Final Report - Colorado State University
Contents
1. 22 P m 24 T 26 26 Chapter I Introduction Flow cytometry is a technique for counting examining and sorting microscopic particles suspended in a stream of fluid that has been around since the mid 1970s It allows simultaneous multiparametric analysis of the physical and or chemical characteristics of single cells flowing through an optical and or electronic detection apparatus beam of light usually laser light of a single wavelength 15 directed onto a hydro dynamically focused stream of fluid A number of detectors are aimed at the point where the stream passes through the light beam one in line with the light beam Forward Scatter or FSC and several perpendicular to it Side Scatter SSC one or more fluorescent detectors Each suspended particle passing through the beam scatters the light in some way and fluorescent chemicals found in the particle or attached to the particle may be excited into emitting light at a lower frequency than the light source This combination of scattered and fluorescent light 15 picked up by the detectors and by analyzing fluctuations in brightness at each detector one for each fluorescent emission peak it 15 then possible to extrapolate various types of information about t2. 5750 Far 08152 2652 Frase Phekeres Farce EA FL du Suse Trpo Pec du da 01822 Massy Ciis France Telephone 33 1 Ea E3 71 00 Fus 33 05 3 74 Lnded Hisce uu Phasia UK Limited 2 Hoard Cani 2 Tein Aad ALT 16 nidi Former 1441 1707 204 828 1441 1707 3257 IT Hare Phat Pinos 12 41 55 DH hs Hala SRL Maa UE 205020 Asma Oan 9 22 035 81 735 Se OS Cat KLEDAC21EUZ Jun 2006 DN J 44 ACKNOWLEDGMENTS We would like to thank Dr Kevin Lear PhD for his help and guidance as well as four of his eraduate students Bob Pownall Hua Linda Shao Sean Pieper and Weina Wang for all of their help 45
3. __ __ _____ Optek Technology Inc 1215 W Crosby Road Carrollton Texas 75006 972 323 2200 Fax 972 323 2396 8 90 27 Type OPF482 Electrical Characteristics TA 25 C unless otherwise noted 0 6 TEST CONDITIONS Flux Responsivity 045 055 AW Vn 50 V Peak Response Wavelength nm m 10 90 __ ns jveetsvmeeson esses 201 5 0 V 50 1096 9096 20 sov ui Typical Performance Curves DARK CURRENT Relative Spectral R vs AMBIENT TEMPERATURE elative Spectral Response _ 10000 lt 1000 2 6 5 10 5 1 o 2 0 25 50 75 100 125 Ta AMBIENT TEMPERATURE Wavelength nm DARK CURRENT Typical Capacitance vs REVERSE VOLTAGE vs Reverse Voltage F BER OPTIC O gt O 2 25 EB 20 ur ul 1 5 lt 5 o 1 0 amp a 3 lt 0 0 0 10 20 30 40 50 60 70 80 90 100 REVERSE VOLTAGE REVERSE VOLTAGE VDC Optek reserves the right to make changes at any time in order to improve design and to supply the best product possible Optek Technology Inc 1215 W Crosby Road Carrollton Texas 75006 972 323 2200 Fax 972 323 2396 8 91 28 D2 DAQ Compariso
4. or ware nalienal corm jeuonejedo 134 jeng 137 LF412 Simplified Schematic Note 1 Available per 305 1071 1905 Detailed Schematic Vee vana m rr 1 2 Dual 31 Absolute Maximum Ratings 2 H Package N Package If Miligtary Aerospace specified devices are required Mote 12 Note 5 570 mW please contact the National Semiconductor Sales Cfflce T max 150 C 115 npa for and specifications Typical meu Uperatnc Temp Harge Note Mote 6 LF412A LF412 Storage Temp Bb C T4s150 C 65 C lt T lt 150 C Supp y Voltage 220 181 Range Differential Input voltage 38V 30 Lead Input voltage Hange Soldering 10 260 C 260 C Note 3 t19V tiS ESD Tolerance Output Short Circuit Mote 13 17009 1700 41 CGontinuaoags Continuous H Fackage Package Power Dissipation DC Electrical Characteristics Mate 7 10 25 19 w of Input As 10 Note E 10 20 m eT loe input Offset Current Tress 25 100 100 pA lotes 7 9 moe Ja _ 2 m Temse J 25 m Input Bies Current 5 75 Tress 50 200 50 200 p 9 95
5. Features Absolute Maximum Ratings TA 25 C unless otherwise noted Component pre mounted and ready Reverse CU o aos a wate acci PU Qoi ESQ Pet Va dora EX o adi 100 VDC to use Continuous Power 5 200 mw High speed low capacitance Storage Temperature 55 C to 100 C e Pre tested with fiber to assure Operating Temperature Range 40 C to 85 C performance Lead Soldering Temperature 1 16 inch 1 6 mm from case for 5 sec with soldering Popular ST style receptacle NOU sais Od ace the t eU FA NEN ok Eri rs 240 Electronically isolated from case Notes 2 RMA flux is recommended Duration can be extended to 10 sec max when flow soldering 3 Test 8 5 V with 50 125 micron 0 20 fiber amp 10 uW optical power Q 850 nm The OPF482 consists of a low cost Responsivity levels apply to 50 62 5 and 100 um core optical fibers plastic cap PIN photodiode pre mounted and aligned in an ST receptacle This configuration is designed for PC board or panel mounting Includes lock washer and jam nut two 2 56 screws and dust cap Description The PIN Photodiodes are designed to interface with multimode optical fibers from 50 125 to 100 140 microns ST is a registered trademark of AT amp T A AA
6. Allowable forward current vs ambient temperature mA AE 5 B Ex lll B SN EU 3 ilis amp s 3 E m AMBIENT TEMPERATURE C AMBIENT TEMPERATURE PLES PLES ra 43 Infrared LED 2791 series B Allowable forward current vs duty rato 25 26 1033 eee T NI SEE 1 21 100 ALLOWABLE FORMARD CURRENT DUTY RATIO 1 Dimensional outlines unit mm IDL2731 2791 22 amp p27891 03 EFORT 54 0 1 COMMUN TO CASE e COMMON TO CASE bo a HAMMAM ATs rhe mp be ili Hoven moon Et p edd pedi es ers cr ere aie autera c bes Pep EOS Peis 5 HAMAMATSU PHOTONICS Sold Stale Division 1126 1 Ichina cno Hamamatsu 435 8558 Japan Telephone 81 053 434 3311 Fac 81 D53 434 5182 www com LES A 382 Rua P Bex ento Bridgsesalm 57 1807 00 HI 152 Tere T PE 251 080 Fus 11 908 231 1218 Penne 52 Hering im Acme Deity
7. e ALr Ti1Le 1 ue s usus a3 un a gr bu i 6 un up al wj Inside Diameter mm Hamilton Hicroliter 0 103 c n c I m H eu Publication 1200 01 49 05 26 06 D5 Infrared LED Infrared LED _ L2 91 series small emission spot LED using current confined chip 12 01 is infrared LED with a lens cemented to the current confined chip surface This combination ensures high directivity and improved emiss on uniformity In particular L2781 02 uses bens cap that delivers even narrower directivity As a variant type not using a microball lens 2701 03 also avaiable with the LED potted with resin which gives a small emission spot of p160 Features Small emission spot Automatic control systems L2781 400 um Optical switches L2781 03 180 Auto focus Uniform emission L2781 02 e Narrow L27891 D2 Absolute maximum ratings Taz25 C Parameter Symbol Condition Value Unit Forward currant D ES SSE _ ESS Reverse voltage Pulsa widthz10 Pulsa forward currant Duty ratios A Operatingtamperature Topr 30 to 85 2 Storage temperature Tstg Cd 40 100 12791 03 is guaranteed to resist temperature cycle test of up to 5 cycles
8. TANT 15 POSITIVE OUTPUT VOLTAGE SWING V OUTPUT SOURCE CURRENT mA Output Voltage Swing SWING Vp p UTPUT VOLTAGE 0 3 10 15 20 25 SUPPLY VOLTAGE VJ war Manone com 34 LF412 Typical Performance Characteristics continued Output Voltage Swing Bendwldth a Sa ic gt _ 2 ER 5 c 50 235 0 25 50 75 100 725 TEMPERATURE 30 20 BRI 10 a 1 E V z E 5 IM 2 30 ip 155 0 1 10 50 235 0 25 50 75 100 125 ae MHz TEMPERATURE C ea undistorted Output Voltage swing 0 2 10 HH ti E z ai EN uu F 5 1 08 zii gt FREQUENCY Hz FFEDUENCY Hz wa National oom 35 Typical Performance Characteristics continued GAIN OPEN LOOP VOLTAGE 1 10 100 Ik 10k 100k M 10M FREQUENCY Hz Power Supply Rejection lo POWER SUPPLY REJECTION RATIO 48 0 ik 30k 1005 1 FREQUENCY H3 Open Loop Voltage Galn LOOP VOLTAGE SUPPLY VOLTAGE W COMMON MODE REJECTION RATIO dB EQUAVALENT INPUT NIHSE WOLTAGE nV Hz OUTPUT IMPEUANTE iF EH comm Rejection Ratlo 8585 10 100 Ck FREQUENCY Hz Equivalent Input Molse Voltage a Mcr uin Hl 100
9. 4 m R lw Posi Large Signal Voltage ek 200 eb Vi mV s FS ere Input Common Mode 16 4195 ttt Ais W Voltage Jangs 16 5 Common Mede lam a0 100 Rejection Ratio Supply voltage i Mots 10 100 mme Hote 2 Absolute Maximum gt indicate limits beyond which damages to the device may Operating Retings indicate conditions Tor which the dewiceis Functional but da not marane perfamnancs limits AC Electrical Characteristics Mata 7 Symba Parameter Conditions LF112A Min Typ Min Typ Amplifier to Amplifier 25 f 1 Hz 20 kHz 20 120 Goupling Input Referred SewHas __ e 3 9 6 DIEA UU DR 3 www naltonal com 32 LF412 AC Electrical Characteristics Continued Mata 71 Symbol THD Ay 10 10k 20 Vp p BW 20 Hz 20 kHz Equivalent Input Moise Ta z25 C 1000 l Equivalent Input Noise Ta4 25 C 1 kHz Note 3 Unless othenvise specified the absolut maximum negative input voltage equal to the negative power supply voltage Note 4 Any of the amplifier outputs can be shorted ground howewer mare than one should nat be simultansously shorted as the maximum junction temperature wil be sxoseded Note 5 For operating at elevated temperatuns these devices must
10. controlled with Labview VI This also took out the guess work for logic chip In specification sheet for a 7400 series logic component there was a large range of voltages that were specified to be undefined logic The DAQ took a lot of the uncertainty out of the logic The DAQ is accurate to within 3 mV DC on its 8 available channels of analog inputs It also can output digital logic at 5 V DC on one of its 18 Digital I O channels This may seem like it s overkill to have so many but the was 2 3 of the price of some of the other data acquisition units that are on the market It has a sampling rate of 13 000 samples per second on a single channel More expensive units were about the same or even worse We designed a Labview VI that would take an analog input compare it to some specified reference and output a digital signal based upon whether or not it was higher or lower than the given reference 18 Here 15 the front panel of the Labview VI Figure C1 2 Front Panel of VI The interface 1s quite simple The triggering voltage is input in the field labeled Comparison Voltage The channel of the analog input 1s easily configured Any errors are easily read on the error out field Since we configured the DAQ to take inputs on analog channel 0 the default value getting a digital output based upon comparing to a preset voltage was as easy as inputting the comparison voltage and hitting t
11. E EE E 4 UC DEP Cup Micro POTIS ____ _ 7 NU 15 E A EE 9 ons sac E E E E E 11 e E _ _____ ________ E T 15 EEE E es se 16 Appendix __________4 ____ 16 auen ___ 6 6 _ __ 66_ _ _ 6_ 6 _ 17 ABDEBOIEL Edad 17 Appendix D Data Sheets User Manuals 27 FIGURES ur PH E 4 n 6 Hm 7 9 M 10 ___ _6 _ _ ___ ___ _ 11 gun depo L 12 13 uunc rr mn 14 lyon co PC 15 N T 18 19 19 M 20 guns O 9 O O 21 P 21
12. T idk 1104 pane 5 5E 8 e a 4 FREQUENCY Output Impedance 3 ae t a 10 1 FREQUENCY H2 Www 36 LF412 Typical Performance Characteristics continued Inverter Time i piil LL e E N SETTLING TIME us TODD Pulse Response gt c 10 pF Small Signal inverting Small Signal Non nverting g E _ 35 a 43 55 3 B TIME 8 2 as DIV TIME 0 2 por conn QOBEERZT Large Signal inverting Large Signal Non nverting DIN VOLTAGE SWIND 88 Div VOLTAGE SWING TIME 7 DIVI TIME 2 47 MY TIHI waa national comi 37 Pulse Response c 10 pF Continued Current Limit 210 062 EE OU TPUT VOLTAGE SWING civ DN 45 5 DIN Application Hints The LF412 series cf JFET input dual op amps are internally trimmed BI FET II providing very low input offset voltages and guaranteed input offset voltage drift These JFETs have large reverse breakdown voltages from gate to source and drain eliminating the need for clampe across the inputs Therefore large differential input voltages can easily be accommodated without a lange increase in input current The maximum differential
13. able to get cells into the chip There are several considerations for acceptable means to accomplish the desired flow rate of cells in the channel of the chip The first and primary requirement is that the flow rate must be less than or equal to 40 um per second 40 um s This requirement comes from the DEP traps on the chip If the flow rate exceeds 40 um s the force of the DEP trap to stop a cell will not be sufficient to stop a cell and hold it in place Another limitation of flow control is that there must not be too much pressure on the nanoport chip seal or in the channel The bonding on glass to glass chips 1s not very strong and high micro fluidic pressure will cause the chip to leak If leaking is severe it is possible for cells to flow outside of the channel Obviously this is very undesirable because all of the traps and optical detection are near the center of the channel It is also worth noting that all of the glass chips have gaps between the two pieces of glass due to an imperfect bonding process This creates flow outside of the channel and different fluid dynamics than the PDMS chips Because the glass to glass chips glass chips essentially have larger channels the flow rate 1s significantly slower than PDMS chips when the same amount of pressure 15 applied to the fluid Most cell samples such as blood are obtained using a syringe Therefore a syringe 15 used to push samples into a nanotube and into the channel Although
14. be derabed based on a thermal resistance al Note These devices are available in both the commercial temperature rangs and the military temperature range 55 CzT4 z125 C temperature rangs is designated by the position just before the package types in the dece number C indicates the commercial temperature range and an ht indicates the military ternperaturs range The t amp mperabure range iz available in package only In all cases the maximum operating temperature is by internal junction temperature rnax Note 7 Unless othenwizs specified the apply over the ful temperature range arid Tor V 2 x20 V for the LFA12A and for 1 for the 2412 Vas lg and 153 are measured at Vcg Note 8 The LF4124A iz 100 tested to this specification The 12412 sample bested on a per amplilisr basis to insure at least BS of the amplifisrz meet this specification Note 9 The input bias currents junction leakage currents which approximately double for every 10 C increase in the junction temperature Dus bo limited production test time the input bias cuments measured cormelated to junction temperature In nomal operation tha junction temperature ness abcye the ambient temperabure as a af inbernal power dissipation where is the thermal resistance from junction to ambient Liss cf a haot sink is recommended if input
15. channel was needed to form the channel This mold composed of a silicon substrate SU 8 resist This resist is much thicker and viscous than 71512 and can be used as a mold when it 15 baked and fully developed Once the mold 15 constructed PDMS is poured into the mold again degassed Once degassed the mold is placed in an oven to bake for at least 4 hours The curing agent transforms this viscous liquid into an elastic solid The PDMS can then be cut and peeled off the silicon substrate A glass cover slip is then placed on the opposite side of the channel which keeps the PDMS chip clean and allows the channel to be transported easier In order for the PDMS channel to be bonded to the DEP trapping circuit one more step in the DEP circuit must be performed A gold and chrome sheet still remains outside of the trapping circuit which is used for other bonding processes This area is removed with the same lithography steps as creating the traps except that a mask covering the trapping circuit is used Now the two chips are ready to be bonded together The DEP chip is taped to a glass slide with the circuit facing out Both chips are then oxygen plasma treated with micro RIE After treatment the two chips are bonded together using the mask aligner The PDMS channel 15 placed on the stage facing up The DEP chip is positioned facing down being held in the mask holder The DEP traps are then positioned over the channel The channel is then brought in
16. the end goal of the project is to pump cells through the channel and analyze them it is impractical to actually use cell samples to develop and test flow control Therefore de ionized water was used with glass spheres ranging from 5 to 26 um in diameter The majority of the time 9 77 um spheres were used This water with glass spheres will be referred to as fluid parts of this section When the project first began it was thought that the rate at which the end of a syringe plunger needed to be pushed could be calculated based on the fact that the volume in 1s the same as the volume out Ideally chips are fabricated with a 200 um wide by 25 um deep channel This gives an ideal cross sectional area of 200 um x 25 um 5000 um If the syringe plunger is pushed at a rate of x um s then the volume of fluid flowing into the nanotube and the chip is 0 5 x diameter of syringe um x x um s 7 um s This flow rate must logically be the same in the channel since the volume in must equal the volume out This result can then be used to calculate the velocity at which fluid will travel through the channel The equation 15 as follows v um s 7 um s x 5000 Substituting the first equation into the second gives a direct relationship between the velocity of fluid in the channel v um s and the velocity at which the plunger of the syringe 15 pushed x um s This resulting equation is v um s x um s x 0 5 x diameter of syrin
17. the question how do you exert a force upon a neutral object using electromagnetics The answer to this question 15 not trivial DEP trapping occurs when a non uniform electric field is passed through a neutral body Internal to the neutral object the molecules within the neutral object polarize much like the depletion region that occurs when a voltage is put across a pn junction The imbalance of localized charges results in a virtual electric dipole being formed within this neutral object Therefore the electric field will apply a force to this neutrally charged object Since the objects we are concerned with are on the order of 10 um in diameter it is easier to exert a significant enough force on these small particles to push them into a suitable area to take a spectral reading 13 Figure 6 1 Picture of DEP trap electrodes In order to achieve a non uniform electric field a time varying voltage must be applied across the electrodes of the DEP trap The approximate force applied to an object being trap can be defined by the formula R Re CM o x VE r 1 where refers to the dipole approximation to the DEP force 21 refers to the permittivity of the medium surrounding the object being trapped R is the radius of the particle r 1s the spatial coordinate c 15 the angular frequency of the applied voltage E 15 the complex applied electric field CM 15 the Claussius Mossotti factor The Clossius Mossotti factor is
18. to move at a rate of 0 0115 um s to achieve the desired flow rate of 40 um s in the channel Because this 15 still slower than what the 15 capable of Labview software was used to create a duty cycle where the actuator 1s only moving for a small percentage of the time For details on how the Oriel was implemented into the system see Appendix C2 To achieve 40 um s velocity inside the channel the Labview VI was set so that the actuator moved for 0 825 seconds at 0 5 um s 0 4125 um and off for 7 seconds When the VI is used the optimal flow rate occurs at this duty cycle of approximately 10 5 This cycle description applies to the movement of the actuator However duty cycle 1s not an accurate comparison because the flow rate 1 the channel takes much longer to decay than the 7 second period during which the pump is turned off This is discussed more later Another consideration is that the stage mounted to the actuator is spring loaded and adds complication to the analysis This spring action actually allows the plunger to be pushed at a rate slower than the 0 5 um s that the actuator is moving actuator Labview program spring loaded 288 stage li EEI syringe Oriel e re controller me 5 l Figure 4 10 Through experimentation it was quickly discovered that the derived equation does not hold true for glass chips but 1s reasonable fo
19. wavelength that differentiates cell types the spectrometer 1s the heart of the system Triggering DEP traps based 15 on light modulation 1 e intensity 15 very difficult and may not be feasible with current project limitations alternative to trapping based on light intensity would be to use the spectrometer and attempt to detect changes in wavelength and trigger the DEP traps accordingly It may even be possible in the future to skip the trapping step entirely and simply take data of the cells spectrums as they pass Such alternatives will be investigated next semester along with other possibilities to improve the rate at which cells can be analyzed REFERENCES http www optofluidics caltech edu optofluidics index html http www laserfocusworld com articles article_display html id 259933 Wikipedia 1 J Voldman R Braff M Toner M Gray and M Schmidt Holding Forces of Single Particle Dielectrophoretic Traps Biophysical Journal Vol 80 pp 531 541 January 2001 2 W Wang H Shao Lear Lab on a Chip Single Particle Dielectrophoretic DEP Traps powerpoint presentation given March 2006 3 Optofluidic Intracavity Spectroscopy of Canine Lymphoma and Lymphocytes Lear Kevin L Shao Hua Wang Weina Lana Susan E LEOS Summer Topical Meetings 2007 Digest of the IEEE 23 25 July 2007 Page s 121 122 APPENDIX OR APPENDICES Appendix A Abbreviations AC Alternating Current DAQ Da
20. 1 high temperatures The temperature in the heater 1s controlled by a programmable unit attached to the heater The temperature 15 then programmed to ramp up to and hold at a certain temperature and then ramp down Once the desire temperature program completes the bonding vice 15 pulled from the heater The channel and the DEP trapping circuit chips are now bonded together A similar bonding procedure 15 gold to gold bonding The advantage gold has over Indium 15 that the height of the material required for bonding is minimal compared to Indium Minimizing the height for bonding 15 critical because the height dictates how leaky the channel will become Indium bonding adds another layer in the bonding area outside of the chip This layer adds an additional height on the scale of microns This will produce a leakier channel than chips bonded with a gold to gold method This method has not successfully been completed on test chips These test chips are half the size of the DEP trapping circuit chips and have gold with no patterning The two gold chips are pressed together and put between the copper plates and into the vice The temperature is programmed to be much higher than indium bonding This is due to the fact that Indium bonds much easier to surfaces than gold does to gold or any other surface The melting point of gold is also very high In order to fuse the two pieces together a very high temperature must be obtained An appropriate te
21. 5 aus 0 130 8 20 3 302 0 127 0 018 0 203 0 457 0 076 0 108 0 010 _ 2 548 0 254 D 1 2701 Fy Dual In Line Package Order Humber LFA12ACH or LFA12CMH NS Package Number NOBE Ta aun 1 UTE a 0 055 1005 1 143 0 381 12 40 D4 Relevant Syringe Pump User Manual Page Mev Era Pump Systemes Irc www Syringe Pump com Model NE LOUP 12 7 Syringe Diameters and Rate Limits Inside Maximum Minimum Syringe Diameter Rate Manutacturer fcc imm iul flab Ro H mim CF ha a a wb ec un i ce Po as en e d d Norn Ject H i D qum i r r m ch i i Ti d i H E 00 qr ex pm ject m us Ln Lil u 1705 5 1 Lu 2 m Cs bab Fs H Ls n Es rm it 4 gu i mo e R 4 E al uz ic i 1 C ial i i 5 e d L m alin als j Ex xd
22. E Electrical and optical characteristics Ta 25 C Parameter symbol Condition rei Unit E Min Dp Max Min Max Min Typ Max T ss Spectral half width Duc 60 L mm Forward voltage 35 17 t5 1T V Pulse forward voltage ey 461 Reverse current LE 10 100 10 Radiant flux 4e e eo 1920 1209 1501 mw Radiant illuminance i18 20 Rise time t 50 1019 00 0 12 021 o12 o2 ow 02 ps Fall time 1 50 00 10 012 02 012 02 012 02 us 42 infrared LED 1 2791 series Emission spectrum Radiant flux vs forward current Tam25 C pm mu Typ tiw 17 ps 01 WA k E 5 E m E c lt 2j 0 100 1000 WAVELENGTH nmm FORWARD CURRENT mA mA E Forward current vs forward voltage B Directivity C feel 00 um 1 E pr per Lj L1 Li 100 an 20 D 22 80 102 RELATIVE RADIANT 19 Lzrg1 n3 Except Sor ingred em of the ase FORWARD VOLTAGE V Radiant output vs ambient temperature
23. Optical Biosensors First Semester Report Fall Semester 2007 by Allan Fierro David Sehrt Doug Trujillo Evan Vlcek Michael Bretz Prepared to partially fulfill the requirements for ECE401 Department of Electrical and Computer Engineering Colorado State University Fort Collins Colorado 80523 Report Approved Senior Design Coordinator ABSTRACT Currently in order to sort and scan cells quickly and efficiently one has to use flow cytometry or some related process One of the major components of flow cytometry 15 the use of labels fluorescent dyes and markers in order to tell cells or cell parts apart from other cells or cell parts Fluorescent dyes are expensive and light sensitive so care has to be used in working with them and samples to be tagged must be kept in the dark Also there must be a large number of cells in order to run flow and depending on the situation that may be very difficult After flow cytometry the samples are contaminated or dead Plus the machine is very expensive with the initial cost and upkeep maintenance so that signing up for flow time 15 quite expensive Then there is the training that 1s needed to operate the flow cytometer of these factors add to the time and trouble needed to analyze the cell samples by flow cytometry If a way can be found to do cell analysis without needing to fluorescently tag the samples the process of analysis would be cheaper and quicker both of which are very
24. a frequency dependent function of the permittivity of the medium outside the particle and the inside of the particle In simpler terms the force to push an object into a trap 1s proportional to the positional eradient of the electric field The implications of such a thing 1s that the particle will experience a force until it reaches an extrema in the intensity of the electric field 1 e the positional derivative of the electric field 15 zero and therefore the force will equal zero This translates into basically a restoring force that keeps the particle trapped at a certain point until the electric field 1s removed or until a much larger force physically removes the particle from the trapping area In the scope of this project the non uniform electric field comes from a 5 Vpp sinusoidal signal across the two electrodes of the traps 14 AC voltage Ground AC Voltage Figure 6 2 a Electromagnetic Modeling of DEP traps 2 Based upon the field lines shown in figure 6 2 the point of highest electric field intensity is approximately in the center of the trap This provides an optimal trapping point for which the spectrometer can take a reading Chapters VII Future Work At the present time only PDMS channels are successful produced consistently for cell detection These channels can be implemented in trapping cells but makes cell differentiation due to frequency modulation impossible A dielectric coated channel is required for
25. ately 6 times the ex pected 3 dB frequency a lead capacitor should be placed from the output to the input of the amp The value of the added capacitor should be such that the RC time constant cf this capacitor and the resistance it parallela greeter than or equal to the original feedback pole time constant Www com 38 LF412 Typical Application ingre ssuppry Sample TTL c INVERTER Oh AMPLE TMH 24 nallenal com 10 39 LF412 Physical Dimensions inches millimeters unless otherwise noted Continued 0 0210 2 220 02 510 0 291 GLASS RHO D23 0 045 0 065 0 230 GLASS 0 320 SERLAHT n TL LR 0 200 E 0 060 F aiso 0 125 0 200 02 4 0 055 r i BOTH I __ 0 008 0 018 0 003 839 i 0 310 or 0 410 0 012 0 100 0 010 Dual In Line Package J Order Number LF4121 833 HS Number 2 3 0 200 7 8 474 16 18 0932 0 00 TE 08170 12 0 250 0 205 RAD 1 DENT 16 35 20 127 PIN 1I0ENT F OPTION 1 HE go ya GRE meae le 7 112 My 1 015 _ 0 099 0 145 0 200 0300 9320 782 8126 0 009 0 015 _ ee LT 10 229 0 381 DIA paz 0040 NOM 3
26. bias curant iz to kept to a minimum Note 10 Supply voltage rejection ratio iz meazured for both supply magnitudes increasing decreasing simukaneousty in accordance with common practice Vr EW to x15V Note 11 Pieter to FETS412X for LF412MH and LFA1281 military specifications Note 12 Max Power Dissipation is defined by the packags characteristics Operating the part tha Power Dissipation may cause the part to operate outside guaranteed limits Note 13 Human body model 1 5 in series with 100 pF Typical Performance Characteristics Input Blas Current Input Blas Current 100 10k ton E z gt 100 lt 40 ES z 2 0 1 10 5 25 0 25 SD 75 100 125 COMMON MODE VOLTAGE V TEMPERATURE 7 TEAD wav 4 Typical Performance Characteristics continued SUPPLY CURRENT NEGATIVE COMMON MODE INPUT VOLTAGE LIMIT V NEGATIVE OUTPUT VOLTAGE Supply Current SUPPLY VOLTAGE V WESE Hegatlve cCommon Mode Input Voltage Limit 5 10 15 25 NEGATIVE SUPPLY VOLTAGE BIH Negative Current Limit OUTPUT SINK CURRENT mA POSITIVE COMMON MODE INPUT VOLTAGE LIMIT V Common Mode Input Voltage Limit 5 10 15 0 25 POSITIVE SUPPLY VOLTAGE V Positive Current Limit SO
27. brication of our chips and how the channels are made and how the traps and leads are added to the chip Chapter three will be on DEP chip micro fluidic ports Chapter four will discuss the flow control of the microfludics we used plus the pump system Chapter five will discuss the detection circuit we designed and some of the components used Chapter six will discuss DEP trapping Chapter seven will cover the future work and what we plan to do next semester Chapter II Channel Fabrication Photolithography was used to fabricate these Dielectrophoreris Traps in the CSU Cleanroom This process allows building contacts wires and effectively traps on a micron scale The DEP chips were built with two distinct components a DEP trapping circuit and a microfluidic channel These two components are bonded together to form a DEP trap The DEP trapping circuit is composed of three contacts and three discrete lines running through the area under the channel The conductive material used was gold and chrome These metals were first deposited onto a glass slide using an Evaporator in the cleanroom A layer 30nm of chrome was first deposited onto the slide This thin layer of chrome 15 deposited because chrome can adhere to glass much better than gold can A 120nm layer of gold 1s then placed over the chrome layer Gold is deposited because of its low resistance Once this deposition 15 completed photolithography follows 71512 Photoresist 15 placed and then spu
28. ch on the pump will run for a total of 2 Delay Before Read the time value ms specified the top left input box When the VI is run with the stop switch turned on it will stop all pumping functions With only the manual command switch turned on a string will be sent to the Oriel controller For example the string V200 n will command the Oriel actuator to move at a velocity of 200 um second There are many commands like this that the VI already uses to control the actuator within the VI itself such as run and stop The way that the pump command works 15 that it will send a run command Then there will be a specified delay time followed by the stop command being sent to the controller then a second specified delay The loop starts over starting with a run command This allows the user to specify a duty cycle for the pump The values that we used for this VI were time 725 ms delay before write 50 ms delay before read 50 ms and time off 7000 ms This roughly gave a flow rate of 40 um per second The VI block diagram shows all of the internal logic that occurs with the switches and with the delays as well as the Run and Stop commands sent to the Oriel actuator 22 Enable Termination Char T M timeout 10sec 10000 Delay Before Write ms TUNMGGUUUUUUUT 1 1 come 9600 Delay Before Read ms 31 Time Off ms Time On ms 2 Delay Befo
29. desirable traits We have attempted to design a system that uses the reflective index of the cells by measuring the difference in the light detected in the cells of interest rather than using a fluorescent dye This technique will solve many of the problems associated with traditional flow cytometry Our technique will save us from having to fluorescent tag our samples and we will be able to retrieve our cell samples alive after the trapping and analyzing of them something which cannot be done with current flow cytometry techniques We will not need as many cells in our sample as well plus this should reduce the cost of upkeep and operation The components of our system are the microscope the cell detection circuit the analog to digital convertor and computer control software and the chip with the channel on it To date we have made a channel to flow our sample solution through This channel has metal leads for setting up the electric field to trap our cell when it is detected by the detection circuit to be analyzed by the spectrometer We first had to build the channels with the traps on them The next step was to determine the proper flow rate of the solution so as to optimize trapping We also made a circuit to detect the presence of a cell to trigger the trap of the control was done with Labview software TABLE OF CONTENTS 3 HE
30. differentiation There are two solutions that must be established before a chip can both detect and differentiate cells First glass channels must be fabricated successfully on a consistent basis New glass has been ordered to reduce the lattice impurities and hopefully will generate channels with uniform height Second an improved method for chip bonding must be discovered Indium bonding has shown to be successful initially but at some costs Depending on the thickness of the Indium layer the channel can become leaky which could result in the loss of cells to outside the channel Indium bonding also provides a weak bond Under high pressure in the channel the chip can break Gold to gold bonding 15 the ideal method for bonding the two chips together and should be further researched The optical system needs to be fine tuned to allow the light modulation to focus on a smaller area This will allow for more intensity modulation when a small object is in the focus area If we are able to obtain any donations a higher output infrared LED could improve the system Also if a high performance photo diode was obtained the optical detection circuit could be improved in the area of time response In the future of the project the spectrometer will become a essential part of the cell analysis Data acquisition will need to be automated using Labview software and resulting data of the cells will allow for cell differentiation Because it 1s the shifting of the
31. e The piece of paper 1 then removed and the microscope stage 1s then positioned over the emitted LED light until the LED light is centered in the middle of the viewing plane The microscope light 15 then turned off and an alignment laser in the infrared region is transmitted into the viewing plane through an optic fiber Light from the laser 15 seen in the viewing plane and the output from the beam splitter 1s adjusted until the laser light is confined to a small area as well as centered over the emitted LED light This ensures that the output from the beam splitter 1s collecting light from where the LED light is focused on Figure C4 2 Example of alignment using a 25 The difficulties encountered in the calibration of the microscope were numerous and were mainly due to the poor maintenance of the entire instrument The second difficulty 15 the basic concept of the optics The microscope used for our experiments has been poorly maintained Dirt and scratches on the lens are present which may alter the light collection but to a lesser degree The beam splitter stage was unstable and poorly fitted onto the microscope and due to budget constraints has to be fastened to the microscope with zip ties If our budget constraints allow we can machine better housing for the beam splitter The focusing plane where the micro fluidic channels sit 1s also difficult to use with the channels Scotch tape 15 required to steady the chip in place and
32. e noise from the photodetector The output from the filter goes into the operational amplifier or op amp The op amp a LF412CN is setup in a non inverting configuration with a gain 1 of 2 500 After the op amp stage we have a voltage buffer or voltage follower used as a buffer amplifier which 15 used to Hon inverting amplifier 2 eliminate loading effects to interface impedances Vout Vin With Zin oo in theory but in reality it is the input impedance of the op amp which 15 usually to ITO Some of the difficulties we ran into Voltage follower 5 were a noisy signal which is why we added a low pass filter having the op amp osculate so we added V some capacitors to eliminate this We also had a week input signal which 15 why we increase resistor to 1 8MQ Below is the final circuit 12 5 VDC ok hann 5 VDC 5 VDC C7 C9 Ne D1 0 0 PHOTODIODE 100n 100n R2 U1B 3 2k ____ 1 R1 250k 9n 70 70 Figure 5 3 Chapter VI Dielectrophoretic DEP Trapping DEP trapping is necessary in this project for one main reason If the sphere cell particle is not held in a particular position without moving a spectral reading of light shined through that object will be extremely difficult to gather data with the equipment and limited budget that we have DEP trapping 15 a method of using electromagnetic forces to hold an object in place One might ask
33. e to 10V over the full temperature range the amplifier is to dive heavier load currents however an increase in input offset Woltage may occur on the negative voltage swing and finally reach an active current limit on both positive and negative swings Preecautiere should ba taken te ereure that the power supply for the integrated circuit never becomes reversed in polarity or that the unit nct inadvertently installed backwards a socket as an unlimited current surge through the resulting forward diode within the lC could cause fusing of the interreal conductors and result in a destroyed unit As with moet amplifiers care should be taken with lead dress component placement and supply decoupling in order to eneure stability For example resistors from the output to an input should be placed with the body clase to the input to minimize pickeup and maximize the frequency of the back pole by minimizing the capacitance from the input to ground A feedback pole is created when the feedback around any amplifier resistive The parallel resistance capacitance from the input of the device the inverting inputi to ground set the frequency of the pole In many instances the frequency of this pole is much greater than the expected 3 dB frequency of the clased gain and consequently there is negligible effect on stability margin However if the feedback pole is less than approxim
34. e to tell when a cell was present p type n type in our system For this we decided to design a silicon silicon detection circuit This circuit would detect a cell by a modification in the light intensity In order to design the detection circuit we started with a photodetector negative terminal positive terminal connected to a fiber optical cable by a ST connection OPF482 The light from an inferred LED goes into the microscope and then through the fiber optical cable into the photodetector A photodetector is a photodiode which is a component with p n junction When a photon light of sufficient energy strikes the diode it excites an electron in the valence band thereby creating a mobile electron and a positively charged electron hole If the absorption occurs in the junction s depletion region or one diffusion length away from it these carriers are swept from the junction by the built in field of the depletion region producing a photocurrent Photodiodes can be used in either the zero bias mode known as the photovoltaic mode or in the reverse bias mode known as the photoconductive mode the mode we are interested the zero bias mode light striking the diode causes a current across the device which leads to a forward bias of the diode which in turn induces dark current in the opposite direction to the photocurrent Dark current 1s the relatively small electric current that flows through a photodiode even when n
35. er achieved in the semester The channel surface must be as uniform as possible to provide better transmission of light Using an alpha step to measure the surface height deep indents were found in the glass One explanation to this result 1s that there are lattice impurities in the glass that etch faster than the uniform lattice If this were the case then higher quality glass would be needed to perform suitable glass etching Once the layer of resist 15 removed the channel chip can be bonded to the DEP trapping circuit chip Two bonding methods have been implemented The more successful method 15 Indium bonding A piece of Indium metal and the channel with gold on the outer area are placed in a solution A positive voltage 15 applied to Indium metal and a negative voltage 1s place on the of the channel chip This creates a current of Indium to be deposited on the gold The channel chip is then ready to bond to the DEP circuit chip The two pieces are roughly aligned by eye and pressed together by hand The chips are then observed under a microscope to view the alignment Fine adjustments are made to optimize the alignment Once the alignment is made the two chips are sandwiched between two copper sheets These sheets are then put in a vice The vice 15 screwed together with four screws This puts equal pressure on the bonding area The bonding vice 1s then placed into a heater vacuum 15 pulled in the C heater to prevent oxidation from the
36. ge um x 5000 um The only syringes on hand at the beginning of the project were 3 cc syringes with a diameter of 8 585 mm 0 008585 um manufactured by B D Using the equation above equation to achieve a 40 um s flow rate in the channel the plunger must be pushed at a rate of 0 00346 um s The original plan for flow control was an industrial syringe pump Borrowed from the chemistry department the NE 1000 syringe pump made by New Era Pump Systems Inc can push this B D syringe at a rate of 2 434 uL hr This 15 the equivalent of 6 761 x 10 um s This is 1 954 x 10 times too fast Obviously this syringe pump is not a valid solution to the flow rate problem In addition when the syringe pump was set to its slowest setting the torque broke the encoder coupler because the pressure in the chip created a force pushing back against the syringe plunger that was beyond what the pump was designed for As a solution the Oriel Instruments Encoder Mike Controller 18011 was chosen to function as a custom pump The Oriel Instruments Encoder Mike Controller 18011 Oriel 1s designed to be used as a precise way to move a microscope stage It can move the actuator at a minimum velocity of 0 5 um which is still about 150 times faster than the desired rate for the larger syringe 8 585 mm diameter To reduce the rate at which the plunger must move a smaller syringe 4 669 mm diameter was chosen to be used in the system This syringe plunger only needs
37. he physical and chemical structure of each individual particle FSC correlates with the cell volume and SSC depends on the inner complexity of the particle for example the shape of the nucleus the amount and type of cytoplasmic granules or the membrane roughness Flow cytometers form images of each cell s fluorescence scattered light and transmitted light Modern flow cytometers are able to analyze several thousand particles every second in real time and can actively separate and isolate particles having specified properties A flow cytometer is similar to a microscope except that instead of producing an image of the cell flow cytometry offers high throughput for a large number of cells automated quantification of set parameters To analyze solid tissues single cell suspension must first be prepared Fluorescence activated cell sorting FACS 15 a specialized type of flow cytometry It provides a method for sorting a heterogeneous mixture of biological cells into two or more containers one cell at a time based upon the specific light scattering and fluorescent characteristics of each cell It is a useful scientific instrument as it provides fast objective and quantitative recording of fluorescent signals from individual cells as well as physical separation of cells of particular interest The term optofluidics defines an emergent research field that combines microfluidics and optics and from this a class of adaptive optical circui
38. he run button in Labview The VI for controlling was not difficult to program Another reason we went with this DAQ was due to the fact that Labview VI s were available on the Hytek Automation website This made it so that it was a plug and chug type of program Here is the VI block diagram DeviceType DeviceInde al error in no error error out 19 Figure C1 3 Block Diagram of DAQ VI This block diagram shows all of the subVI s that were used to retrieve data from the DAQ which were available on the Hytek website C2 Oriel Instruments Encoder Mike Controller 18011 E b Figure C 2 1 a Control Panel of Oriel Controller b Microscope stage being controlled The Oriel Instruments Encoder Mike Controller 1s normally used as a microscope stage controller to move a microscope stage very slowly Our application of this piece of equipment was as a syringe pump The main reason that this was used 1s because it was able to move a syringe plunger slow enough to get reasonably slow flow within the channel of the chip The minimum rate at which the actuator can move is 0 5 um s Remember that the chip only had a cross sectional area of approximately 5000 um The syringe that we are using is approximately 0 5 cm in diameter This makes it so that the stage must be moving extremely slow in order to get a flow rate within the channel to be as slow 40 um per second which 15 the flo
39. input voltage is independent of the voltages However neither of the input voltages allowed to exceed the supply as this will cause large currents to flow which can result in a destroyed unit Exceeding the negative common moda limit an either input will cause a reversal of the phase to the output and force the amplifier cutout to the corresponding high low state Exceeding the negative common mode limit on both inputs will farce the amplifier output to a high state Im neither case dosa a latch occur since raising the input within the common mode range again pute the input stage anc thus the amplifier in normal operating mode Exceeding pasitve common mode limit on a single input will not change the phase of the output however if both inputs exceed the limit the output of the amplifier may be forced to a high state The amplifiers will operate with a comrnon moede input walt age equal to positive eupplky however the gain bang width and slew rate may be decreased in this condition When the negative common mode volt age swings to within of the negative supply an increase in input offest v altace may occur Each amplifier individually biased by a zener reference which allows normal cir uit operation cn 36 0 power sup Supply voltages less than these may result in lower gain bandwicth amd slew rata The amplifiers will drive a 2 load resistanc
40. ked Then a drill press 15 used with a small drill bit Great care must be taken when drilling the holes because glass cracks and chips very easily Water 15 applied at the drill area and only very small depths are drilled at a time This process 1s very dependent on minimal human error Many of the fabricated chips have non ideal holes when drilling 1s complete It is also very important to have a way to securely place the nanoports on the DEP chip so that there 1s no leakage as cells and liquids are pushed into the channel and pulled back out of the channel This process is quite simple Adhesive o rings are used to create the bond between the nanoport and the chip Adhesive o rings are a sticky glue like material in an shape that act as double sided tape These rings are very carefully placed around the center of the drilled holes and pressed firmly into place After this the nanoports are cautiously placed on the adhesive o rings making sure to center the nanoport over the hole Again this 15 pressed firmly into place Once complete the chip 1s clamped to the nanoport for one hour to allow the adhensive o ring to dry and seal The figure below shows the placement of a nanoport TOP VIEW SIDE VIEW drilled hole EHE adhesive d O me channel J adhesive from pump ring nanotube Figure 3 2 Chapter IV Flow Control An essential part of the project 15 to be
41. leveled with the focusing plane The use of tape leads to oils from the fingers being deposited onto the chip which alters the light characteristics of the chip Again if our budget would allow we could machine a better focusing plane that would allow us to secure the chip into place and avoid taping it The fiber optic output from the beam splitter 18 also difficult to use at time as there 15 no relief at the ST junction for the fiber optic cable A relief at the ST junction would allow for the focal length from the optic fiber to the beam splitter to be changed without causing stress to the fiber optic cable or having to dissemble the output of the beam splitter The knowledge of optics was one of the easier difficulties to overcome with the help of Dr Lear Understanding why we needed a 62 5 125 optic fiber as opposed to a much larger optic fiber is an example of the problems he helped us overcome The reason why a 62 5 125 cable 15 used to minimize the area in which the light from the beams splitter 15 collected 26 Appendix D Data Sheets and User Manuals D1 482 Optic Fiber Photodiode OPTEK Product Bulletin OPF482 August 1996 Fiber Optic High Speed PIN Photodiode in ST Receptacle Type OPF482 086 56 UNC 28 E 235 5 97 ANODE CATHODE 100 2 54 DIA NOM 375 9 53 500 12 70 375 24 UNF 2A THREAD 155 3 94 410 10 41 810 20 57 790 20 07 DIMENSIONS ARE IN INCHES MILLIMETERS
42. mperature and pressure have no been obtained which prevent acceptable bonding Small bonding areas have been obtained but not large enough to be a reliable method for chip bonding Chapter III DEP Chip Micro Fluidic Ports In order to get cells into the DEP chip with a micro channel there must be a way for cells to physically enter the chip It is also important that there 1s a way for the cells to exit the chip This 1s accomplished through the use of nanoports There are two nanoports on every chip one for the cells to enter into the channel and one for cells to exit the channel Nanoports are cylindrical shaped parts that on the order of 1 2 cm in diameter and 2 3 tall They composed of a plastic like material On the bottom of the nanoports there 1s a small circular opening with a rubber seal surrounding it From the top nanoports are hollow with threads so that a nanotube can be secured into the nanoport using a hollow screw like adapter These adapters are included as a kit when nanoports are purchased Nanotubes are very small tubing that are 150 um in diameter that are frequently used in micro fluidics The figure below shows the basic structure of a nanoport nanotube adapter nanoport Figure 3 1 Before nanoports can be placed on a chip holes must be drilled into the chip in the appropriate places To do this a pattern of the chip fabrication 1s placed over the chip and the two places for holes are mar
43. n Mu Channels Measurement range prae __ bG O DI 158 internal gain setting NUSB608 O LabJack U3 iUSBDAQ U120816 0 4 096 MM minimum S high voltage of 2V and a max anda max logic low voltage of O BV low voltage of 0 8V _ had LabView drivers then had aprcecutoffot 150 160 __ O A D Characteristics A Characters __________ throughput sample pM Time 10 70s 14400 4294 rule ree eer 4294 ___ 2550 Ss 7 poo Required Digital EN MESE gt Yes 6 bi directional hi directionalports 4 bi EN ports Yes 20 Ports ves 16 bi directional ports iN D3 LF412 Operational Amplifier National Semiconductor LF412 August 200 Low Offset Low Drift Dual JFET Input Operational Amplifier General Description Tese devices are low occat high spesd JFET input opera tional amplifiers with very low input ofset voltage and quar atteed input offset voltage drift They require low supply current yet maintain a lare gain bandwidth produet and fast slew rate In addition wel matched hgh voltage JET input provide wory low nput and offaot Tho 2412 dual is pin compatible with LM1558 allowing dasigners immediately upgrade the overall performance of exiti
44. n on the chip This resist 1s thinner and has very good resolution A soft bake at 110 degrees Celsius for 1 minute follows Next the chrome DEP mask 15 used to pattern the resist A chrome mask is chosen for this exposure because of the small features it encompasses Chrome masks are expensive but have a high resolution and have an antireflective coating to enhance the resist found and exposure time with the soft bake of 10 Chrome exposure This mask contains two different trapping circuits to select from A characterization of the AZ1512 seconds The resist 1s exposed for 10 seconds which maximizes the resolution of the resist The exposed resist Glass is then developed leaving the unexposed area covered A two step wet etch removes the uncovered gold and Figure 2 1 chrome from the glass slide The DEP trap chip 15 then ready for two of the three types of bonding The DEP channels were composed of either polydimethylsiloxane PDMS or glass PDMS channels were the majority of the channels prepared since the fabrication success rate of these channels were much higher than glass The success of PDMS is attributed to its exceptional adherence to glass The PDMS used was made of bulk PDMS and the PDMS curing agent with a mass ratio of 10 1 respectively This PDMS is then placed in a vacuum and degassed Degassing will remove the impurities in the PDMS which will improve the optical properties of the mold A mold of the
45. ng designs amplifiers may be used in applicationg such as high speed integrators fast DWA converters sample and hale crcuits and many other circuits requiring kw input offset voltage and drift low input bias current high input imped high slew rate and wide bandw dth Typical Connection Ordering Information LFA12XYZ Indicates electncal grade indicates temperature range M for military for commercial indicates package type H or N i2 ederak of Haero Copernic 200d Halenal Semiconductor Corporation Des Ce 656 Features Internally trimmed offset voltage 1 mV max Input offset voltage drift 10 max Low input bias current 50 pA Low input noise curent 0 07 pA Hz Wide bandwidth 3 MHz min High slew 10 ps min Low supply current 1 8 mA Amplifier High input impedance 10120 Low total harmonic distortion Low 1 f noise comer 50 Hz Fast setling time 00 01 218 50 02 Connection Diagrams Matal Can Package RAIT Note Pin 4 connected case EW Order Number LF412MH LF412CH or LF412MIB 902 Note 1 See NS Package Humber Dusl In Line Package A inu Enim buFFUTB INPUT IAFUT AN INVERTING iAPUT y TOP VIEN BHH Order Number LF412 ACH LFA12CN or LF412h1J 883 Plate 11 NS Package Number
46. o photons are entering the device This 15 called the photovoltaic effect and 1s the basis for how solar cells work which are just a large number of big photodiodes Onto reverse bias which only induces a little current known as saturation or back 11 current along its direction But a more important effect of reverse bias is widening of the depletion layer therefore expanding the reaction volume and strengthening the photocurrent Circuits based on this effect are more sensitive to light than ones based on the photovoltaic effect and also tend to have lower capacitance due to the greater separation of the charges which improves the speed of their time response because so the smaller the capacitance the smaller the time constant On the other hand the photovoltaic mode tends to exhibit less electronic noise Another type of photodiodes 15 the avalanche photodiodes which have a similar V structure but are operated with a much higher i 7 reverse bias which allows each photo generated C carrier to be multiplied by avalanche breakdown resulting in internal gain within the photodiode which increases the effective responsively of the Dy dn device Our photodetector is used in reverse bias with a large resistor 1 8M to have the majority of the current flow into the circuit The next step of v dn the circuit is a low pass filter with a cutoff frequency of 1 000 Hz to reduce th
47. pe and the light source is vital for any reasonable data collection from a micro fluidic sample In this section the calibration process of the microscope will be covered The difficulties learning how to calibrate the microscope will also be explored 24 To begin the basic knowledge of how our system works 15 essential Below is a figure on the general set up of the entire system The size of the optic fiber coupled into the beam splitter should be around 50 125 to 62 5 125 and needs to camera be a multimode fiber 50 125 The percentage of light sent through the optic fiber 15 90 not while the remaining 1096 of light goes to a used camera To calibrate the microscope the use of a LED that emits light in the visible spectrum 5 micro as well as a ocusing lens above the LED and below sample the microscope s focus plane By placing a d white of Or fi infrared on the focusing plane source the microscope is alignment LEDs focused while the laser focusing lens for the Figure C4 1 Diagram of microscope being coupled with light source LED is also focused on the same plane The goal of the lens above the LED 15 to make the light emitted from the LED confined to a very multimode fiber beam splitter IRL Ew A 2x2 fiber 1 1 1 1 1 I i 1 small area in a circular shap
48. r PDMS chips This 1s because the actual cross section in a glass chip s channel 1s larger and cannot be accurately estimated Experimentation also revealed that due to complex pressure dynamics the flow rate the channel does not follow the syringe plunger To be more precise when the plunger is moved and then stopped the flow 1 the channel does not stop immediately but rather decays over the course of several minutes This behavior 1s analogous to the behavior to an RC circuit Both have a time constant associated with how long it takes the flow rate to die down The larger syringe 8 585 mm diameter builds up a higher pressure and the time constant 1s several times longer than the time constant for the smaller syringe 4 699 mm diameter This suggests that the cross sectional area of the syringe is proportional to the time constant for the flow rate inside the channel of these more complex dynamics make experimentation the best indication of actual flow inside the channel There was no precise way to obtain the velocity of spheres moving in the channel but the duty cycle described earlier is estimated to produce a flow rate of 40 um s in a PDMS channel Due to limited fabrication of glass chips near the end of the semester the duty cycle for the desired flow rate 1n a glass chip has not been found However it is known that the duty cycle must be higher than that for the PDMS channel Chapter V Detection Circuit We needed to be abl
49. re Read 1 Iunumnumnuuuuuu ISA 1 i 2 IManual Cmd E Figure C2 5 23 C3 Ocean Optics HR2000 High Resolution Fiber Optic Spectrometer The overall goal of the project 1s to differentiate cells as they pass through the channel In order to do this the method of intracavity spectroscopy will be used In order to detect the changes in wavelength as a cell passes through a spectrometer is used to analyze the diffraction of the LED light source At this point in the project the spectrometer has only been used to assist with the setup of the optical system The spectrometer was used to maximize the light intensity from the LED light source as well as the focused LED light after 1t has passed through the lens The resulting spectrum also provided verification that the microscope optics were properly aligned Narrow peaks in the spectrum indicated proper alignment from the LED light source to the microscope C4 Optical Calibration At the heart of the OFIS process 15 the microscope This 15 the instrument that we use to eather light to use for spectroscopy cell detection and for the camera No other instrument needs to be calibrated as frequently as the microscope and no other instrument in the process can dramatically change the resulting data by a slight adjustment Therefore the correct calibration of both the microsco
50. ta Acquisition Unit DC Direct Current DEP Dielectrophoretic LED Light Emitting Diode OFIS Optofluidic Intracavity Spectroscopy 16 OpAmp Operational Amplifier PDMS Polydimethylsiloxane VI Labview Virtual Instrument Vpp Volts Peak to Peak Appendix Budget e ADG 452 Digital Switch 15 e Various circuit elements including Op amps and digital chips 10 e Hytek IUSBDAQ 0120816 105 TOTAL EXPENSES 130 Starting Budget 500 over 2 semesters Money left 500 130 370 With the remaining money we will research things we can buy to optimize our system Appendix C Peripherals The need for peripherals in this project is abundantly apparent We used many different measurement and control systems to be able to achieve our goals These peripherals were Data Acquisition Unit 10SBDAQ U120816 from Hytek Automation Ocean Optics Spectrometer HR2000 Oriel Instruments Encoder Mike Controller 18011 Microscope heavily customized Olympus 230997 Logitech USB camera Beam splitter Newport parts manufactured in house 17 C1 Data Acquisition Unit Figure C1 1 Hytek Data Acquisition Unit The data acquisition unit was brought on board due to a need for triggering a circuit based on amount of light collected by the photodiode This made it so that instead of creating a new logic circuit every time we changed the gain stage of the amplifier the logic could be
51. to contact with the DEP circuit chip The two are now bonded together The mask holder 15 then unscrewed and pulled out vertically from the aligner Glass channels were experimented with as well These glass channels are more desirable than PDMS because a dielectric coating can be deposited on the surface of glass producing a light modulating cavity necessary for cell differentiation Pyrex glass 1s used for channel etching and chrome and gold are deposited on the surface thick layer of 400 positive resist 15 deposited onto the surface This resist was chosen because it is much thicker than the 21512 resist which will be necessary with HF etching The tradeoff with using P4400 15 that 1t doesn t have as good a resolution as AZ1512 In this application giving up resolution 15 certainly acceptable The soft bake for this resist 15 110 degrees Celsius for 2 minutes The resist 1s then exposed with the channel mask for 35 seconds The exposed area is washed away in development The chip is then baked at 110 degrees Celsius for 10 minutes This turns the resist into a hard film which can withstand the wet etching process The gold and chrome are etched away The next step 15 to etch the glass This 15 done using a wet etch of HF The chip 15 placed in HF for approximately 3 4 minutes The chip is removed from the HF and washed with deionized water The chip 15 then placed in an acetone bath to remove the hardened resist Acceptable glass etching was nev
52. ts that integrate optical and fluidic devices In many biological applications the two technologies are used in combination microfluidics for sample delivery and optics for sensing The introduction of liquids in the optical structure enables flexible fine tuning and even reconfiguration of circuits such that they may perform tasks optimally in a changing environment The use of fluid as a medium for transport is appropriate for a significant class of clinically important biological entities ranging from DNA strands viruses and bacteria to cells and microorganisms In addition microfluidics based devices require very low input sample volumes nanoliters or less and can be very conservative in terms of the samples The implementation of optics in the microfluidic platform enables an unprecedented level of integration Moreover optofluidic devices are easily and highly reconfigurable which can be a significant advantage for manipulating and handling biological samples The goal of our project 1s to design a system of cell detection and analyses without the need for fluorescent markers or tags This will help reduce the cost as well as the time involved in sample preparation Another benefit of such a system 15 the ability to keep the samples for further use all of which cannot be done with tradition flow cytometry If we are successful there is a large market for our product mostly in the biological fields Chapter two will discuss the fa
53. w rate by which it 1s suitable to trap cells Again we used Labview to control the actuator This was much more difficult to control because there were no VIs available on the internet This made for a much bigger challenge because we were unfamiliar with RS 232 communication that was required for control of the Oriel actuator Using a wiring diagram that was 1n the User s manual of the Oriel actuator we created our own RS 232 to 9 cable to interface between the control panel and a computer 20 Block Diagram DB9 gt 5 232 Controller Yes Yes Parameters Pump OffCycle Stop Pump a b Figure C2 3 a RS232 input to Oriel Controller Figure C2 3 b DB9 input to computer 21 A Labview VI controls the signals that are put on the different wires of the RS 232 Cable The front panel for the Labview VI looks like this String to Write IDN rin Time On ms 2 Delay Before Read 71500 Delay Before Write ms 2 1500 I Delay Before Read ms 7 500 7 Read String Time Off ms 1500 Pump Stop Manual Cmd ON ON ON Figure C2 4 Front Panel of Oriel Controller VI The switches in the lower right portion of the front panel control what the switch actually does Only one switch can be on at one given time in order to function properly When the VI is run with only the pump swit
Download Pdf Manuals
Related Search