt 1 p RARA A A NN A RS A AA Extract the X Y Z values from the Robot communication string M JA A AAC EIER NR E ON A econ ui void GetCoorVal char strVal double pdX double pdY double pdZ char pstr_temp int i idx pstr temp _strVal while pstr temp pstr_temp pdX atof pstr temp while pstr temp pstr_temp while pstr temp pstr_temp pdY atof pstr temp while pstr temp pstr_temp pdZ atof pstr temp Callback function to handle the emergency button 75 int CVICALLBACK EmergencySwitch int iPnlHdl int _iCtriHdl int iEvntHdl void pvCallBackData int iEvntData1 int iEvntData2 switch iEvntHdl case EVENT COMMIT _EmgyProc return O break void EmgyProc void
37. NS Suganya Parthasarathy Dr Timothy N Chang Thesis Associate Professor of Electricaland Computer Engineering NJIT Dr Patricia Soteropodlos Committee Member Date Managing Director Center for Applied Genomics PHRI Dr Sui hoi E Hou Committee Member Date Associate Professor of Electrical and Computer Engineering NJIT BIOGRAPHICAL SKETCH Author Suganya Parthasarathy Degree Master of Science Date August 2005 Undergraduate and Graduate Education e Master of Science in Electrical Engineering New Jersey Institute of Technology Newark NJ 2005 e Bachelor of Engineering in Electronics and Instrumentation Engineering J J College of Engineering and Technology Trichy India 2003 Major Electrical Engineering Presentations and Publications Chang T N Parthasarathy S Wang T Gandhi K Soteropoulos P Automated Liquid Dispensing Pin for DNA Micro array Applications to appear in the IEEE Transactions on Automation Science and Engineering iv To my family for their unconditional love guidance encouragement and support To JILLU PERI ACKNOWLEDGEMENT I would like to express my deepest gratefulness and respect to Dr Timothy N Chang who not only served as my thesis advisor providing valuable and countless resources insight and intuition but also constantly gave me support encouragement and reassurance Special thanks are given to Dr Patricia Soteropoulos and Dr Sui ho
38. NULL Fmt strErrMsg s lt CreateThread failed i n GetLastError MessagePopup CreateThreadt Message strErrMsg return 1 SetXMode iCommPort 0 iXMode 0 SetCTSMode iCommPort 0 iCTSMode 0 SetComTime iCommPort 0 dTimeOut 0 if iPortOpen 1 0 CommPort 1 2 strZabPortName 0 0 strcpy strZabPortName COM4 iBaudRate 1 9600 Parity 1 0 DataBits 1 8 iStopBits 1 1 ilnptQ 1 512 iOuptQ 1 512 DisableBreakOnLibraryErrors Commerr 1 OpenComConfig CommPort 1 strZabPortName iBaudRate 1 iParity 1 iDataBits 1 iStopBits 1 ilnptQ 1 iOuptQ 1 EnableBreakOnLibraryErrors if iCommerr 1 _DispRS232Err 1 return 1 iPortOpen 1 1 FlushInQ iCommPort 1 61 FlushOutQ iCommPort 1 Allow for only one installation InstallComCallback CommPort 1 LWRS RECEIVE ZABCNT O _ZabRplyEOLFunc 0 if NCommStartEvnt 1 NULL hCommStartEvnt 1 CreateEvent NULL TRUE TRUE NULL if hCommStartEvnt 1 NULL Fmt strErrMsg s lt CreateEvent failed i n GetLastError MessagePopup CreateEvent Message strErrMsg return 1 ResetEvent hCommsStartEvnt 1 if RCommEOLEvnt 1 NULL hCommEOLEvnt 1 CreateEvent NULL TRUE TRUE NULL if nCommEOLEvnt 1 NULL Fmt strErrMsg s lt CreateEvent failed i n GetLastError MessagePopup CreateE
39. OnLibraryErrors Commerr 0 OpenComConfig CommPort 0 strRobPortName iBaudRate 0 iParity 0 iDataBits 0 iStopBits 0 ilnptQ 0 iOuptQ 0 EnableBreakOnLibraryErrors if iCommerr O _DispRS232Err 0 return 1 iPortOpen 0 1 FlushinQ iCommPort 0 FlushOutQ iCommPort 0 Allow for only one installation InstallComCallback iCommPort 0 LWRS RXFLAG 0 int ROBEOL _RobRplyEOLFunc 0 if NCommStartEvnt 0 NULL hCommsStartEvnt 0 CreateEvent NULL TRUE TRUE NULL if NCommStartEvnt 0 NULL Fmt strErrMsg s lt CreateEvent failed i n GetLastError MessagePopup CreateEvent Message strErrMsg return 1 ResetEvent hCommsStartEvnt 0 if hCommEOLEvnt 0 NULL hCommEOLEvnt 0 CreateEvent NULL TRUE TRUE NULL if hCommEOLEvnt 0 NULL 60 Fmt strErrMsg s lt CreateEvent failed i n GetLastError MessagePopup CreateEvent Message strErrMsg return 1 ResetEvent NCommEOLEvnt 0 if hcommEndEvnt 0 NULL hCommEndEvnt 0 CreateEvent NULL TRUE TRUE NULL if RCommEndEvnt 0 NULL Fmt strErrMsg s lt CreateEvent failed i n GetLastError MessagePopup CreateEvent Message strErrMsg return 1 SetEvent NCommEndEvnt 0 if RCommThrd 0 NULL I hCommThrd 0 CreateThread 0 0 RobCommMonProc 0 0 amp dwCommThrdlD 0 if NCommThrd 0
40. RobStep 0 dRobTarg 1 dRobCurr 1 dRobTarg 2 dRobCurr 2 break case 2 Minus Y dRobTarg 0 dRobCurr 0 dRobTarg 1 dRobCurr 1 dRobStep 1 dRobTarg 2 dRobCurr 2 break case 3 Minus Z dRobTarg 0 dRobCurr 0 dRobTarg 1 dRobCurr 1 dRobTarg 2 dRobCurr 2 dRobStep 2 break case 4 Plus X dRobTarg 0 dRobCurr 0 dRobStep 0 dRobTarg 1 dRobCurr 1 dRobTarg 2 dRobCurr 2 break case 5 Plus Y or Nex Column dRobTarg 0 dRobCurr 0 dRobTarg 1 dRobCurr 1 dRobStep 1 dRobTarg 2 dRobCurr 2 break case 6 Plus Z dRobTarg 0 dRobCurr 0 dRobTarg 1 dRobCurr 1 dRobTarg 2 dRobCurr 2 dRobStep 2 break case 7 Next Row dRobTarg 0 dRobCurr 0 dRobStep 0 dRobTarg 1 dRob1stSpot 1 dRobTarg 2 dRobCurr 2 break case 8 Get up dRobTarg 0 dRobCurr 0 dRobTarg 1 dRobCurr 1 dRobTarg 2 dRobCurr 2 50 0 break case 9 Elongation GetCtrlVal iPnIHdl PANEL ROBLIMITZ amp dRobLim 2 dRobTarg 0 dRobCurr 0 dRobTarg 1 dRobCurr 1 dRobTarg 2 dRobLim 2 ROBLIMGAP break default break strRobCmd 0 O GetCtrlVal iPnIHdl PANEL ROBSTEPX amp dRobStep 0 if dRobTarg 2 gt 0 0 dRobTarg 2 0 0 if dRobTarg 2 lt dRobLim 2 dRobTarg 2 dRobLim 2 ResetEvent hCommsStartEvnt 0 ResetEvent RCommEOLEvnt 0 _SendRobCmd
41. RobWell 0 dRobWell 1 dRobWell 2 SetEvent hCommsStartEvnt O Callback functions to move the Robot to the 1st spot int CVICALLBACK _Go1stSpot int iPnlHdl int iCtriHdl int iEvntHdl void pvCallBackData int iEvntData1 int iEvntData2 switch _iEvntHdl case EVENT COMMIT _RobGo21stSpotFunc break return 0 void RobGo21stSpotFunc void SetCtriVal iPnIHdl PANEL EXPMSG1 ROBOT GOING TO THE 1ST SPOT SetCtriVal iPnIHdl PANEL_EXPMSGZ2 SetCtriVal iPniHdl PANEL_ROBMSG COMMA GetCtriVal iPniHdl PANEL 1STSPOTX amp dRob1stSpot 0 GetCtrlVal iPniHdl PANEL 1STSPOTY amp dRob1stSpot 1 GetCtrlVal iPnlHdl PANEL ROBLIMITZ amp dRobLim 2 dRob1stSpot 2 dRobLim 2 ROBLIMGAP SetCtriVal iPniHdl PANEL 1STSPOTZ dRob1stSpot 2 strRobCmd 0 0 ResetEvent hCommsStartEvnt 0 ResetEvent RCommEOLEvnt 0 _SendRobCmd 2 dRob1stSpot 0 dRob1stSpot 1 dRobCurr 2 _SendRobCmd 2 dRob1stSpot 0 dRob1stSpot 1 dRob1stSpot 2 SetEvent hCommsStartEvnt 0 r HE SS STEEN EE E AA Ta AA AA At ine EA EE Callback functions to Form one spot qe oe NT COUP A A A MN DE et ST int CVICALLBACK _FormSpot int iPnlHdl int _iCtriHdl int iEvntHdl 66 67 void pvCallBackData int _ EvntData1 int iEvntData2 switch _iEvntHdl case EVENT COMMIT break return 0 I A ETE rae NER RE AR En A RSEN NER ON E er S NEN EEN re SRL
42. To run the program and move robot arm to T40 position press START Pressing START executes the current program in RAM 3 Serial communication concept The Seiko D Tran controller has two RS 232 ports for serial communication with any device It is a 25 pin female connections RS 232 is a standard that describes a common method of serial signaling using positive voltage for logic 0 and a negative level for logic 1 When RS 232 line is powered up but idle not conveying data it is in its lower voltage stage When data is sent RS 232 jumps to its higher voltage state momentarily to alert the receiver and this is called the start bit The communication takes place through the American Standard Code of Information Interchange ASCII The Seiko controller s Tech Terminal or the keyboard s keys are coded in to 7 bits ASCII which 1s normally the character length Parity is generally added for authentication This 1s followed by one or two stop bits Typical ASCII transmission is shown for transmitting a character say A Character A Decimal 065 Hexadecimal 041 Binary 01000001 48 Format START CHARACTER LENGTH OOO Space 0 1 0 0 0 0 0 1 1 0 The bit time is expressed in samples per second known as baud The common baud rate used 1s generally 9600 baud A bit time is defined as 1 bps For example at 9600 bps the bit time is 1 9600 104 microseconds Protocol and use of DARL RS 2
43. al that remains in the pins is lost when the pins are washed This waste of valuable resources significantly increases the cost of chip production Present fabrication cycle time with a 48 pins and 40x384 well microplates 15360 spots is about 8 hours which must be significantly reduced Finally due to the design of the impact pins they tend to get clogged by viscous liquids such as those containing glycerol which is useful in proteomics Existing drop generation mechanisms can be categorized into contact printing with pins quill solid or pin and ring or non contact printing with microsolenoid thermal ink jet piezoelectric or acoustic printing heads Due to the impact at contact pin structure deformation and clogging from contaminants collected at contact pin based contact printing is prone to suffer from slide to slide inconsistency A novel contact printing device is the fabrication of a micromachined silicon pin MEMS technology offers advantages such as freedom of pin and slot design both in size and shape possibility silicon pin can pick up lul of DNA solution One of the big challenges of droplet size control is preprinting It has been reported that after loading sample into microchannel the commercial pins must be spotted a number of times onto a slide to create consistent spots due to the adherence of additional DNA solution to its hydrophilic surface outside the slit Silicon microarray pin also has a similar problem but to a les
44. arse platform The Seiko D Tran robot communicates with a PC via LabVIEW driver developed in the lab The product specifications and their definitions are given below e Resolution The resolution or addressability is the distance equivalent to the smallest incremental move the device can be instructed to make In other words it is the linear or rotational displacement corresponding to a single microstep of movement e Repeatability The repeatability is the maximum deviation in the position of the device when attempting to return to a position after moving to a different position e Accuracy The accuracy is the maximum deviation of the actual position of the device from the requested position over the full range of motion e Backlash As was seen in the repeatability section backlash is the deviation of the final position that results from reversing the direction of approach The Seiko D Tran robot has a 10 micron repeatability on all three axes The positioning accuracy is augmented by a piezoelectric positioner with a 15 micron range and better than 0 1micron repeatability The Zaber Stage with a 8 micron accuracy and 0 3 micron repeatability drives a fiber probe within the SmartPin assembly Figure 2 2 SmartPin cross section view and prototype As shown in Figure 2 2 the SmartPin assembly comprises of the following subcomponents 1 A fluid reservoir containing the sample liquid materials This reservoir
45. canned as stated under SET 2 it was left to dry for 30 min and scanned again It can be noticed that there is edge effect The molecules gravitate towards the edge of the well and accumulate around it and fluorescence can be seen in the form of a ring SET 4 After scanning as in SET 3 the wells are re hydrated using distilled water While scanning a homogeneous pattern of fluorescence can be observed as before in SET 2 results unlike the ring effect in SET3 Again the fluorescence increases as the concentration of MB increases from 100 nM to 500 nM As the concentration of the Molecular Beacon increase the Composite Pixel Intensity CPI observed under the scanner also increases pronouncing more fluorescence The CPI is a representative composite intensity calculated for each pixel based on the intensity values The slope of the plot in Figure 4 18 is almost linear with 6600 CPI increase with every 100nM increase in concentration of the Molecular Beacon Molecular Beacon Concentration Vs Scanner Composite pixel Intensity y Q o o e o 25000 20000 15000 10000 5000 0 Composite Pixel Intensit 0 100 200 300 400 500 600 Concentration of Molecular Beacon nM in the sample with 500nM of target Figure 4 18 Concentration of Molecular Beacon Vs Composite Pixel Intensity Apart from efficient material handling and spotting in the wells the smart pin can handle various materials without cross contamination after cleani
46. cron SPOtS wewwewenwenenea 38 4 15 Molecular Beacon combining with the target to produce UO AA e iets MUNDUS 39 4 16 Fluorescence exhibited by the buffer MB T and MB and T together for varying Quantities id 40 4 17 Fluorescence observed by the scanner 4 4 18 Concentration of Molecular Beacon Vs Composite Pixel Intensity 42 4 19 Alternate columns of buffer with 10 Cy3 and distilled water spotting showing no cross contamination of material handled by the pin 43 A 1 Reduction of RS 232 characteristics into integers in DARL 49 A 2 Communication port pin configuration 51 A 3 Communication port 1 SEttINBS oooocococcncnccconnrocononcnnnnnn eee canoso 5 A 4 ASCII settings for communication pott 52 A S MTI 1000 Fotonic sensor characteristicS 53 xii CHAPTER 1 INTRODUCTION Genomic research is undergoing an industrial revolution with automation and high speed computation being two of the enabling factors responsible for the explosive growth in sequencing efforts reducing the cost from 5 per finished base to about 0 10 in just ten years The next phase gene expression profiling is equally significant especially in clinical studies of genetic diseases such as cancer The basic steps of determining gene
47. ction on Control Systems Technology Special Issue on Smart Materials January 2001 9 Chang T N Jaroonsiriphan P and Sun X Integrating Nanotechnology Into Undergraduate Experience A Web Based Approach International Journal of Engineering Education Volume 18 5 August 2002 10 Chang T N and Jaroonsiriphan P Web Based Distance Experiments for Real Time Control and Signal Processing Proceedings of the 2002 ASEE Annual Conference 11 Chang T N Servo Control Design Encyclopedia of Life Support Systems United Nations Educational Scientific and Cultural Organization UNESCO 2004 82 83 12 Chang T N and Tolias P P Delivery of metered amounts of liquid materials US and International Patents pending 13 Introduction to Molecular Beacons Public Health Research Institute n d Retrieved July 14 2005 from http www molecular beacons org Introduction html 14 Brochure for MTI Fotonic Sensor MTI Instruments Inc n d Retrieved July 14 2005 from http www mtiinstruments com pdf mti2 1 00 pdf 15 Instruction Manual of MTI 1000 Fotonic Sensor MTI Instruments Inc 16 Instruction Manual of SEIKO D TRAN Robot 17 Zaber Technologies Inc T Series Positioning Products User s Manual 18 Products microarray hardware TeleChem International Inc n d Retrieved July 21 2005 from http arrayit com Products Printing 946 946 html 19 Affymetrix GeneChip techn
48. d by again checking for maximum column number occurrence If the Z limit of the robot has not been reached then the spotting decision 1s transferred to the decision controller and a YES or NO decision is awaited from the decision controller A YES from the decision controller results from a successful spot formation and then the robot has to back up and has to go to next column in the same row for spotting A NO decision corresponds to unsuccessful spot formation and then the robot Z axis has to be jogged down by another 10 um and again the Z limit is checked and continued in the same fashion as depicted in the below flow chart Figure 3 3 24 START INITIALIZE THE START SPOTTING BY GOING TO SPECIFIED FIRST SPOT LOCATION ROBOT ZABER MOVE ZABER TO MAXIMUM LIMIT 20 000 UM GET DROP AT THE TIP OF NEEDLE BY ROBOT X X DOING ZABER STEP 20 UM X STEP Y WAIT TO FIT THE NEEDLE OVER THE SENSOR PROBE NO qun fe ae STOP COLUMN ES ss COLUMN 1 SET THE LIMIT OF X Y Z ACOORDINATES OF THE ROBOT AND ROBOT Y Y Y STEP INPUT THE ROBOT X Y ZANDA FOR 1 STEP VALUES 2 WELL POSITION 3 FIRST SPOT LOCATION COLUMN lt COLUMN MAX BACK ROBOT Z BY ZSTEP UPTO 120 UM FROM Z LIMIT ROBOT Z lt Z INTHE ZABER ROBOTZ lt Z LYE PANEL SET LIMIT pe THE LIMITS NO AND STEP YA S
49. dized with Cy3 and CyS labeled RNAs prepared from human liver and kidney respectively oooooocoonccconccoconnonanoonacononaconanananos 2 DETECTIVE spot FOL MAO Ka 3 Prototype microarrayer with SmartPin overnnrnnrnnrnnvvenvevevnsvnsnnnnnnnnnn 6 SmartPin cross section view and prototype 7 SE SO D Tr an RODO sene 9 MTI 1000 Fotonic Sensor with a MTI 3802 sensor module 11 Receiver illumination and instrument Output 11 Fiber distribution in the sensor probe sees 12 The optical lever principle realized on a bundled fibre probe 12 Calibration of Fotonic sensor up and front side and back side slope charts of Fotonic sensor dOWN wmwwn wewe 14 T LA Series Zaber hnear actuator ii ds 15 Front Panel and LabView program for Zaber stage 18 Mechanical integration of Zabi 18 SPOUT se eer cts isc 20 Front Panel of spotting experiment cece ee eeee cece ee eee cee tens 22 Flow chart for the entire automatic spotting sequence 24 LIST OF FIGURES Continued Figure Page 3 4 Standard spot formation Graph showing the four different regions 26 3 5 Controller action flow chart for spotting ezperiment 27 4 1 Intensity plot for 100 glycerol spot formation 29
50. e JJ seann ee P x o e o ef us ma f g et 0 Q v m A Y ends t j Dl x e p ss s L y aia RA a 8 Wa Ya Races z m a m em iV i d A E re 3h E gt a gh gt t A 2 t we A G tt Qs 4 ee gt f x E aks an I f xx Eh ra 3 La pA vm HA mms E ze ANI Be mm gt e ay a AS Y v ES y 2 gt pb 1 aT fom WT PIG aw B ae saw v he t e WI We ee ee ae EA pi 2 Pase BI 1 se p Ny gt fe t a WA da t ne e tk41 gt e t WA uvenn L AL Onde x p t H nes PE tt a A ma A bm Figure 4 11 Spot matrix formed out of 100 glycerol each spot measures 160microns left and 50 glycerol each spot measuring 190 microns right 3 GenePix Pra 2 25 05anonomous tif Image Histogram Lab Bock Batch Analysis Resis I Scatter Plor Aspon Image Festure Viewer Block Feature Previews 535 532 X Y fam 3850 41930 Preview 22 Name Prewew 83 Wavebengih 535 Wavelengin 532 Wavelengh 23 Wavelength 714 E Ratio 535 537 Rook Ratio M3 Tooks zem eg arar 4 Li ra ES A SY Ges e eoe Hardware Kes not ho Dik 2505 68 No barcode Figure 4 12 Microarray with 100 glycerol and 80 microns diameter spots Figure 4 13 and Figure 4 14 show that the spots made by the smart pin are uniform in size with uniform spacing in between the spots Different spot sizes can be achieved b
51. echnique impact or inkjet as shown in Figure 1 1 on the other hand is flexible and less expensive For the impact printing method a hollow pin draws up DNA sample fluid and prints a series of small dots on a glass slide as shown in Figure 1 2 Figure 1 1 Microarrayer with impact printing technology left and contact printing pin right 18 Figure 1 2 Image of a section of a Microarray Experiment using the Center for Applied Genomics CAG Human 19K DNA oligo array The chip was hybridized with Cy3 and Cy5 labeled RNAs prepared from human liver and kidney respectively However minimum spot size and reliability remain key limiting factors that impede the competitiveness of the printing technique Presently a standard microscope slide can hold up to 40 000 spots To accommodate the entire human transcriptome this density must be increased to well over 120 000 spots per slide implying that the current spot size of 100 microns must be decreased accordingly Furthermore both impact and inkjet printing methods are open loop and require careful calibration for each run where uneven spot formations are not uncommon A scan of a defective print is shown in Figure 1 3 where blurred and missing spots are present Figure 1 3 Defective spot formation Furthermore the printing technique generally deposits only about 30 of the liquid material that is drawn into the pins by capillary flow during each 100 chip print session The materi
52. ed by the user to microsteps before sending it to the device Figure 2 9 T LA Series Zaber linear actuators 16 Table 2 1 Product Specifications of Zaber Stage Part Range Resolution Repeatability Cyclic Backlash Number Accuracy o TT The control is through the RS232 port The communications settings must be 9600 baud no hand shaking no parity one stop bit The amber LED will light when there is activity on the RS232 lines After power up the units in the chain will each initialize themselves as unit number 1 and thus they will each execute the same instructions To assign each unit a unique identifier a renumber instruction as specified in Table 2 3 must be issued after all the units in the chain are powered up and every time an unit is added or removed from the chain All instructions consist of a group of 6 bytes They must be transmitted with less than 10 ms between each byte If the unit has received less than 6 bytes and then a period of more than 10 ms passes it ignores the bytes already received It 1s recommended that the software behaves similarly when receiving data from the devices especially in a noisy environment The following Table 2 2 shows the instruction format The first byte is the unit number in the chain Unit number 1 is the unit closest to the computer unit number 2 is next and so forth If the number 0 is used all the units in the chain will execute the accompanying command simultaneously
53. elements allows higher element density and increases printing tip life over the customary slotted stainless steel pins But implementation of ceramic tips requires custom tooling consisting of a modified tip holder printing pin assembly and air reservoir Moreover the ceramic tips are more sensitive to inaccuracies in leveling of the printing tip holder to the arrayer slide platter As a result the tip holder must be carefully aligned to prevent an increase in deposition failure Another issue with ceramic tips is they are more difficult to dry after cleaning and if they are not completely dry the next DNA samples do not aspirate completely 23 CHAPTER 2 HARDWARE AND SOFTWAR DESCRIPTION 2 1 Overall Hardware Description In this work a smart pin liquid dispensing system is designed and constructed to integrate a number of critical tasks spot formation via active sensing and control fine positioning and spot characterization The SmartPin combines sensing actuation and feedback control it is capable of regulating spot size and providing robust non contact delivery at a high speed SEIKO D TRAN ROBOT AN CONTROLLER D f SEIKOD TRAN E SMART PIN e ue PCLAB VIEW ASSEMBLY INTERFACE 77 RABERSTAGE K PIEZOELECTRIC N POSITIONER Figure 2 1 Prototype microarrayer with SmartPin The prototype system is shown in Figure 2 1 where a Seiko D Tran robot serves as the co
54. eosiving Fiber Transmitting Fi Figure 2 6 Fiber distribution in the sensor probe 14 Each MTI 1000 Fotonic probe contains a set of light transmitting and light receiving fibers which can be arranged in three different configurations random hemispherical or concentric as shown in Figure 2 6 As shown in Figure 2 7 a tungsten halogen lamp acts as the light source and feeds light down the transmit fibers where 1t exits the probe tip and hits the target Light that is reflected from the target 1s captured by the receive fibers and processed by the console The light intensity is monitored which 1s proportional to the distance between the probe tip and the target being measured OPTICAL SENSOR TRANSMITTING BUNDLE OPTICAL FIBRE PROBE PHOTO DETECTOR RECEIVING BUNDLE Figure 2 7 The optical lever principle realized on a bundled fiber probe 13 Referring to the Figure 2 5 the front side of the curve is defined as the operating area between sensor probe to target contact and the optical peak and it is characterized by a positive slope and higher sensitivity on the response curve The front side slope is plotted on the most linear rise of the curve s front side and indicates the sensor s front side operating range The back side of the response curve is the area from the optical peak on out to large gaps The back side slope is the most linear portion of the back side of the curve and indicates the sen
55. equences that are located on either side of the probe sequence A fluorophore is covalently linked to the end of one arm and a quencher is covalently linked to the end of the other arm Molecular beacons do not fluoresce when they are free in solution However when they hybridize to a nucleic acid strand containing a target sequence they undergo a conformational change that enables them to fluoresce brightly In the absence of targets the probe is dark because the stem places the fluorophore so close to the nonfluorescent quencher that they transiently share electrons eliminating the ability of the fluorophore to fluoresce When the probe encounters a target molecule it forms a probe target hybrid that is longer and more stable than the stem hybrid The rigidity and length of the probe target hybrid precludes the simultaneous existence of the stem hybrid Consequently the molecular beacon undergoes a spontaneous conformational reorganization that forces the stem hybrid to dissociate and the fluorophore and the quencher to move away from each other restoring fluorescence 13 as shown in Figure 4 15 E o s QAI o E P FIN Molecular Bisang Target Hybrid Figure 4 15 Molecular Beacon combining with the target to produce fluorescence 13 40 Determination of Sensitivity of Fluorescence by the Scanner The smart pin can handle a metered quantity of the Molecular Beacon MB and can be used to deliver the MB to wel
56. he material contact condition can be derived by continuously monitoring the sensor intensity For example 100 glycerol has a refractive index of 1 47 while that of water is about 1 33 both of which are sufficiently high to induce total internal reflection at the pin tip due to the liquid convex curvature Once the liquid contacts the slide the internal reflection is replaced with transmission due to the change of refractive index from 1 air to 1 57 glass resulting in a sudden drop of sensor signal intensity as most of the light is transmitted away from the sensor This effect is shown Figure 4 7 where ray tracing is utilized to show the light transmission patterns Before the droplet at the pin tip contacts the slide a major portion of light rays are trapped within the pin and reflected back to the sensor as show in Figure 4 7 up Once the contact is made most of the rays are transmitted into the slide and backscattered resulting in a lower sensor signal This signal intensity drop is compared to the threshold stored in the database so that a disengagement signal is synthesized and communicated to the robot and the piezoelectric stack An internal timer provides the predetermined contact delay time which is one of the dominant influence factors on spot size 33 iV i IA fi i M ALD JA i li i j N M M i als E M i VN D 4
57. i ES bh 500 nM T 300 nM BOO SOnnM k g KE s 0 nM 500 nMISO0 nM T4200 nM nM 500 nM EN nM 300 nM 400 nM 1500 nMIT 400 nM 400 nM 500 nM T 400 nM m j ET zA 400 nM 500 nM S00 nM T 500 nM Figure 4 17 Fluorescence observed by the scanner MB Molecular Beacon with the following sequence Fluorescein CGCAG ACC ATG ATC GGC GGC CTGCG BlackHole Quencher 2 The underlined sequences are the arm sequences of the Molecular Beacon and are not related to the target of the MB T Target always 500 Nano Molar nM Complementary Oligonucleotide target with sequence TT GCC GCC GAT CAT GGT TT T MB Target Molecular Beacon The four sets of experiments can be described as follows SET 1 The plastic slide was scanned to check for fluorescence effect There was no fluorescence noticed 42 SET 2 The molecular beacon target and the combination of target and molecular beacon of the specified concentration were spotted by the use the Smartpin metered to deliver lul and the slide was scanned immediately before drying A near background fluorescence is observed in the MB alone or the target alone But appreciable fluorescence is observed in the combination of the target and the molecular beacon The lower the concentration of the MB the weaker is the fluorescence So it can be noticed that the pattern of fluorescence increases as the concentration of MB increases from 100 nM to 500 nM SET 3 After the slide was s
58. i E Hou for actively participating in my committee I owe much to the members of Public Health Research Institute PHRI Sincere thanks go to Dr Patricia Soteropoulos Mr Tongsheng Wang Dr Salvatore A E Marras and Dr Sanjay Tyagi for their invaluable help I appreciate having had the chance to get to know them Iam thankful to the National Science Foundation Grant 0243302 High resolution high density microarrayer for genetic research for partially supporting my work Many of my senior colleagues in the Real Time Controls Laboratory are deserving of recognition for their support I would like to thank my colleagues Biao Chen Puttiphong Jaroonsiriphan Yuan Ding Qiong Shen Paiboon Sriwilai Jaroen and Kunj Gandhi for their immense help during practical difficulties Also many thanks to Ms Brenda Walker and the entire staff of Electrical and Computer Engineering Department at NJIT Last but not the least I would like to thank Aravind Parthasarathy and Sowmya Vedhartham Sampath all your understanding and support saw me through difficult times vi TABLE OF CONTENTS Chapter 2 HARDWARE DESCRIP TION costaba e 2 1 Overall Hardware Descrip Ola sisas Wi 2 2 Description of SEIKO D Tran Robot eese 221 Seiko Robot Manipulacion 22 2 COMMONER tire 2 3 Description of Optical Fibre Displacement Sensor Fotonic Sensor 2 4 JDescrp omot Zba SA sv 3 CONTROLLER AND SOFTWARE DESCRIPTION
59. if i n 4 dive PURO li Nt Y A m Y W i i t a N N LE FUY S BEEN ia Men aa il gr Figure 4 7 Ray Tracing result for SmartPin before Up and after Down droplet engagement A high frequency vibration can be generated by the fiber driver on the piezoelectric positioner to facilitate separation of the droplet from the fiber This way extremely small droplets can be formed at high speed For example a 50 micron diameter fiber can deliver droplet size as small as 0 1 nL resulting in a 25 micron spot and a ten fold reduction of use of the DNA materials These reduced spots can be reliably detected by scanners with 0 1 fluor micron and 2 5 5 micron resolution Currently the pin diameter is about 200 microns and spots size can be controlled from 80 microns to 220 microns in diameter Throughout the process the SmartPin does not come into contact 34 with the slide and therefore does not encounter the standard wear out and slide damage problems of the impact pins 4 2 Spot Detection The SmartPin not only monitors and controls spot formation but also doubles as a spot sensor This function is useful for aspiration of sample as well as spot quality check A rapid raster scan algorithm is stored in the PC and in turn controls the Seiko D Tran robot to scan in an x y plane 60 microns over the slide An example droplet formed by 100 glycerol
60. int 5 along X axis and programming to move by 10um increment along the Y axis on either sides of point 5 the row scan is performed The position of the robot is noted as points 3 and 4 at the start and the end point of intensity change during the row scan The difference between these two positions of the robot is the diameter of the spot in microns And the mid point of these two positions namely point 6 in Figure 4 8 will give the midpoint of the spot Now that the size of the droplet is well defined the raster scan to get the geometry can be carried out The X direction movement will be column wise scan The edge of the spot namely point A as in figure can be made as the start point of the scan by just incrementing the position of point 4 by the radius of the spot in the X axis The Y coordinate value and Z coordinate values are fixed and the pin is moved along the X direction by 10um each time till point C 1s reached Then the next column is scanned by incrementing 10um along the Y axis and fixing it The X coordinate is incremented in steps of 10um for the range of the diameter of the spot and Z value is fixed 60um above the slide as before In the same fashion all the columns are scanned until points B to D are covered The intensity values recorded in each column scan is arranged in the form of a matrix This intensity value will be one of the dimensions of the 3D plot and the other two dimensions will correspond to the diameters of the spo
61. intensity pick up by the sensor 6 Controlling dwell time After the spotting has been observed the pin has to be pulled back immediately so that the spot size does not grow bigger Just after the spot formation 1s observed the pin is reversed meaning the robot moves back towards home with a maximum speed of 10 1um ms Downloading the program to the robot reduces the time delay involved in spotting and pulling up of the pin and hence the spot size will not grow in size 7 Surface preparation The slides are coated with PLL Poly L Lysine so that the entire sample at the tip of the pin could be used in spotting The coating spreads the spot uniformly and makes it feasible to get regular spots Uncoated slides will not spread the spot uniformly and hence results in irregular spotting 3 1 Description of Spotting Experiment The robot and the Zaber positions are initialized meaning the robot 1s taken to the home position and then the Zaber stage is moved to its maximum limit of 20000um Now a fine cut and polished borosilicate pin of outer diameter 1 35mm and inner diameter 0 59mm is fitted to the end of the sensor driven by the Zaber stage The maximum safe limit of robot X Y Z axis are specified to prevent the needle to come in contact with the experimental glass slide The center to center distance between the spots can be fixed by the step incremental values of X and Y coordinates of the robot The position of the well containing the sample li
62. lopment of a new liquid dispensing aspiring system that is capable of producing micro sized spots droplets for molecular biology research and analysis In particular the application is focused on DNA microarray fabrication with the goals of smaller spot size higher yield more efficient usage of biological materials and capability to handle high viscosity liquids The new system is based on active sensing and control and it is part of a fully integrated robotic microarray system for genomic and proteomic applications The prototype system handles water as well as thick liquids such as 100 glycerol and generates spots in a contactless manner with controllable spot size ranging from 80 microns to 200 microns Microarray technology is enhancing many areas of biological research including stem cell cancer and infectious disease research This new method of microarray production will afford hospitals and laboratories the system necessary to help detect and study genetic changes in cells in a more efficient and cost effective manner AUTOMATED LIQUID DISPENSING PIN FOR DNA MICROARRAY APPLICATIONS by Suganya Parthasarathy A Thesis Submitted to the Faculty of New Jersey Institute of Technology in Partial Fulfillment of the Requirements for the Degree of Master of Science in Electrical Engineering Department of Electrical and Computer Engineering August 2005 APPROVAL PAGE AUTOMATED LIQUID DISPENSING PIN FOR DNA MICROARRAY APPLICATIO
63. ls There will not be any waste of material as a metered quantity is taken in the pin and is fully delivered into the wells lul of each of the solutions shown in Table 4 1 is delivered into the wells machined into a plastic surface and the fluorescence of each well is determined with a GenePix microarray scanner The focus is set to 50um towards the slide so that the scanner can look into the wells Table 4 1 Sample Solutions of Molecular Beacons MB Molecular Beacon T Target nM nano Molar LE lr 7 IX PCR IX PCR Em pU o f 2 A BUFFER i lul P f B Molecular Beacon lul C TARGET lul 00 nM 5 nM 500l50nM 500 f500nM 500 D MB TARGET lul lis d A Figure 4 16 Fluorescence exhibited by the buffer MB T and MB and T together for varying quantities 41 Figure 4 16 shows the fluorescence pattern observed by the scanner It can be seen that the Molecular Beacon and target combination produce fluorescence as expected In the following experiment one microliter of Molecular Beacon target and the combination of target and Molecular Beacons are spotted in plastic wells Each time the concentration of the materials are varied in steps of 100nM from 100 to 500nM as indicated in Figure 4 17 and the fluorescence intensity is measured by the scanner SET I SET 4 Slide Scanned immediately Dried and Scanned Hydrated 100 nM S00 nMET 100 nM mete 100 n500 nMT 100nM 200 nM S00 nM T 200 nM di
64. mple of Z in EDIT Mode 10 Z 78 20 END Example of Z in MONITOR Mode DO Z 78 press enter In the Computer Side as shown in Figure A 2 the COMMI port is used 51 Figure A 2 Communication port pin configuration Hyper terminal is a terminal program that will enable a PC to communicate directly with a Communication port Eg Comml On Windows 95 98 98 SE Windows 2000 Windows ME or NT4 the Windows HyperTerminal program can be used as it is included as a part of the operating system After a new connection is opened by going to CONFIGURE the parameters on the computer side can be set as per the settings on the robot side as shown in Figure A 3 Data bits le Parity None Stop bits 1 Flow control Hardware v EE vr UA vn ee need Restore Defaults Figure A 3 Communication port 1 settings Now to set the other parameters go to PROPERTIES gt SETTINGS leave the EMULATION to auto detect and go into ASCII set up as shown in Figure A 4 ASCII Setup ASCII Sending V Send line ends with line feeds a Iv Echo typed characters locally Line delay fo milliseconds Character delay fo milliseconds ASCII Receiving v Append line feeds to incoming line ends APARECIA RCA AAA ISI EOE AA ILI v Wrap lines that exceed terminal width SD Fs Min hum Figure A 4 ASCII setting
65. nal reflection principle due to the difference in refractive indices of air glass pin sample material and the shape of the material contact condition can be derived by continuously monitoring the sensor intensity 2 2 Description of SEIKO D Tran Robot The Seiko D Tran Robot is four axis Cartesian robot Other robots in the family of Seiko D Tran are RT Cylindrical Coordinates TT Multi Articulated SCARA Robots The entire robot assembly consists of three visible parts as seen in Figure 2 3 1 Seiko Robot Manipulator 2 Controller 3 Key Board Console Seiko robot manipulator Controller Key board console Figure 2 3 SEIKO D Tran Robot The Seiko D Tran robot offers high precision repeatability and speed The Seiko D Tran robot is modularly constructed and its high precision accuracy repeatability and speed make applications with close tolerances possible 2 2 1 Seiko Robot Manipulator The Seiko Robot has four manipulators that are based on closed loop DC servo motors which provide different motion in the robot envelope The A or the Alpha Axis allows rotation for the End Effecter It can be mounted with the Tool Fang downward or upward The X Axis allows front back motion in the X plane The Y Axis allows side ways motion The up down motion is provided by the Z Axis 2 2 2 Controller The Robot comes with a well equipped controller which is easy to operate and has an emergency stop button and a
66. ng with distilled water each time The sample to be spotted 3XSSC with 10 Cy3 dye for fluorescence is 43 picked up by the pin and the spotting is carried out in one column on a glass slide Six spots are spotted in a column as shown in Figure 4 19 Then the sample is emptied by moving the Zaber to the maximum limit Now the pin is immersed in distilled water and the Zaber is jogged up and down 4 to 5 times to draw and release distilled water in the pin Standard ultrasound sonicating is not necessary Next spotting is carried out with distilled water in the next column and six spots are made Following this again a fresh supply of the buffer and Cy3 dye is filled in the pin and spotting of the next column is carried out Thus an alternate column of buffer with Cy3 and distilled water spotting is done It is evident from the Figure 4 19 that there is no contamination in the pin after cleaning and refilling The spots in every alternate column water only do not produce fluorescence Only the spots made with the buffer and Cy3 exhibit fluorescence Do prin weh hulter cyd ant w EY E JR E A an Pew 1 P X wet MES An i Pre a Kama i o Pr ne H1 ada EM 0 Wam Y k Hi i GNI air 522 amp 0 i ire 1 i d ng 34 Ww if i Toe P sv i hen DAY E 1 23 Fans Ei 2 6 9 con w o i P q wt f i HE 3 o0 amp D pu iw t ty l li A 3 A i i P f 4 0 6 HE s Ry 70 m g
67. nsity in volts Air gap in um Figure 4 1 Intensity plot for 100 glycerol spot formation 29 30 The corresponding time series plot with X axis scaled as 1 unit 5 ms and the corresponding spot imaged by a 10 micron GenePix scanner are shown in Figure 4 2 Downward trend of the intensity curve indicates the pin s approaching the slide and the abrupt transition mirrors the changes in the intensity plot in Figure 4 1 It is also observed that the spots are round and uniform t CLR pe guns 1 50 000 100 000 150 000 200 000 250 000 305 0 Figure 4 2 Graph depicting the intensity change during 160um droplet formation for 100 glycerol The second and third sets of tests dispense 50 glycerol and de 1onized water respectively Cy3 1s added to the sample liquid The intensity plot time series plot with X axis scaled as 1 unit 1 ms and the corresponding spot imaged at 10 micron resolution in a GenePix 4000B scanner for 50 glycerol sample are shown in Figures 4 3 and 4 4 Those for de ionized water are shown in Figures 4 5 and 4 6 During spot formation the intensity drops by 56 for 50 glycerol and 30 for water The intensity change 1s less pronounced than that for 100 glycerol sample because the effects of total internal reflection decrease with the refractive index Furthermore a low viscosity and weaker interaction with poly L lysine also result in slightly large elongation and subsequently larger spots
68. o move to a well which contains the test liquid The pin is immersed into the well and the Zaber stage is withdrawn back mak ng the Fotonic sensor probe act as a plunger and takes in metered quantity of test sample After cleaning the side walls of the pin the robot is moved 120um above the glass slide and the Zaber stage is pushed by 20um so that the Fotonic sensor probe plunges a metered quantity of sample at the tip of the pin Now the robot is jogged 10um each time until a sharp decrease in the intensity of the sensor is noted indicating the formation of spot This process is a non contact method as the robot stops jogging further when the liquid on the pin tip contacts the slide surface In this method as shown in Figure 3 1 there are four stages namely Pull down Contact Pull up elongation and Pull up separation The pin travels down with the sample droplet at the tip and makes a contact with the slide when it is approximately 10um above the slide and then the robot is reversed back during which the droplet tends to stick to the needle and elongates as a column before it detaches fully from the pin Some part of the droplet is left behind in the pin Analysis has been done with different liquids with varying viscosities 19 20 SAMPLE LIQUID LIQUID ELONGATION SEPARATION PULL DOWN CONTACT Figure 3 1 Spotting sequence The factors that affect the spotting include the following l Pin size and sha
69. obLim 2 SetCtriVal iPnlHdl PANEL WELLZ dRobLim 2 ROBLIMGAP SetCtriVal iPniHdl PANEL 1STSPOTZ dRobLim 2 ROBLIMGAP break return 0 F NE a a a a aaa aaa e a a ee Se pt AA AI Callback function to automate the 1st part of the experiment J SA EE ANEEL E IE A O PCI AN DE int CVICALLBACK _AutoExpStep1 int iPnlHdl int iCtriHdl int iEvntHdi void pvCallBackData int iEvntDatai int iEvntData2 switch _iEvntHdl case EVENT COMMIT if RExpStartEvnt 0 NULL hExpStartEvnt 0 CreateEvent NULL TRUE TRUE NULL if RExpStartEvnt 0 NULL I Fmt strErrMsg s lt CreateEvent failed i n GetLastError MessagePopup CreateEvent Message strErrMsg return 1 ResetEvent hExpStartEvnt 0 SetEvent hExpStartEvnt 0 if NExpFuncThrd 0 NULL hExpFuncThrd 0 CreateThread 0 0 AutoExpMonProc1 0 0 amp dwExpFuncThrdID 0 if RExpFuncThrd 0 NULL Fmt strErrMsg s lt CreateThread failed i n GetLastError MessagePopup CreateThread Message strErrMsg return 1 j break return 0 77 Thread for the 1st part of the experiment DWORD WINAPI AutoExpMonProc1 LPVOID IpParam while 1 Trigger the process WaitForSingleObject hExpStartEvnt 0 INFINITE Get ready for getting liquid ResetEvent NCommEndEvnt 1 _ZabJogZFunc 3 0 WaitForSingleObject RCommEndEvnt 1 INFINITE Go to liquid
70. ology Affymetrix n d Retrieved July 26 2005 from http www affymetrix com index affx 20 Hsieh H B et al Ultra High Throughput Microarray Generation and Liquid Dispensing UsingMultiple Disposable Piezoelectric Ejectors Journal of Biomolecular Screening 2004 21 Jane T Chang J and Kim C J A Silicon Micromachined Pin for Contact Printing Proceedings of the IEEE Micro Electro Mechanical Systems MEMS 2003 Pages 295 298 22 Smith J T and Reichert W M The optimization of quill pin printed protein and DNA microarrays Annual International Conference of the IEEE Engineering in Medicine and Biology Proceedings 2002 Pages1630 1631 23 George R A Woolley J P and Spellman P T Ceramic capillaries for use in microarray fabrication Genome Research Volume 11 Issue 10 2001 Pages 1780 1783
71. on 0 010 mm Speed 500 mm sec Axis dimensions 100 mm deep X 150 mm Wide X 441 mm high 45 46 2 DARL language overview The standard language used by the Seiko D Tran Robot is DARL Version 2 3 It is easy to use and similar to BASIC language Steps for running a program using DARL from tech terminal Make sure the TERMINAL AUTO switch is in TERMINAL position Turn the main power on Switch the main power to ON position Turn on the power on the servo motors by pressing the GREEN button One will see M DARL XY 3000 VER 2 3 COPYRIGHT 1984 SEIKO I amp E M refers to the Monitor mode it is the default mode of the robot Type HOME which will get the robot in the home position Home position is necessary before the start of any operation to have the robot know its workspace and initial transitional points Type EDIT to go to EDIT mode from the Monitor mode One will see E This is EDIT mode and this mode is used for typing own programs and feeding them to the controller The following is an example program to move the robot arm in place from home position to a position described by the summation of two translation points T20 and T 30 NEW 10 T20 100 0 20 0 300 0 0 0 20 T30 20 0 30 0 100 0 0 0 30 MOVE T40 T20 T30 40 END 47 Program execution is done in MONITOR mode To get back in to MONITOR mode type QUIT in EDIT mode This will bring back to MONITOR mode from EDIT mode
72. pe The size and the shape of the glass pin are major factors affecting the spot formation Finely cut and polished borosilicate pin of outer diameter 1 35mm and inner diameter 0 59mm must be chosen to fit around the Fotonic sensor driven by the Zaber stage The geometry of the pin tip affects the intensity pick up drastically Uneven ends may cause disturbances in the voltage pick up and affect the spotting The size and shape of the spots will be irregular and would not be repeatable Viscosity of samples The types of sample used have different viscosities and hence the filling of the pin without air bubbles also becomes an issue for flawless spotting Zaber increment The spot size drastically depends on the Zaber push given to release a metered quantity of the sample at the pin tip If the Zaber increment is more then naturally the spots formed are bigger in size Air gap control The size of the spots can be controlled by positioning the pin properly over the air gap distance Even with larger pin openings smaller size spots can be produced making spotting independent of pin geometry 21 5 Intensity enhancement The voltage pattern observed can have better resolution with more intensity pick up and this can be facilitated by proper sample level filling in the pin by the Zaber stage The Zaber has to be pulled just by 1000um above the maximum limit By doing so there will be sufficient sample for spotting and there will be more
73. quid 1s specified by the three coordinates relative to the robot Also the first spotting position coordinates are fed into the robot panel In the Zaber panel the maximum and minimum safe limits are to be mentioned along with the step increment which dictates the metered quantity of the liquid that will cling to the tip of the needle In the spotting panel the maximum numbers of rows and 22 columns to be spotted are mentioned All the parameters shown in the front panel Figure 3 2 are specified before the beginning of spotting process EJ Spotting Experiment ROBOT PANEL e ZABER PANEL of well mm Y of well mm Z of well mm ert ts Pd Inte T RUE S 260 3 7272 750 4 1 HOME X Tst spot mm Y 1st spot mm Z 1st spot mm fa Up Lima um 2 Zabet Z um J lt I 7580 E 4 2000 E Ue eror Fes net Z limit mm 2 SJ 000 Y 1500 JF 7600 2 EE G0 WELL step mm Y step mm Z step mm 2POTTING PANEL vene GET LIQUID om 4 ox om Max Row Curent Row Spots formed Target X mm Target Y mm Target Z mm a 2 uo 2 50 PUSH DROP 000 000 000 Max Column Current Column Droplet every ls Is FIRSTS POT Robot mm Robot Y mm Robot Z mm zl E 4 op n5 DOO M AA ES SPENT Har T es di QIONG SHEN Ver 1 0 0 05 22 2005 eM EXPERIMENT MESSAGE EMERGENCY STOP NEXT SPO T Graph
74. r temp 60 IZabTargTemp1 long int pstr_temp 2 IZabTargTemp1 floor double IZabTargTemp1 ZABCONVFACT SetCtriVal iPnIHdl PANEL ZABTARGZ IZabTargTemp1 iBytsSent 1 ComWrt iCommPort 1 pstr temp iStrSize 1 WaitForSingleObject hCommEOLEvnt 1 INFINITE pstr temp ZABCNT i idx ZABCNT SetCtrlVal PniHdl PANEL ZABMSG OK ResetEvent hCommStartEvnt 1 72 SetEvent RCommEndEvnt 1 return 1 ce ETT AA Callback function to deal with the ending of communication 3 According to the communication protocol ae command and reply have only 6 bytes respectively y BMC EDT T Susa mi void CVICALLBACK ZabRplyEOLFunc int iPortNum int iEvntMask void pvCallBackData strTempZabRd 0 0 ComRd iCommPort 1 strTempZabRd GetlnQLen iCommPort 1 IZabCurrTemp long int strTempZabRd 2 IZabCurr floor double lZabCurrTemp ZABCONVFACT SetCtriVal iPnIHdl PANEL_ZABCURRZ IZabCurr if strTempZabRd 1 60 SetCtriVal iPnIHdl PANEL ZABTARGZ IZabCurr SetEvent NCommEOLEvnt 1 ICD ER ENE A A A DR BESIDE mf Get liquid from the well ga PE ANA IA IO RR TS aa E E EA AA E MY coe Se T int CVICALLBACK GetLiquid int iPnlHdl int _iCtriHal int _iEvntHdl void pvCallBackData int _iEvntDatat int iEvntData2 switch iEvntHdl case EVENT COMMIT _ZabJogZFunc 3 0 break return 0 P R
75. s for communication port Important Points about ASCII set up e The DARL protocol for communication transmits all transmission blocks other than a STOP character with a Carriage Return CR and a Line Feed LF e An Enter Key on the Keyboard would automatically do that if Send line with line feed 1s enabled e Echo type characters locally will display the characters on the screen being typed and outputted e Enforce the incoming data to 7 Bit ASCII This is used because the Robot s Keyboard is fully expressed in 128 bit characters of ASCII so we require 2 128 bits e The Character length is set to 8 bits so that all the bits are 0 s and contribute towards a START BIT 52 53 The program is as given below In EDIT mode NEW COMM 1 78 TUNIT 1 OUNIT 1 END QUIT START Press Start The LCD is as good as numb and one can see 0M gt on the HyperTerminal Screen Now the computer is the new I O terminal 4 Specifications of the Fotonic sensor The plug in module used is MTI 3802 with the probe outer diameter of 504um and inner core diameter of 150um The Figure A 5 shows the MTI instruments Fotonic sensor gap calibration chart MTI Instruments Fotonic Sensor Gap Calibration v Wa ee Instruments MTI 1000 S N Slope 1 0 65405 uin mv Plug In MTI 3802 S N 2028 Slope 2 3 74905 uin mv Probe O20R Date 04 13 05 Noise 55 0 mv pk pk 10 9 8 7 2 6 3 5 5 O
76. ser degree And it also needs to be packaged in a way that can fit into a pin holder on a commercial arrayer A molding process combined with silicon micromaching is needed to manufacture the pin and cap combination 21 Another contact printing technique would be the quill pin technique though it is optimized as a function of substrate wettability and composition of the buffer to improve microarray density it still suffers the other draw backs of the contact process 22 The commercial non contact arrayers have lower numbers of print heads per rack than pin based arrayers Part of the reason is due to the complexity of plumbing required with their designs and the inherent tendency for clogging 20 The non contact piezoelectric systems uses piezo ceramics located adjacent to the fluid near each nozzle A tiny electric charge is forced into the piezo ceramic causing it to change shape and displace fluid When the piezo ceramic displaces in and out it shoots a droplet of ink out of the nozzle Piezo ink jet has advantages of small drop sizes and high ejection rate In this method though the cell solutions were successfully ejected until reservoir depletion with no observed adverse effects or clumping one potential problem was the inhomogeneous cell concentration in the ejected fluids possibly due to cell settling within the reservoirs 20 Another printing method using hollow cylindrical ceramic tips improves the morphology of microarray
77. sor s back side operating range Calibration Procedure of the Fotonic sensor l Ze Perform instrument warm up for 20 minutes and electrical zeroing Insert the probe into a notch of the fixture attached to the robot arm Position the probe by jogging the robot so that the probe tip lightly contacts the target mirror surface and set the zero gap Tighten the probe clamp evenly and ensure that the probe remains aligned with the target and centered in the notch Check the panel meter the indicator should read 0 Turn the displacement vibration control to the READ position Check the panel indicator it should remain at 0 when switching to the READ position but actually the panel indicator has a different intensity read out due to the polished surface being at the underside of the target mirror used Now since the probe does not touch the polished surface of the mirror the variation from true zero read out 1s observed Move the robot upwards and observe the panel indicator The indicator will move up scale as the target moves away from the sensor probe The indicator will stop moving when the optical peak 1s reached Turn the intensity fine control clockwise to move the indicator to the set cal mark on the scale If the intensity fine control reaches full stop and the indicator has not reached the set cal mark then the light intensity of the probe must be increased by changing the intensity coarse
78. st the formation of a spot BEP A RR PR RE A A AA SO ERN To RON I int TestSpotForm void int idx i idx1 idx2 double d temp d max d min double pd temp data double d inti d int2 if SensDAQPosDataldx1 0 amp amp iSensDAQPosDataldx2 0 pd temp data dSensDAQData i idx1 iSensDAQPosDataldx1 i idx2 isensDAQPosDataldx1 d inti double SensDAQPosDataldx2 iSensDAQPosDataldx1 for i idx iSensDAQPosDataldx1 1 idx lt iSensDAQPosDataldx2 1 i_idx if pd temp data i idx lt pd temp data i idx 1 i idx2 7i idx continue else i idx1 1 Idx idx2 i idx continue j if i idx2 1 i idx1 d int2 double i idx2 i idx1 d temp pd temp data i idx1 pd temp datali idx2 y pd temp data i idx1 d temp d temp 1000 0 d int1 4d int2 10 0 if d temp gt SENSSPOTTESTTH return 1 return 0 j int CVICALLBACK _ZabGoHome int iPnlHdl int iCtriHdl int iEvntHdl void pvCallBackData int iEvntData1 int iEvntData2 switch iEvntHdl case EVENT COMMIT 81 _ZabGoHomeFunc break return 0 void ZabGoHomeFunc void SetCtriVal iPnIHdl PANEL ZABMSG COMMA GetCtriVal iPniHdl PANEL ZABUPPZLIM amp lZabTargTemp IZabTarg ceil double lZabTargTemp ZABCONVFACT Cmdidx 1 0 ResetEvent NCommsStartEvnt 1 ResetEvent NCommEOLEvnt 1 _SendZabCmd 20 amp lZabTarg
79. t in X and Y directions Then a mesh plot is done to obtain the geometry of the spot A mesh plot gives the 3D view of the droplet as shown in the Figure 4 10 There are three usage of the scan First the newly formed spot can be checked for acceptability and if necessary re dispense Second the pin is capable of locating the spot for 36 hybridization e g hybridization on the chip Third the pin can locate sample materials in e g microwells for aspiration This is significant if the sample material is limited in volume Raster Scan of droplet 3 204 n 500 iie 400 n 200 02510 intansity height x axis scan i um in volts in Figure 4 9 Graph depicting the 3D view of a 220micron diameter droplet and a cross sectional measurement of the spot which is about 18 microns high at the time of scan a infernity in volts tv I ta m0 O y comi SO um Figure 4 10 Graph depicting the 3D view of a 400micron diameter droplet of 100 glycerol 4 3 Production of Uniform Size Microarrays Production of uniformly spaced microarray spots is carried out with the SmartPin for 100 50 glycerol and de ionized water Sample results are shown in Figure 4 11 and Figure 4 12 where uniform and round spots are clearly visible 37 a C o Loba bd Vare pg o me iL on moron mt etn Wa oa gt e n Lo te e o om AA TIP X ed M ew P ORTA a Tied na EP e urat m o an
80. t were the spot gets detached from the pin Figure 3 4 shows the graphical pattern observed during spot formation for buffer 3XSSC The following Table 3 1 shows the regions of the graph and the action to be taken when that region is being plotted in the graph The slope of the voltage is calculated as Previous data Present data 1000 Gap distance Data refer to the intensity in volts along the Y axis in Figure 3 4 Table 3 1 Look up Table for the Controller ABOVE THRESHOLD BELOW THRESHOLD HIGH VOLTAGE REGION II OF GRAPH REGION I OF GRAPH JOG PIN BY 10UM JOG PIN BY 30UM DOWN DOWN LOW VOLTAGE REGION Ill OF GRAPH REGION IV OF GRAPH PULL THE PIN UP BY PULL THE PIN UP BY 10UM LARGER STEP OF 30UM 26 Figure No 1 x File Edt View Insert Tools Window Help su a Rh AAS Y ec e Gap Vs heno buffer 3XSSC Zaber level 219920 zabar push 20um t t 4 i 1 i 4 i i t intensity in volts gap in um Figure 3 4 Standard spot formation Graph showing the four different regions The indication of droplet formation is given by the reach of the highest point The highest point will be the point after which there is more than the threshold voltage slope and the data points lie in the low voltage range zone After the highest point is reached the needle is commanded to be pulled back The controller action is depicted the flow chat given in the Figure 3 5 where D
81. ted to find the nature of the material used Experimental results show that more elongation occurs in less viscous sample In the future more efficient control strategy can be employed in the controller to detect the spot formation and the commands can be downloaded to the robot to draw the pin back with the maximum speed of 10 1um ms so as to achieve smaller dot sizes The current Fotonic sensor can be replaced by more sensitive sensor and the current Zaber stager with accuracy 8 um used can be replaced by a more accurate actuator A platform with accuracy 1um and higher speed will enhance the technique 44 APPENDIX A HARDWARE SPECIFICATIONS The specifications of the robot software used connection establishment between the robot and the computer terminal and the specifications of the Fotonic sensor is discussed below 1 Specification of SEIKO D TRAN robot Mounting Area Work envelope x axis is 300mm Y axis is 200mm and Z axis is 100mm Base plate dimensions 470mm deep X 420 mm wide Seiko D Tran robot specifications Repeatability or 0 008mm 0 0003 in Speed 1500 mm sec 59 in sec Accuracy 0 030 mm 0 0012 in Payload 1 kg Seiko D Tran dimensions 554 mm wide X 1084 mm deep Seiko D Tran weight 105 Kg The Z axis is located between the A axis and the X axis of the Seiko D Tran robot The power train of the axis 1s a rack and pinion assembly Z axis specifications Stroke 100 mm 3 94 1n Resoluti
82. the ending of communication p According to the communication protocol when gt letter is received one communication is over A O RI A A IR RI TER A dr void CVICALLBACK RobRplyEOLFunc int iPortNum int iEvntMask void pvCallBackData char pstr temp m char pstr temp d char pstr temp int idx strTempRobRd 0 0 ComRd iCommPort 0 strTempRobRd GetInQLen CommPort 0 FlushinQ CommPort 0 strcat strRobRply strTempRobRd pstr temp m strstr strTempRobRd M n pstr temp d strstr strTempRobRd D Wn if pstr temp d NULL pstr temp strstr strRobRply XYZA if pstr temp NULL i idx strcspn pstr temp n 1 pstr temp i idx _GetCoorVal pstr temp amp dRobCurr 0 amp dRobCurr 1 4dRobCurr 2 SetCtriVal iPnIHdl PANEL ROBCURRX dRobCurr 0 SetCtriVal iPniHdl PANEL ROBCURRY dRobCurr 1 SetCtriVal iPniHdl PANEL ROBCURRZ dRobCurr 2 65 j if pstr temp m NULL pstr temp d NULL strRobRply 0 0 SetEvent NCommEOLEvnt 0 I EEPE A A ESTAS EIN Se REED Callback functions to HOME the Robot Y SORRENTO MERETUR int CVICALLBACK RobGoHomef int _iPnlHdl int iCtriHdl int _iEvntHdl void pvCallBackData int _iEvntData1 int iEvntData2 switch _iEvntHdl case EVENT COMMIT _RobGoHomeFunc break return 0 void RobGoHomeFunc void dRobTarg 0
83. time counter that counts the hours of operation Its basic components consist of a Z80 Microprocessor and DC servo amplifiers The Seiko Controller supports the DARL robot language which may be programmed via a RS 232 10 link on a PC or the Tech Terminal The Tech Terminal is ideal for altering the program in an industrial arena and useful for trouble shooting and maintenance The output unit is a Liquid Crystal Display LCD with 40 characters per line and 4 lines per view More features of the Tech Terminal can be found in the user s manual provided by the manufacturer The Seiko D Tran controller has two RS 232 ports for serial communication with any device 2 3 Description of Optical Fiber Displacement Sensor Fotonic Sensor The Fotonic sensor as shown in Figure 2 4 consists of a console and a sensor module The console processes sensor signal and sensitivity while the probe consists of light transmitting fibers and light receiving fibers that are bundled together realizes the optical lever principle Displacement measurement is based on the interaction between the field of illumination of the transmitting fibers and the field of view of the receiving fibers As the probe to target distance decreases increasing amounts of light are captured by the receiving fibers This relationship will continue until the entire face of the receiving fiber is illuminated with reflected light This point is called the optical peak as shown in Figure 2
84. uitApp int iPniHdil int iCtriHdl int _iEvntHdl void pvCallBackData int iEvntDatat1 int iEvntData2 switch iEvntHdl case EVENT COMMIT QuitUserinterface 0 break return 0 void MyExitApp void int i idx for i_idx 0 i_idx lt 2 i_idx if hCommStartEvnt i_ idx NULL CloseHandle hCommsStartEvnt i idx END IF if iPortOpen i idx iOutQLen i idx GetOutQLen CommPort i_idx if OutQLen i_idx gt 0 MessagePopup RS232 Message The output queue hasin data in it Wait for device to receive the data or flush the queue n break END IF Commerr i_idx CloseCom CommPort i_idx if CommeErr i_idx _DispRS232Err i idx END IF END IF ENF FOR ja WA AA ty do se NN s I Initialize Experiment i Je Bn A A AA AA AA SE SD re JE EU el int CVICALLBACK _InitializeExp int iPnlHdl int _iCtriHdl int iEvntHdl void pvCallBackData int iEvntData1 int iEvntData2 59 switch iEvntHdl case EVENT COMMIT _MylnitExp break return 0 int MylnitExp void SetCtriVal iPnIHdl PANEL EXPMSG1 INITIALIZING SetCtriVal iPnlHdl PANEL EXPMSG2 if iPortOpen 0 0 iCommPort 0 1 strRobPortName 0 0 strcpy strRobPortName COM1 iBaudRate 0 9600 iParity 0 0 DataBits 0 8 iStopBits 0 1 ilnptQ 0 512 iOuptQ 0 512 DisableBreak
85. vent Message strErrMsg return 1 ResetEvent hCommEOLEvnt 0 if NCommEndEvnt 1 NULL hCommEndEvnt 1 CreateEvent NULL TRUE TRUE NULL if hCommEndEvnt 1 NULL Fmt strErrMsg s lt CreateEvent failed i n GetLastError MessagePopup CreateEvent Message strErrMsg return 1 SetEvent hCommEndEvnt 1 if NCommThrd 1 NULL hCommThrd 1 CreateThread 0 0 ZabCommMonProc 0 0 dwCommThrdID 1 if hcommThra 1 NULL Fmt strErrMsg s lt CreateThread failed i n GetLastError MessagePopup CreateThread Message strErrMsg return 1 Read Robot Position SetCtriVal iPniHdl PANEL_ROBMSG COMM1 strRobCmd 0 M0 ResetEvent hCommsStartEvnt 0 ResetEvent hCommEOLEvnt 0 _ SendRobCmd 3 0 0 0 0 0 0 SetEvent hCommsStartEvnt 0 Read Zaber Position SetCtriVal iPniHdl PANEL ZABMSG COMMA iCmdldx 1 0 ResetEvent hCommsStartEvnt 1 62 ResetEvent hCommEOLEvnt 1 _SendZabCmd 60 NULL SetEvent hCommStartEvnt 1 if SensDAQNumID 0 isensDAQNumID DAQ_Numeric_ConvertFromNumeric iPnIHdl PANEL SENSCURRV Sensor DAQ Numeric SetAttribute iPnIHdl isensDAQNumID ATTR DAQ NUMERIC SCAN FREQUENCY SENSDAQNUMRATE return 0 f VERTRETEN a A IE a i Display error information to the user DOT A E TGE N AE PEENES Ss Se ee eee ee IUE SEE void DispRS232Err
86. y properly positioning the same pin above the slide surface and by controlling the Zaber push 38 3 GenePix Pra 6 10 05 test tif kwaya Histogram Lab Book BeichAnabe Fonts Scatter Plot Ropon image Festure Viewer Block Fuse C Preview 1635 5221 X Y pnd 15310 22806 C Preview 82 Hana C Presea i ory 8 cog T Wang 35 C Wevelengh 532 C Wavelength 83 O Wavelength 14 f Reto 635 6229 Rate 22 Rew 33 z EEE mm 5 og f am 9n E Toots 3 1 E 07 ooo ARPS HEHE 4j By gj ES ON WE g nyg 8 3 PIGA E 52 32 pod 4 fe Hardvan Kay rot lo Dk 402368 No bamoda Image Histogram Lab Book Gatch Analysis Results Scatter Plot Report Image Feature Viewer Block Fest Preview 525 522 2x C Prenew 82 Preview 2 5 C Wavelength 535 Wavelength 522 C Wavelength 43 Wavelength 4 5 P F E Rao 654522 Hato 82 Rato amp 3 Unirked Hardware Key not hound Disk 40 22 GB No barcode Figure 4 14 Microarray with spotting solution 3XSSC with 160 microns diameter spots and 130 micron spots 39 4 4 Experiments with Molecular Beacons Molecular beacons are single stranded oligonucleotide hybridization probes that form a stem and loop structure The loop contains a probe sequence that is complementary to a target sequence and the stem is formed by the annealing of complementary arm s

njit-etd2005-112 - New Jersey Institute of Technology

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