Home

A Parylene Real Time PCR Microdevice

1. essen 22 Figure 2 4 POMS micromolding esses eene eene eene 36 Figure 2 5 Surface qnie FrOmaoc DUITI oc ids ouo on Gua uds i aeo o pepe qoe Vo edo eua ease gure 38 Fig te 2 6 Chemical Structure OF ParyIene eon oed ber Pe Te Ue tino eee UNS Ubera E dS 40 Figure 2 7 Schematic of parylene CVD deposition eeeeeeeeeeeer 42 xiil Figure 2 8 Chemical structure of di p xylylene the dimer precursor to parylene N 42 Figure 2 9 Surface micromachined parylene channel sess 46 Figure 2 10 Embedded channel technology creron e E T N 49 Figure 2 11 Microfluidic components fabricated using parylene technology 51 Figure 2 12 Thermal isolation by parylene stitches cccccccccccccceeeeeeeeeeeesesseeeeees 52 ligure 2 13 Integrated HPLC SyStefriza iced a a a e 53 Figure 2 14 QPCR on low volumes in parylene coated tubes 53 Figure 2 15 Amplification of 0 5 ul QPCR solution eeeeeeeeeeeee 54 Figure 2 16 QPCR with various S A volume ratios of Parylene C 55 Figure 2 17 High SA vol ratios of Parylene on a 0 5uL RTPCR sample 57 Figure 2 18 Concept of an effective distance h in which PCR is inhibited 58 Figure 2 19 QPCR with various S A volume ratios of Parylene HT 59 Figure 2 20
2. Holes of various sizes 1 7 mm were punched into an unpatterned sheet of PDMS while another PDMS sheet with a larger 5 mm hole was placed on top The bottom holes contained the QCR fluid while the top sheet contained the mineral oil placed on top of the QPCR solution to prevent evaporation Using a fluorescent microscope and a CCD camera QPCR reactions were performed The advantages of using PDMS are its ease of use and flexibility Disadvantages of using PDMS arrive from its porosity and surface properties Evaporation of liquids is common as solvent vapors penetrate the material Surface treatments are necessary to prevent bio fouling and protein adsorption The PDMS glass interface is not capable of supporting high pressures over 30 psi Furthermore integration into a standard MEMS process or pre fabricated CMOS chip 1s limited to placing a finished PDMS piece on top of the chip 2 3 3 Surface Micromachining Surface micromachining involves building structures onto the surface of the substrate Channel formation in this case does not require a second bonding step with 38 another wafer Instead the structural material such as polysilicon is deposited on top of a sacrificial layer such as silicon oxide See Figure 2 5 eee Sacrificial Layer Structural Layer Figure 2 5 Surface micromachining In this way all four walls of the channel are already present and the inside is hallowed by dissolution of the s
3. Figure l l Basic concept of PCR ampliHe lon soot dente edo ta at dete eee l Figure 1 2 Chemical structure of nucleoside triphosphates eeeeesssss 3 Figure 1 3 Typical thermal recipe for PCR sse 8 Figure 1 4 PCR schematic illustrating selective amplification of the target region Delween the PUIG Dall Sonen a E Lund MU Lu Ut UD ane D EA ee DC dU ELE 11 Figure 1 5 MJ Thermal Cycler from Bio RAD ssssssesesssesseseeeeeeeererrerrererererrrrrerrerrrrereees 13 Figure 1 6 Chemical Structure of SYBR Green I eene 16 Figure 1 7 Fluorescence spectrum of SYBR Green ccccssessssssesssesssssssesseeeeens 17 lieure 1 5 Schematic or Lag Man pro DS acoso o bte e nea Bet o NRI Ee Ged 18 Figure 1 9 Amplification plots for a calibration curve Replaces 16 20 are 10 fold serial SPITWIRTO I NERONE 20 Figure 1 10 Calibration curve for an M13 virus DNA sample 21 Figure 1 11 Strategene MX3005P benchtop RTPCR system eee 22 Figure 1 12 Serial dilutions to determine sensitivity of assay eeeeeeeeeeeeeesee 24 Figure 1215 Assay Specificity Sesso vester ta anta tasca sausage er da nasa een etes 25 Figure 2 1 Basic schematic of photolithography sese 28 L1601e 2 2 Photolitho PrapBy USING a SIEDDSE uin eet qme eaten eet Ge RA 3l Figure 2 3 Example of bulk micromachining
4. more details 4 5 3 Optical Detection Protocol The same optical protocol was used except a 4X objective instead of 10X was used at a shutter speed of 350 ms The same normalization scheme was also used Please see Chapter 3 for details 123 4 5 4 Results and Discussion dps 1 08 B No Primers M13 Virus Sample e o 1 06 o o g o e E 1 04 e o e N e E 1 02 zc e u e E a 1 C 3 i mu n B E 0 98 T T T T T T T T T T T T T T l 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Cycle Number Figure 4 19 Detection of M13 virus on chip Free Standing Chip Versus Conventional Machine 1 1 9 6 mm e A 66 mm e dun e free standing chip ee e 8 di S 1 06 e D li e 2 9 e 1 04 e e e A D A D A i Aa A e 444 4 A E 102 2 4 O R A AAAA 1 A c 1 amn A4 o e e eo e 0 98 T T T T T T T T T T T T T T 1 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Cycles Figure 4 20 Comparison of chip versus conventional machine Sample volumes and surface area to volume ratios of parylene were comparable 124 The resulting amplification Figure 4 19 1s very similar to the previous version The M13 virus sample shows an increasing fluorescence signal compared to the no primers control To compare the results to a conventional machine total volume and surface area to volume ratio were considered The chip had an esti
5. 22 ay oL oet beue oit 95 PIOUS 2 21 Tempertdt te TeCIDE S58 eate S TO ie tudoeus 96 Figure 3 32 Filter block for SYBR Green I detection sse 97 Figure 3 33 SYBR Green fluorescence in microchannel sess 100 Figure 3 34 Detection of M13 virus Data normalization described above 101 Figure 3 35 Air gap chip versus conventional QPCR machine suusse 102 Figure 4 1 Overall device fabrication steps seen 104 Fig re 4 2 Bare Silicon Ci ps aseo one brunette taa een x ehe ea edet te p rel e erue s 105 Figure 4 3 Oxide layers Notice the back side shows silicon etched by the DRIE Back side also shows the legs of the front side oxide pattern for clarity Actual silicon is not TANS PALI NER Um 105 Figure 4 4 First parylene layer with representative holes The holes are actually present thirouehoutthe outhied Hanneles ION oat e Reda tbe t diam 106 Figure 4 5 Channels etched into silicon Bottom right 1s 1 0 hole 107 Figure 4 6 Second parylene layer deposited sese 108 keur Ael Platnum DOE CEDE cuo o Coupon oes ien iwaace ondes T aus E du ee 109 Figure 4 8 Back side finishing View from back side Left After DRIE Right After XVI Figure 4 9 Finished chip front and back essent 111 Figure 4 10 Platinum traces directly on parylene Left contact pads R
6. 3 1 3 Analogues to Electrical Engineering An alternative way to thermally analyze a system is using an electrical engineering analogy to construct thermal circuits This is particularly useful in simplifying time varying temperature changes Below is an RC circuit Figure 3 17 EE analogue for thermal characterization where V voltage difference U temperature difference Vo voltage difference at time 0 I current source J heat source R electrical thermal resistance C electrical thermal capacitance The equation describing this RC circuit under constant current 1s V t Ve c t IRI ec Equation 3 5 T RC 81 Please see the appendix for detailed derivation This model can be used to characterize a system such as a thermally homogenous solid with an internal heat source surrounded by air In the analogous thermal circuit the current source 1s the heat source the voltage differences are the temperature difference between the solid and room temperature the resistance becomes the thermal resistance from the solid to the air by convection and the capacitance 1s the heat capacitance of the solid Hence the solution to the thermal circuit is U t U eR JR 1 e RC U T T Equation 3 6 T RC Connecting the thermal circuit with physical values 1 hA Equation 3 7 C pC V where h heat transfer coefficient A heat transfer area p density of the solid C heat capacity
7. 5 um min depending on plasma parameters such as oxygen flow rate pressure power and configuration of the plasma chamber For increased anisotropy a Bosch like process of C4Fg plasma alternated with an O plasma can be used The mechanism for etching of parylene in an O plasma is not well understood however based on studies using a remote microwave oxygen plasma R R A Callahan et al proposed a mechanism involving hydrogen abstraction oxgygen absorption and ring opening resulting in formation of an aldehyde or carboxylic acid group The most convenient masking materials for plasma based etching are photoresist and metal Photoresist has a 1 1 selectivity with parylene which is suitable for etching thin parylene films however since mass transfer effects are prevalent in the small feature sizes used in MEMS a conservative guideline is to use a photoresist mask that is twice the thickness of the parylene etching required For photoresist layers thicker than about 10 um photoresist cracking can become problematic due to the thermal stress resulting from the high temperatures used in a plasma system despite water cooled wafer platters Cracks originate at stress concentration sites such as small circular holes or sharp corners and propagate along the length of the wafer Avoiding such features in design helps reduce this problem Hard baking may also help although at extreme temperatures depending on the photoresist in general above 140 C
8. I knocked on the door of the chairman of the bioengineering department Professor Morteza Gharib during Spring Break of 2003 without an appointment nor any advanced warning After 5 knocks and 2 minutes waiting I figured he was out of town and took 6 steps towards the exit when his door cracked open he stuck out his head and he welcomed me into his office Already with two back to back letters of rejection from Caltech I pleaded my case to him in 5 of the most life changing and legendary minutes of my professional life Thank you Professor Gharib for seeing in me the talent and desire to purse this highest level degree and personally accepting me into the highest quality institute of technology vi The most enjoyment amusement and awe I have experienced during my time at Caltech has come from my interaction with the campus community I would like to thank my advisor Professor Yu Chong Tai for his advice guidance and enlightening and entertaining answers to my technical questions I would also like to thank him for demonstrating how to be a leader and pursue excellence To this day he remains the only person in the world with whom I can explain all my technical worries and questions and in return I receive an out of this world solution or explanation that is based on fundamental physics but points to amusing directions that tickle your brain Nowhere else can I get the experience of being in his office and saying hmmm maybe you re
9. QPCR machine and parylene coated tubes amplification can occur in reaction volumes from 28 uL down to 0 5 uL There does not seem to be any adverse effects due to low volumes except increased noise from the optical measurement system For very low 1 ul reactions consistency 1s decreased In the above figure D10 refers to a reaction with 1 63E9 molecules of DNA d100 1 63E8 and dlk 1 63E7 The dlOk sample was 1 63E6 however it was indistinguishable from the no primers controls 55 2 4 6 2 Biochemical Compatibility of parylene with QPCR To study the effects of Parylene C on the PCR reaction from a biomaterials standpoint the reaction tubes were coated with parylene and additional parylene sheets were included at different surface area to volume ratios To fit inside the tubes these sheets were crunched with tweezers until their entire area was submerged in the PCR solution Measured in inverse millimeters the ratios were 0 15 mm and 60 mm The parylene coated reaction tube itself contributes about 1 5 mm to the surface area vol ratio The ratios of the QPCR chips in this work are about 50 mm oe T 8 n pu o LL Cycles Figure 2 16 QPCR with various S A volume ratios of Parylene C 56 Figure 2 16 shows that a ratio of 15 mm did not have any effect on the biochemistry nor the fluorescent readings of QPCR At 60 mm what appears to be inhibition did occur relative to the other two samples As sh
10. V volume of the solid 3 3 2 Device Thermal Design A metal trace comprised of 2000 A thick platinum over a 300 A thick titanium adhesion layer was used as both a temperature sensor and heater The room temperature 82 resistance of the stack was dominated by the thicker platinum portion and about 2 3 kOhms Characterization of both temperature sensor and heater functions is given in this section For fast chip performance temperature cycling must occur quickly To achieve this end the chip was designed with a thermally isolated silicon island so that only the area of the chip containing the fluidic channels was heated Originally designed for use in temperature control for high performance liquid chromatography chips the technology works nicely for this RTPCR chip as well Reaction chamber Traversing channel eater Temperature sensor Air gap OOOO kgs sto m j ely af a amine LELLLITLTLTELTLLTIHEITEUTI ji mm E m ATA TT A PPL CULL LLL TLE CELT LLEL DDD DANDINI Figure 3 18 Thermally isolated island 83 zn i A Ka ETT x Sigaal A s SEF Date 28 Jun 200 Phote Mao 4453 Time 185358 Figure 3 19 Parylene stiches The thermal model described in Figure 3 17 would characterize the temperature on the thermally isolated island This island however can still exhibit a non zero leak of heat To account for that leak into the chip body the model can be extended by adding an
11. a T T Equation 3 9 R T where To is a reference temperature not necessarily 0 C and a is the temperature coefficient of electrical resistance Linear regression of a set of resistances at different temperatures will yield suitable value for a From Equation 3 9 it can be seen that resolution of such a device improves by increasing the product of a and Ro For the RTPCR chip the following R versus T behavior was observed 85 2700 2650 y 5 7163x 2149 3 2600 R 1 2550 2500 R Ohm 2450 2400 2350 20 30 40 50 60 70 80 90 100 Temperature C Figure 3 21 Temperature sensor calibration The calibration curve shows the expected linear relationship between R and T with an a value of 2 3x10 C This value is lower than the literature value of a for platinum 3 7x10 C perhaps due to differences in atomic or crystalline configuration of the thinner thermally evaporated platinum used in this work Previous group members have reported a 200A Ti W adhesion layer with 1000 A platinum with a TCR value of 1 0 x10 SC and a 300 A titanium 2000 A platinum hybrid layer with a TCR value of 2 1x10 C Thus the values reported in this thesis are consistent with those of previous group members Resolution of 0 1 C difference can be sensed by a measurable 0 571 Ohm change in resistance Resistance in lead wires is less than 1 Ohm The largest source of inaccuracy 1s likely the calibration s
12. active valves pumps check valves and nozzles This wide array of components demonstrates the versatility of parylene and shows the potential for integration of many components into one multifunctional device 51 See TETTE HIT ETT E e gall P RH EG T L c Normally closed in channel check valve i Electrostatically actuated valve top left and ae WS rq NC N FR cm with vacuum collapsed sealing diaphragm middle with thermal flow sensor top right and d Electrospray ionization ESI nozzle for coupling on chip analysis to a mass spectrometer b Electrostatically actuated peristaltic pump Individual chambers also can serve as pressure sensors Figure 2 11 Microfluidic components fabricated using parylene technology One interesting component of interest to this thesis 1s the thermal isolation island Such an island is a small mm cut out of silicon that is thermally isolated from the main chip yet mechanically connected to the chip by a gap composed of air and strips of parylene Since these stitches are made of parylene and can be as long as 100 um they make good thermal insulators us m a os 150 um wide E Riri MEE L Ir iJ A 1 v z k p Se i Wed re pac E e i Figure 2 12 Thermal isolation by parylene stitches 2 4 5 3 Integration A key advantage in using the parylene microfluidics platform is the promise of system integration resulting in a sophisticated ch
13. are desired If high aspect ratio channels are desired a plasma can be used to etch the silicon if the overhang material is properly designed e g thick enough even after non specific etching by 50 the plasma Wet etching techniques are generally not recommended as the surface tension from the drying process can pull the overhang structures down and break them Thus the bulk material should be one that can be etched by dry processes e A conformally coating layer light grey such as parylene is deposited and coats all surfaces creating a microfluidic channel that is embedded into the bulk substrate Parylene is one of the only materials that can be deposited conformally and is also chemically inert and biocompatible for microfluidic applications Theoretically the parylene thickness needs to be only half the width of the opening in the overhand structure A conservative thickness would be equal to the width of the opening The extra parylene ensures channel is completely sealed and the top surface is still flat enough for further processing e For the inlet and outlet holes a cross section of the channel with a larger overhang structure opening will result in a partially open section of the channel No sacrificial material is used here so excessively long channels can be fabricated as easily as shorter ones 2 4 5 2 Parylene Microfluidic Components Many microfuidic components have been fabricated using parylene technology including
14. by etching a 8 um wide trench around the channel which in subsequent steps were completely filled with parylene and acts as an etch stop for isotropic XeF etching This 67 feature can be seen in Figure 3 5 as black line forming an outline of a channel The channel itself is shaded gray indicating that it has not been etched yet The stitches are formed by 8 um wide trenches across the air gap Upon filling with parylene these trenches are 200 um x 8 um x 100 um parylene slabs that connect the island to the main body A thicker photoresist is used for DRIE 15 um of AZ 9260 which is patterned and developed in undiluted AZ developer Following hard bake this resist sustained 333 loops of the Bosch process Photoresist etching for current conditions is 2 um per 100 etching loops in the inductively coupled plasma ICP etcher used Thus 6 um minimum is required for 300 loops 15 um was chosen as a conservative thickness After etching the photoresist was stripped using ST 22 Acetone stripping may work but residual fluorocarbons and excessive photoresist heating during the Bosch process make the photoresist more resistant to dissolution in acetone 1 IT f Figure 3 6 First parylene deposition 68 First parylene deposition An adhesion layer of A 174 gamma methacryloxy propyltrimethoxysilane was applied followed by parylene deposition to fill the trenches taking advantage of its conformal coating properties The adhesion layer
15. formation of a more robust thin free standing structure sheet This also allows for micro actuation for use in devices such as on chip valves and pumps Since parylene is electrically insulative metal wires which serve as heaters and temperature sensors can be placed directly on top of it making thermal management more efficient and temperature measurement more accurate Parylene also has low permeability to gases which prevents evaporative loss of the pcr mixture during thermal cycling Finally parylene is compatible with various MEMS processing techniques which means the usual well established techniques such as metal deposition and photolithography can be used in conjunction with parylene on the wafers Parylene is not a new material It has been used in electronics industry as protective and insulation coatings for circuit boards wiring assemblies and as dielectrics for capacitors because of its resistance to moisture penetration and ability to coat devices conformally It has also been used in the biomedical field as microencapsulation for controlled drug release devices and medical instruments because of its inert properties when interfaced with human tissues It is thus a natural extension to use parylene in MEMS and in particular bio MEMS applications 40 2 4 2 Parylene Chemical Structure The proper chemical name for parylene is poly para xylylene Many variants exist with varying substitutions on the benzene ring three of wh
16. m K Water 0 6 S1 130 Parylene 0 082 Still Air 0 026 3 3 1 2 Convective heat transfer Convective heat transfer or convection 1s a mechanism of heat transfer by which heat is moved by the bulk motion of fluids It is broken down into two categories natural 78 heat convection and forced convection In many cases both these affects are present In natural convection bulk fluid movement occurs due to density differences caused by temperature differences within a fluid In forced convection an external force such as a fan supplies the bulk fluid motion Analytical solutions to such fluid flow with the exception of a few simple cases are extremely difficult thus engineers routinely rely on correlations to quantify the extent of heat transfer using the heat transfer coefficient AQ hAAT Equation 3 2 where AC rate of heat flow h heat transfer coefficient A heat transfer area AT temperature difference between the solid surface and the bulk fluid Thus in convective heat transfer much of the analysis involves choosing an accurate value for h General correlations are available in the form of nondimensional numbers For example if we wish to calculate the heat transfer coefficient for the 2 mm x 2 mm thermally isolated island on the chip we can refer to correlations under similar conditions To use these correlations we must determine the Rayleigh and Nusselt nondimensional numbers 79 Rayleigh number Ra
17. must be biocompatible so it does not interfere with the reaction In particular it must not cause adsorption and denaturalization of DNA molecules or Taq polymerase The surface must also be transparent to allow optical fluorescence detection during thermal cycling Transparency is also convenient for monitoring the fluid while inside the chamber Similarly the surface must not be excessively fluorescent itself otherwise a high background emission may conceal the desired signal The surface must also provide sufficient heat transfer This property is a function of both material and design By applying a thin layer even low thermal conductivity materials can be used Figure 3 14 Bubble trapped in reaction chamber from early chip designs From the design standpoint it is also important that the channels limit the formation of bubbles and facilitate the removal of the formed bubbles Variable cross sections make bubble flushing difficult as fluid simply flows around bubbles even at 75 high fluid velocities see Figure 3 15 The channels should also be sufficiently deep to allow enough optical cross section for measureable fluorescence Quantifying this depth is difficult as many additional parameters such as focusing effects of the channel geometry thickness of the top parylene layer and intensity of the excitation light source contribute to the minimum depth required Device volume is chosen to suit the application Small volumes down
18. of oxygen plasma at 400 watts for about 8 minutes The process was broken into 2 3 minute etching intervals to allow etching progress monitoring and wafer cooling After complete etching the photoresist mask was removed by dissolution in acetone then cleaned with isopropanol The wafer was then exposed to a 200 W 2 min 200 mTorr O plasma to remove remaining photoresist residue The exposed silicon area was dipped into a 10 solution of hydrofluoric acid to remove native oxide and further clean the surface in preparation for XeF gas etching 20 loops of XeF2 gas etching were performed isotropically undercutting the parylene and creating an overhanging structure Each loop consisted of exposure of the wafer to 2 5 mTorr of XeF gas for 1 minute followed by evacuation and an additional 30 sec of exposure from a storage chamber In the regions with the inlet outlet holes the XeF was etched deep enough to form a continuous path with the previously etched inlet outlet holes This can be achieved by designing the process such that the sum of the heights etched by the back side DRIE and front side XeF sum are greater than the thickness of the wafer Second layer parylene deposition and patterning 71 a yi Figure 3 10 Second parylene patterning Underlying oxide is once again the top layer A 16 um parylene layer second parylene was then deposited Since parylene coats substrates conformally the second parylene seals off the gap in
19. silicon etching required towards the end of the process After etching the photoresist 1s stripped using acetone followed by isopropanol Deposit pattern 1 parylene Figure 4 4 First parylene layer with representative holes The holes are actually present throughout the outlined channel region An adhesion layer of A 174 gamma methacryloxy propyltrimethoxysilane was applied then 12 um of parylene is deposited onto the wafer The adhesion layer provides a 107 hydrophobic surface for improved adhesion between parylene and the substrate A simple layer of tape 1s placed on the back side of the wafer to prevent parylene deposition there After deposition 10 um holes are etched into the parylene layer using a photoresist mask and reactive ion etching RIE with an oxygen plasma at 350 mTorr and 65 W applied power for 25 minutes These holes allow the silicon underneath to be later etched away forming the channel Their arrangement and thus channel layout can be seen in Figure 4 4 inlet outlet Front side DRIE XeF2 Figure 4 5 Channels etched into silicon Bottom right is i o hole 108 Using the parylene layer as a mask silicon is etched about 80 um deep using a modified DRIE process followed by isotropic XeF etching These steps allow the formation of the channel Figure 4 5 The inlet and outlet holes bottom right in Figure 4 5 are larger diameter holes that cannot be filled with parylene in the next ste
20. the photoresist becomes increasingly difficult to remove by simple room temperature dissolution in acetone If tall features 20 um already exist on the wafer the flat area photoresist thickness must not only be about twice as tall but care must be taken to ensure good step coverage Out 45 of plane corners caused by such tall features are a site of stress concentration as well as nucleation sites for solvent bubble formation Such bubbles can be reduced by optimizing the resist baking heat flow direction temperatures times and ramp rates To address these limitations a metal film can be used instead or in addition to a photoresist mask A 2000 A layer of metal such as Al Cr or Au is sufficient to etch more than 30 um of parylene Metal masks may not have the required step coverage however if the metal 1s deposited in a thermal evaporation chamber It also requires an additional lithography step to pattern the metal Evaporation of metal itself is a time consuming process due to the high levels of vacuum 3x10 Torr required 2 4 5 1 Parylene Microfluidic Channels One of the most fundamental components of microfluidics is the channel Here two types of channels made from parylene are presented surface micromachined channels and embedded channels surface micromachined channels are built on top of the substrate surface using photoresist as a sacrificial layer The method is outlined in Figure 2 9 below 46 Figure
21. the primer it ends when the polymerase labeled with a P simply runs out of template to replicate For the template molecules this end can be incredibly far from the primer binding region creating extra long products These products when used in the next cycle however produce products whose length is the region between the primers step 4 It can be seen that after the first few cycles the majority of products also called amplicons will contain only the target region while the original templates and extra long products become a minority A more quantitative description will be provided in the next chapter 1 1 4 Equipment The main piece of equipment required to perform PCR is a thermal cycler Such a machine attempts to cycle between the relevant temperatures as quickly as possible In early implementation of PCR the scientist would manually transfer the PCR mixture from one water bath to the next Furthermore before the Taq DNA polymerase came into usage PCR required addition of fresh E coli DNA polymerase after each denaturing step Modern machines have one computer controlled heating block that cycles through temperatures at programmed times The machines also have a heated cover that keeps the caps of the reaction tubes at 105 C This prevents condensation of the water vapor on the cap which in turn keeps the concentrations and pH of the reaction solution constant 13 A typical machine is the Bio Rad MJ Mini Gradient Therma
22. the virus simply includes more g8p proteins into the capsid This property makes the M13 virus a good cloning vector Foreign DNA can be inserted into the M13 genome then transfected into a suspension of E coli If sequencing of the genome with foreign DNA is desired the viral particles can be isolated from the E coli cells by centrifuging the solution and keeping only the supernatant Various mutations of the M13 virus have been produced each containing useful DNA sequences built into the genome Thus usage of this virus allows others to 96 clone pieces of DNA into its genome to be amplified by PCR The target length for this study 1s about 180 bp 3 5 2 Thermal Cycling Protocol 94 72 55 30 s each Although not optimized the following temperature protocol was used e 95 C for 15 seconds e 55 C for 15 seconds e 72 C for 15 seconds Each temperature transition used 5 seconds Further reduction in temperature soaking times can be made experimentally especially denaturation time A reduction in transition times can be made by optimizing different PID control constants Temperature Control First 10 Cycles Temperature Control Typical Cycle O 85 o o 5 Temperature ET temperaiurs o x Desired o Desired a 65 Temperature E Temperature o o 55 a 0 20 40 60 Time seconds Time seconds Figure 3
23. time PCR Journal of Environmental Monitoring 8 153 160 2006 P Same as ref 12 l4 B Liss Improved quantitative real time RT PCR for expression profiling of individual cells Nucleic Acids Research 30 17 pp e89 2002 P Image from Hitequest website www hitequest com l6 S A Campbell The Science and Engineering of Microelectronic Fabrication Second Edition Oxford University Press 167 2001 17 Image from Connexions website http cnx org 18 M Madou Fundamentals of Microfabrication CRC Press 1997 I G T A Kovacs N I Maluf and K E Petersen Bulk Micromachining of Silicon Proceedings of the IEEE 86 8 Aug 1998 127 G Beheim Chapter 21 Deep Reactive Ion Etching for Bulk Micromachining of Silicon Carbide The MEMS Handbook Chief Editor M Gad el Hak CRC Press 2002 1 p Wilding M A Shoffner and L J Kricka PCR in a Silicon Microstructure Clinical Chemistry 40 1815 1818 1994 2 J Chen M A Shoffner G E Hvichia L J Kricka and P Wilding Chip PCR II Investigation of different PCR amplification systems in microfabricated silicon glass chips Nucleic Acids Research 2 2 1996 3 M A Burns B N Johnson S N Brahmasandra K Handique J R Webster M Krishnan T S Sammacro P M Man D Jones D Heldsinger C H Mastrangelo and D T Burke An Integrated Nanoliter DNA Analysis Device Science 282 484 487 1998 T Erill S Campoy N Erill J Barbe and J Aguilo Bioch
24. to SYBR Green I are the family of TaqMan probes These have the advantage of fluorescing only during the synthesis of the target DNA thus adding another level of specificity A schematic of the TaqMan probe system is shown in Figure 1 8 Denatumne Ane f E E Pa E mMm WITITIT Extend Figure 1 8 Schematic of TaqMan probes In its free state the green and red fluorophores are connected via the DNA bases between them These bases are designed to be complementary to a sequence within the target DNA The proximity allows Forster resonance energy transfer FRET to occur the energy from the optically excited green fluorophore donor molecule transfers to the red fluorophore acceptor molecule which accepts and dissipates the energy as heat or 19 light in a longer wavelength During the annealing step of RTPCR the TaqMan probe binds to its complementary sequence on the target DNA step labeled as anneal above During the extension step as Taq DNA polymerase extends the target DNA it destroys the seemingly disruptive Taqman probe as it synthesizes the new strand This allows the green fluorophore to become spatially separated from the red quenching molecule thus emitting its green photons instead of participating in FRET This increase in fluorescence is then measured by the RTPCR machine This mechanism is key to the added specificity provided by the Taqman system If the target DNA does not exist the probes will not bind a
25. to tens of nanoliters have been reported which can be useful for highly parallel arrays of PCR reactions in small areas or when sample volume itself is small e g DNA from a small number of cells Smaller volumes also mean lower materials consumption lower materials cost and faster thermal cycling Large volumes on the order of tens of microliters however are still important even in chips Detection of very dilute samples and off chip gel electrophoresis are two examples where large volume is advantageous In this case the surface area to volume ratio is reduced which reduces material surface interference effects Figure 3 15 Channel layout Figure 3 15 shows the channel layout for the QPCR device Parylene is chosen as the surface material because it 1s known to be biocompatible and deposited using 76 methods that are compatible for current MEMS processes It can also be deposited in thicknesses from 0 1 30 um which is thin enough to allow good heat transfer and limit effects of auto fluorescence A serpentine design was used rather than a large chamber design to reduce bubble formation and facilitate bubble flushing should any trapped air appear during sample introduction Designs with large chambers changes in channel cross sectional area allow bubbles to become trapped in the chamber as fluid flows around the bubble The channels are 110 um deep 60 um wide and about 8 mm long making the total sample volume
26. triphos phate R dR Uracil Thymine Figure 1 2 Chemical structure of nucleoside triphosphates The two DNA primers are short 20 100 bases single stranded pieces of DNA that flank the target region within the DNA template See Figure 1 3 and Figure 1 4 for an illustration as to why the primer pair defines the target region When they bind to the template they define the starting point of amplification The starting point of one strand is the ending point of its complimentary strand Thus after a few cycles all the products start and end at the DNA primers These primers can be synthesized in house or easily obtained from a vendor such as Integrated DNA Technologies Design of these sequences can be complicated but software such as MIT s Prime3 exists to facilitate the primer design process Some guidelines are given below o The sequences should be designed such that the target DNA 1s contained between the primers and is about 100 1000 base pairs bp in length o Sequence length should be about 15 30 bases long Sequences that are too short lack specificity To illustrate this point consider that the complementary sequence of a primer of length 1 can be found in 1 4 of the sequences of length 1 For length 2 about 1 16 of the length 2 sequences are complimentary For length 16 only one in set of 4 3 X 10 45 about the length of the human genome random 16 mers will match Thus sequences should be larger than abou
27. used underneath AZ 1518 for metal lift off applications The undercut ensures the metal film will be discontinuous at the features thus lifting off cleanly Without the LOR3B layer the metal may provide good step coverage over the thin AZ 1518 resist resulting in a continuous layer metal After hard baking the wafer for 2 hours at 120 C metal was deposited using an electron beam thermal evaporator A tri layer of 30 nm titanium 200 nm platinum and 200 nm gold was deposited The lift off resist layers were then dissolved in ST 22 photoresist stripper a commercial product 66 based on N methyl 2 pyrrolidone NMP This results in a patterned tri metal layer A serpentine design was used to obtain the desired 2 kOhm resistance in a small area The gold is subsequently patterned using photoresist and gold etchant to remove gold everywhere except contact pads for improved adhesion between the metal and the gold wires used in wire bonding The platinum layer serves as the heater and temperature sensor while the titanium 1s for better adhesion to silicon oxide DRIE trenches for sidewalls and stitches Channel sidewall Stiches Figure 3 5 DRIE etching of the bulk silicon The sides of the channels and the slots where parylene will fill and make stitches are etched The parylene stitches and channel sidewalls were defined simultaneously by etching the silicon wafer 100 um deep using deep reactive 10n etching The sidewalls are defined
28. 009 Denver Colorado 2009 35 i Paratronix website Wwww paratronix com J Shih T D Lee and Y C Tai Surface Machined Parylene Microfluidics Lab on a Chip Technologies and Applications Horizon Scientific Press UK 2008 3 E Meng and Y C Tai Parylene Etching Techniques for Microfluidics and BioMEMS Proceedings of the IEEE International Conference on Micro Electro Mechanical Systems MEMS 2005 Miami Florida pp 568 571 2005 38 R R A Callahan G B Raupp and S P Beaudoin Effects of gas pressure and substrate temperature on the etching of parylene N using a remote microwave oxygen plasma Journal of Vacuum Science and Technology B 19 725 731 2001 K Walsh J Norville and Y C Tai Photoresist as a sacrificial layer by dissolution in acetone Proceedings of the 14 IEEE International Conference on MicroElectroMechanical Systems MEMS 2001 Interlaken Switzerland p 114 117 2001 J Xie J Shih Q He C L Pang Y C Tai Y Miao and T D Lee An integrated LC ESI Chip with Electrochemical based gradient generation Proceedings of the 17 IEEE International Conference on MicroElectroMechanical Systems MEMS 2004 Maastricht The Netherlands 2004 P J Chen C Y Shih and Y C Tai Design fabrication and characterization of monolithic emb edded parylene microchannels in silicon substrate Lab Chip 6 803 810 2006 129 C Y Shih Y Chan and Y C Tai Parylene strengthended the
29. 084 mm or 8 4 um This value represents an effective distance h from the surface of the parylene sheets within which PCR was inhibited Thus the minimum radius for parylene C microchannel to be a reaction vessel for RTPCR is 8 4 58 um It should be emphasized that this is an effective metric and no physical effect is claimed here 3 d 0 o E O gt 800 1000 1200 1400 1600 1800 SA Added um2 Figure 2 18 Concept of an effective distance h in which PCR is inhibited 59 2 4 6 3 QPCR with Other Materials ral a m L a LL Figure 2 19 QPCR with various S A volume ratios of Parylene HT Parylene HT has lower auto fluorescence thus provides lower background fluorescence for superior optical signal Its interaction with the components of the QPCR solution however made it a less attractive material As seen in Figure 2 19 even small S A Vol ratios of 3 6 mm and 14 6 mm of Parylene HT had a noticeable adverse effect on QPCR At 58 9 mm similar to the chip s ratio QPCR was totally inhibited At has been shown that perfluoroalkoxy a highly fluorinated hydrophobic material can absorb DNA and SYBR Green The similarly highly fluorinated Parylene HT might inhibit QPCR by a similar mechanism as they both have a contact angle of about 105 Adsorption of Taq 60 Polymerase is also a possibility Thus from a biomaterials standpoint untreated and unpro
30. 18 20 22 24 26 28 30 32 34 36 38 40 42 Cyde Figure 1 12 Serial dilutions to determine sensitivity of assay 25 To test for specificity the authors performed the same assay on different types of fungi Figure 1 13 The various species of Cladosporium gave a typical signal while other types of fungi showed virtually no signal 10000 10000 r 9000 d 9000 z 8000 7000 7000 6000 6000 5000 5000 4000 3000 4000 3000 PCR Base Line Subtracted CF RFU 2000 2000 1000 1 1000 Ors 0 1000 1000 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 Cycle Figure 1 13 Assay specificity 1 2 5 2 mRNA Expression Profiling As described in the earlier section real time PCR allows quantification of initial template DNA amount when compared against a calibration curve An extension of this technique is to quantify the amount of specific messenger ribonucleic acid mRNA expressed in a cell or tissue by first reverse transcribing the mRNA into complementary DNA cDNA This technique is referred to as reverse transcription quantitative PCR RTqPCR Some authors also call this technique RTPCR so care must be taken to distinguish between real time PCR and reverse transcription PCR Upon stimulation cells will undergo a signal transduction process resulting in the transcription of mRNA which travels from the nucleus where the DNA is into the cytoplasm to be translated into proteins and enzymes Qua
31. 2 9 Surface micromachined parylene channel An outline of the 6 steps shown above The substrate s surface is properly cleaned and treated An adhesion layer such as A 174 gamma methacryloxy propyltrimethoxysilane the roughening of the lower level or a molten layer of parylene may be used to improve adhesion First layer of parylene is deposited forming the bottom of the channel This layer can be thin 2 um however should be sufficiently thick 15 um if the underlying substrate will be eventually removed for a free standing channel Sacrificial photoresist 1s deposited and patterned This step is possible because adhesion between photoresist and parylene is good For many cases this resist must be baked at high enough temperatures and long enough times to evaporate any remaining casting solvent or water from development to prevent bubble formation in later steps 47 A second layer of parylene is deposited forming the top of the channel For improved adhesion a thin portion of the first parylene layer can be etched away using an oxygen plasma This serves two purposes 1 it cleans the surface by etching organic contaminants such as residual material from the photoresist processing and lifting off inorganic contaminants in the top layer and 2 it leaves a rough surface with increased surface area for better adhesion Following the plasma treatment a 20 second dip in a 10 HF solution is performed to clean off any remai
32. 27x 22 8 F 0 9995 75 65 O0 Steady State Temperature C 45 35 0 20 40 60 80 100 Applied Power mW Figure 4 14 Thermal resistance to heat transfer into environment 4 3 3 2 Thermal Capacitance To determine the thermal capacitance heating and cooling experiments were performed For the cooling experiments the zero applied power model was used U t ZU eR Equation 4 6 Using this model a thermal time constant T RC was fitted to the data in Figure 4 15 The fitted model below uses t 3 seconds corresponding to a thermal capacitance C 3 25 mJ C Using published data on specific heat capacities of water and parylene the calculated thermal capacitance was 3 03 mJ C which is fairly consistent with the experimental results 119 These same parameters also fit the heating experiments well Figure 4 16 maintaining a consistent model for both cooling and heating data In this case the zero initial temperature difference case of Equation 4 2 was used U r JRQ e C Equation 4 7 4 4 Interface with Housing To interface with the new chip design modifications were made to the housing unit Using the same CNC machining techniques and materials described in the previous chapter a housing unit was designed and fabricated with back side fluidic inlet outlet ports along with back side electrical contact pins Figure 4 17 To prevent the pins from penetrating the thin platinum and paryle
33. 31 Temperature recipes As seen in Figure 3 31 the temperature control was quite good as there was good agreement between desired and actual temperatures 977 3 5 3 Optical Detection Protocol 3 5 3 1 Background Optical measurements were performed using a fluorescence microscope and the SYBR Green fluorescent dye With this dye fluorescence increases as the amount of double stranded DNA increases 3 5 3 2 Equipment Fluorescence measurements were made using a microscope CCD camera and image analysis software The microscope was a Nikon Eclipse E800 fluorescence microscope with a mercury arc lamp USH 102DH and Nikon B 2E C filter block see Figure 3 32 The excitation filter allows wavelengths of 465 495 nm to pass to the sample while the emission filter allowed wavelengths 515 555 nm to pass from the sample to the camera A long pass dichromatic mirror with cut off wavelength 505 nm was used to control light paths c B 2E C Medium Band Blue Excitation 5 8 amp Transmission Percentage e 35 450 550 650 750 Wavelength Nanometers Figure 3 32 Filter block for SYBR Green I detection 98 The CCD camera was an RT KE from Spot Diagnostic This is a one CCD based camera with an LCD based optical filter that cycles between red blue and green for color images Thus each image is the composite of 3 separate images taken in succession For optical measurement purposes only one LCD mode was used
34. 50 nL EHT 10 00 kV SignalA SE2 Date 28 Jun 2007 WDs 25mm Photo No 6657 Time 19 22 20 Figure 3 16 Channel cross section 3 3 Device Thermal Engineering 3 3 1 Heat Transfer Background The three heat transfer mechanisms are conduction convection and radiation Radiation is not relevant for this thesis and will not be discussed 3 3 1 1 Conduction Conduction occurs through movement of molecules and their electrons on the molecular scale Conductive heat transfer 1s described by Fourier s law TT db kVT Equation 3 1 Where q is the heat flux k is the material thermal conductivity and T is temperature If the material is anisotropic k becomes a tensor In isotropy it is a scalar This equation is useful in thermal engineering as a low k material is an insulator preventing heat from flowing despite a large temperature gradient while a high k material is thermally conductive allowing heating to easily flow thus creating a more homogenous temperature distribution Thermal conductivities of materials important to this thesis are given in Table 3 1 It should be noted that the thermal conductivity of parylene is only 3 15 times that of air and 6x10 times that of silicon making it a relatively insulative material for a parylene based chip system Silicon on the other hand maintains a homogenous temperature distribution in this work Table 3 1 Thermal conductivity of selected materials Material k W
35. 8p T T I7 Equation 3 3 Vo The Nusselt number L Nu i Equation 3 4 We know that the island cycles temperatures between 95 C and 55 C in air at a temperature of 23 C For this situation definitions and values for the parameters for the nondimensional numbers are given below Table 3 2 Values for calculation of Rayleigh number for air Variable Definition Value Symbol g gravitational acceleration 9 8 m s p thermal expansion coefficient 3 1x10 C V kinematic viscosity 1 8x10 m s a thermal diffusivity 2 7x10 m s Ts temperature at the solid surface 75 C 95 55 2 ds room temperature of air 23 C Liop bottom ratio of solid s surface area to perimeter top bottom 5x10 m sae ratio of solid s surface area to perimeter sides 2x10 m k Thermal conductivity of air 27 8x10 W m C From Equation 3 3 the Rayleigh number is Ra 0 41 for the top and sides and Ra 0 026 for the sides Using empirical correlations such as those found in the CRC Handbook of Mechanical Engineering this Ra correlates to various Nusselt numbers depending on the surface orientation 80 Table 3 3 Nusselt numbers All correlations from CRC Handbook Surface Nu h Top surface 2mm x 2mm 1 23 68 3 W m C Bottom surface 2mm x 2mm 1 15 64 1 W m C Side surfaces 0 5 mm x 2mm x 4 0 842 117 W m C By scaling each heat transfer coefficient with its area an overall coefficient can be found overall 83 W m C 3
36. 9 nm diameter with a cylindrical shape V E hal A LS f AA LA DA AN N 3 e v Figure 3 29 Structure of the M13 virus It is categorized as an Ff phage because it requires the E coli F pilus for infection The bulk of the capsid 1s composed of about 2800 copies of a 50 amino acid polypeptide protein derived from gene 8 g8p This protein has an alpha helix structure with three domains The hydrophilic amino end is negatively charged comprised mostly of acidic 95 amino acids and forms the outer surface of the virus The inner surface is positively charged so that it 1s stable next to the negatively charged DNA genome This charge is a result of basic residues near the carboxyl terminal The middle region 1s hydrophobic which allows interactions with other g8p proteins to form a stable membrane structure These hydrophobic interactions are key to the use of the M13 virus as a model virus for PCR At 95 C the thermal energy is high enough to disrupt these interactions causing lysis of the virus and allowing its DNA genome to participate in the PCR reaction Figure 3 30 Genome of the M13 virus The capsid has an interesting plasticity If the genome is artificially made to be longer by insertion of DNA into the non essential intergenic regions of the genome the resulting capsid is automatically assembled longer to accommodate this longer genome This is possible because of the modular design
37. A Parylene Real Time PCR Microdevice Thesis by Quoc Brandon Quach In Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy California Institute of Technology Pasadena California 2010 Defended December 4 2009 2010 Quoc Brandon Quach All Rights Reserved ii To Cuong Quach and Nga Huynh 1V It never ceases to amuse me that I was once a 7 year old weekend factory worker a 10 year old dry cleaner and now a Caltech PhD graduate all within a 10 mile radius Cheers to the American Dream Acknowledgements I would like to acknowledge my parents whose courageous journey as Chinese refugees from war torn Vietnam to the United States serves as an inspiration for everything I do in life Their dangerous flight in small boats off the coast of Vietnam under the cover of darkness followed by years of hard work raising a family in uncertain conditions as immigrants to the United States was all done to provide a better life for their family and especially their children It is from my desire to show my deep appreciation and endless gratitude that I have been able to produce and present this thesis and earn my PhD from Caltech To my parents such 1s the level of accomplishment I have been able to achieve from the sacrifices you made on my behalf Right next to the turtle pond Guggenheim Laboratory is a special place on campus for me Armed with only a dream and a desire to achieve it
38. One convenient way is get this information 1s to analyze an area of the image where quantum yield is constant region B in Figure 3 33 Figure 3 33 SYBR Green fluorescence in microchannel In region B Equation 3 13 still applies and since is constant relative I can be obtained by directly looking at relative X r gx I X Equation 3 15 I x TX where I Fluorescence intensity at time t Io Fluorescence intensity at time 0 first cycle of PCR X Excitation intensity at time t Xo Excitation intensity at time 0 qx Qo constant quantum yield 101 Now if regions A and B are spatially close enough and the intensity distribution of light is fairly uniform we can assume X XP This assumption links our equation together to obtain A J I E 2 E Equation 3 16 0 0 t 3 5 4 Results A raw sample of M13 virus from ATCC was detected using the qPCR device Normalized Fluorescence 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 Cycle Number Figure 3 34 Detection of M13 virus Data normalization described above Normalized Fluorescence The no primers control and M13 virus sample are distinguishable Although the data shows proof of concept some imperfections exist linear and not exponential This might be caused by incomplete reactions in each step Although parylene is known to be an inert surface extensive studies on the effect of parylene on micro PCR reaction
39. QPCR with glass added into reaction tubes sseeeessussess 60 Figure 312 Overall process HOW Sousa Scu pda tpm IU DURER mU DU mu e m seewtededawn 62 Dieure 32 5 16 ON CMI congesta d opu lind t aa de aia ERU a de sere Ga eso cet pM dei Du 63 Figure 3 3 Patterned oxidation layer essen 64 Figure 3 4 Metal deposition and patterning Oxide layer purple underneath the metal orange acts as relectrical THSUTAEOE ons eet tt ho dmt matti b Dd tod m ews 65 Figure 3 5 DRIE etching of the bulk silicon The sides of the channels and the slots where parylene will fill and make stitches are etched esesessssssss 66 Fig re 3 6 Tirst parylene deposi Mise in paese euet ieu et etetu s Up Re Me t tens E tu aiu eR Iud 67 XIV Figure 3 7 Inlet outlet formation Notice the back side etching shaded in brown overlaps the channel etching region ensuring a continuous path when the channel 1s cela cod m E C T P A E E 68 Figure 3 8 Etching of first parylene layer light blue and XeF etching of underlying SUT RII PR rtr 69 Fissure 329 Inlet outlet holes edt dob i bia baa detta de tllo a aad de 69 Figure 3 10 Second parylene patterning Underlying oxide is once again the top layer 71 Figure Sail At Sap TOP MAU ON eoe hire oae n Eee NS 74 Figure 3 12 Zoom showing the parylene stitches used to connect the island to the main Figure 3 13 Wire bonding on the completed chip
40. The wire bonds provide electrical continuity across the parylene stitched air gap eessssssseeeeeernnnn 73 Figure 3 14 Bubble trapped in reaction chamber from early chip designs 74 Fiure oslo Channel Ay QUU st ea a dp Qe n Sum me Reed Mee 75 Figuren lo Channel Cross SecHOTI os sen cma sas o oot adde A aa 76 Figure 3 17 EE analogue for thermal characterization see 80 Ligure 2 L5 Thermally slated 15 lan doi iiri nce ete d endo e e desde e esa eost 82 Pigute 25 19 Paty le tie Sti Decus dots 19311 dna at edo m altace at Gaede 83 19ure 5 20 Extended RC Mode lisica RENM 83 Figure 3 21 Temperature sensor calibration esses 85 Figure 3 22 Temperature control hardware arrangement cccccccceeeeeeeeeeseeeeeeeeeees 87 F1eure 2929 Steady State tempera e sto n vo beo t tete muet ed apa e duS 88 Figure 3 24 Heating with step function applied power cccccccccecceeeeeeeeeeeeeeseeeeees 89 XV Figure 3 25 Temperature cooling dynamic with zero applied power 90 Figure 5 26 Chip housing assembly 25 ooi et Vioc E iesu oc Eg Vaud oun 9 Figure 2 27 C ip hous me compone Bs 5 599 9992 0 9 c minata da ad orDu coastal wi oae Ra 92 Figure 3 28 Chip housing with external valves sese 93 Figure 3 29 Str cture or the MTS VIUS iique ves eroe eaa taada rorat 94 Figure 3 30 Genome of the M ES Vitus
41. acrificial layer If the finished device uses only surface methods the substrate may be a cheaper material such as polycarbonate If polysilicon is used as a structural layer biocompatibility will be poor requiring surface treatments Flexibility and ability to integrate movable parts will also be difficult An alternative set of materials are parylene for the structural material and photoresist as the sacrificial layer This scheme will be explored in detail in the next chapter 2 4 Parylene MEMS Technology 2 4 1 Why Use Parylene There are many reasons to use parylene to make microfluidics devices A brief list of advantages will be given here followed by more detailed discussion in the sub sections Parylene is a biocompatible material as shown by its USP Class VI classification which means the FDA has previously approved its usage in long term human implants This 39 elite classification translates well into the biochemical arena as it has been shown as a good material for in vitro experimentation as well Parylene is chemically inert able to withstand various solvents ranging from organic to acidic This is convenient for testing purposes as well as during device fabrication where organic solvents are used for example to dissolve photoresist Parylene is optically transparent allowing direct measurement of fluorescence signals a requirement for real time PCR It is flexible Young s Modulus 4Gpa rather than brittle allowing
42. adtlee toda a E aie Ge en a 93 S Real Time Polymerase Chain Reaction Components 93 3 5 2 Thermal Cycling Protocol 94 72 55 30 s each sssses 96 3 5 3 Optical Detection Protoe OL eie eo eaaet uo Eon n Ae noe ode Luci guttas en Eds 97 3 5 4 Roo ane ee Nue LM MEL LI eM D D ECT 101 Xl 3 6 Barre desinis tr c 103 4 RIPCR Microdevice Free Standing Version 104 4 DLabric adosados donatos ene cree ee era nee er ay ree nee ec ba ete 104 4 2 Pidie channel DeSIOfl 2 itor rotto eode ies e eo dete Soie ttas ee into 112 4 3 Device Thermal Bne9Ttice EID ince nueve D etae E lee se T 112 4 3 Heat Prans ler Bae ke routid seseris n Open i M een 112 4 3 2 Device Therma DE SIDI zoe neoa A 112 4 3 3 Thermal Performance Results eoe a Diog teens 116 44 Interface With MOUS ise oett innui eta deti ne tenuit ehe de eu 119 4 5 Device Pertform atioe oet d a Eos out E Quae ite de 122 4 5 1 Real Time Polymerase Chain Reaction Components 122 4 5 2 Thermal velimbs ProtOCOl ns eoe bera pte rite ebbe S 122 4 5 3 Opca Detection Protocol koirienne ee eo ELE EE trn eese 122 4 5 4 Results dnd DISCUSSIOTI iut oor e evo on Hugo du totu S 123 4 6 CaP er SUA y E m T I m 124 5 COBCIUSIO 62 009 iae vercoveesavesssvesssesesesesevs 12D References eee ee eee eee ee ee eese se sesesesecccseseseses 1 20 Xll List of Figures
43. aine and DMSO Enhancing Agents for PCR Promega Notes 65 p 27 1998 gt http www microbiologybytes com virology Phages html 130 D Greenstein and R Brent Vectors Derived from Filamentous Phages Current Protocols in Molecular Biology 1 14 1 1 14 5 1990 W S Rasband ImageJ U S National Institutes of Health Bethesda Maryland http rsb info nih gov 1j 1997 2007 F Kreith Editor in Chief The CRC Handbook of Mechincal Engineering Section 4 16 1998
44. art polymerase Invitrogen s Taq polymerase contains anti Taq polymerase antibodies that serve this function One major disadvantage of Tag polymerase is the lack of a proofreading ability resulting from random errors in replication about 1 per 10 nucleotides Since the errors are random they are not significant for most applications of PCR since a given site has an overwhelming number of correct nucleotides compared to the erroneous ones For applications where even slight errors are not tolerable DNA polymerase from other organisms such as Pyrococcus furiosus and Thermococcus Litoralis can be used The buffer solution maintains the optimal pH and salt concentration values for efficient amplification This solution is often purchased from a vendor in a pre mixed form It is typically a Tris HCl buffer system around pH 8 4 with KCl and some MgCl already added Of particular importance is the concentration of Mg ions that are often supplied separately since each reaction condition requires a different concentration The ions function as a cofactor for Taq polymerase and also enhance the ability of primers to bind to the target template DNA Excess Mg however causes Taq DNA polymerase to become more error prone The buffer solution may also be tested for the absence of DNase and RNase enzymes that degrade DNA and RNA respectively 1 1 2 Procedure The PCR reaction solution is assembled to a total of 20 100 uL in a plas
45. ate a photosensitive photoresist layer 1s deposited This 1s often performed by placing the substrate on a spinner pouring the photoresist suspension onto a wafer then spinning between 1000 8000 rpm depending on the desired thickness and viscosity of the suspension The casting solvent is then removed by evaporation in an oven or hot plate 29 Exposure A light source such as a mercury lamp is used to supply energy while a mask is used to supply the pattern This mask itself is usually a glass plate with patterned chromium as the reflecting layer The photoresist reacts to the light in a way that changes its solubility in a developer solution One example of this process is the DON family of photoresists They are comprised of a photoactive diazoquinone ester DQ and a phenolic novolak resin N Upon exposure the DQ undergoes a photochemical reaction that makes the DON soluble in a basic developer solution while the unexposed regions remain insoluble Some types of resists also require a post exposure bake to speed up reactions that initiated during exposure Development During this step the wafer 1s exposed to a solution that selectively dissolves only the portions of the photoresist that has been exposed to UV For negative photoresists the portions that were not exposed are dissolved The wafer 1s then rinsed and dried resulting in a patterned photoresist layer At this time the resist 1s often hard baked or post baked by placin
46. ate is a 100 mm diameter silicon wafer 500 um thick oriented in the 1 0 0 direction and polished on both sides 64 Figure 3 3 Patterned oxidation layer Thermal oxidation and metal patterning A 1 um layer of thermal oxide was grown in a furnace at 1050 C The oxide layer was then patterned using 1 um of AZ 1518 photoresist as the mask It was etched for 8 minutes with buffered hydrofluoric acid The buffered acid was used instead of diluted hydrofluoric acid for a more consistent etching rate The oxide layer was preserved everywhere except the locations of the channels and air gap since their formation involves etching the bulk silicon After etching the photoresist was stripped by submersing the wafer in an acetone bath followed by an isopropanol bath 65 For metal patterning a lift off technique was used A 0 5 um layer of LOR 3B resist was spun onto the wafer then baked at 170 C for 10 minutes on a hotplate Serpentine heater Contact nad Figure 3 4 Metal deposition and patterning Oxide layer purple underneath the metal orange acts as in electrical insulator A um layer of AZ 1518 photoresist was then spun onto the wafer on top of the LOR 3B Both layers were then patterned by UV exposure and development in AZ 351 photoresist developer LOB 3B is a lift off resist based on polydimethylglutarimide PMGI that dissolves isotropically in AZ 351 photoresist developer solution providing desired undercut when
47. be provided followed by the procedure equipment and applications 1 1 1 Components To prepare the reaction the components of PCR are mixed together in a thin walled for improved heat transfer test tube Oftentimes this tube will be certified as DNAse free to prevent degradation of the template and products by the DNAase enzymes The components are commonly placed inside a bucket of crushed ice to minimize reactions while mixing Below is a list of components The order of the list does not reflect the order in which reactants are added e The DNA template is the original source DNA that contains the target region This can be as simple as a synthesized strand of single stranded DNA or the entire genome of an organism In reverse transcriptase PCR the template is a strand of RNA which before the reaction starts is reverse transcribed by the reverse transcriptase enzyme into the corresponding complimentary DNA cDNA Depending on the assay some sample preparation may be required to obtain a suitable template material For example tissues have to be treated to access cells which then are lysed into a suspension that undergoes DNA extraction using a glass solid phase chromatography Care must also be taken to ensure contaminants from the sample preparation steps do not inhibit the PCR reaction e Deoxynucleoside triphosphates dNTPs are the monomeric building blocks of DNA The standard set of dNTPs used consists of deoxyadenosine tri
48. ber c Thus enh C Equation 1 6 logd E logd E If m is the slope of a c versus log To plot the efficiency E can be calculated m E 10 Equation 1 7 logd E 1 2 4 Equipment There are many manufacturers of real time PCR machines including BioRAD Applied Biosystems Roche Cepheid and Strategene The entry level models from these manufacturers are very similar most featuring an LED or halogen light source 23 peltier based heating and cooling CCD or photodiode photodetectors with rotating filter wheels and a thermal block that fits standard 48 or 96 well plates These machines typically cost around 30 000 weight 20 kg use max 10 Amps at 120 VAC and have a length scale of about 40 cm Temperature ramping times are typically 10 C second and results are obtained in about 1 5 hours Some companies seek to differentiate their machines with slight modifications The Roche Light Cycler 2 0 has a rotating carousel of capillary tubes instead of a 96 well plate thermal block In this design temperature distribution is more uniform as a fan blows heated or cooled air past the rotating carousel The Applied Biosystems StepOne model is a standalone unit that does not require a computer and has its own touch screen interface The Strategene MX 3000P and MX 3005P models shown below feature a scanning photodetector unit comprised of a fiber optic cable leading to a photomultiplier tube wi
49. ble 3 5 3 3 3 2 Heating Rates Analysis of time dynamics of temperature were performed using data from heating and cooling experiments For heating the island is heated from room temperature by applying heat as a step function 120 100 e e e 90 oe 9 9 99300 PT 80 l g ae o Pu L 25 e e e eoe 4 Time s e Temp Leak Model Applied Pow er Figure 3 24 Heating with step function applied power Here the important parameters are the thermal capacitances 1 e heat capacity of the island and the body These were calculated using known values for specific heat and multiplying by the appropriate mass Values are shown below 90 Table 3 5 Parameters used for thermal model R from island to room R from body to room 0 131 R from island to body Re 0 175 Heat capacity of island Heat capacity ofbody n7 3 3 3 3 Cooling Rates Analysis of temperature drop with zero applied power shows the island can drop from the denaturing temperature 95 C to the annealing temperature 55 C in about 1 5 seconds 100 350 9999 90 300 804 N 70 bw 250 D E ve 200 a 50 na eee 4 S 150 F I 40 30 100 20 50 10 0 e o o o 90 1 0 5 0 0 5 1 1 5 2 2 5 3 Time s Temp Leak Model e Applied Power Figure 3 25 Tempe
50. bombard the substrate High densities mean the plasma is more chemically reactive while lower kinetic energy means nonspecific mechanical etching erosion of mask materials is reduced This means higher specificity which allows for thinner masking materials which then allows better in plane patterning of mask materials since aspect ratios are reduced 35 To transform the silicon trenches into channels the fourth wall must be introduced This usually occurs by bonding the silicon to either another silicon or glass wafer A common technique is anodic bonding where the silicon 1s placed in contact with a special high sodium glass at 200 500 C and a high DC voltage kV across the bond This combination allows the sodium ions in the glass to migrate away from the interface causing a negative charge on the glass side and a positive charge on the silicon side Electrostatic force then holds the two pieces in place to create a water tight seal Early pioneers using this technology to create PCR devices include Peter Wilding et al In these early chips the silicon was etched about 40 80 um deep and anodically bonded to Pyrex glass to create reaction chambers between 5 10 uL An external Peltier heater and cooler was used for thermal cycling M A Burns et al published an early integrated DNA analysis system with PCR chamber elctrophoresis and optical detector by etching glass slides and bonding them to the electrical and optical component
51. cessed Parylene HT would not be a good choice for a QPCR material b T E D c o e E B 3 Z E 8 n E S iL Figure 2 20 QPCR with glass added into reaction tubes Glass was tested for QPCR inhibition Figure 2 20 Compared to the curve with various amounts of parylene the glass curves have noticeable shifts to the right These shifts resemble the phenomenon observed when less templates are added into a QPCR reaction Thus it appears that glass is causing the adsorption of template DNA molecules onto its surface This is consistent with the fact that glass is used to bind and purify DNA in chromatography format although those occur in different salt and pH values Thus it seems materials that are too hydrophilic such as glass contact angle 15 or too hydrophobic such as parylene HT contact angle 105 inhibit PCR whereas 6l the slightly hydrophilic Parylene C and polypropylene have the least inhibitory effect Based on current literature it is postulated that hydrophilic materials cause the adsorption of DNA and Taq polymerase whereas hydrophobic materials cause adsorption of Taq polymerase and SYBR Green Although these hypotheses are offered by the author based on these simple experiments definitive rules and literature are lacking in this area of materials interaction with QPCR 2 5 Chapter Summary Parylene MEMS technology has been shown to enable fully integrated microfluidic devices becau
52. device visible from the back Figure 4 8 right side Back Figure 4 9 Finished chip front and back The finished chip is shown schematically in Figure 4 9 Here the channels are shown in green as they appear during testing with green fluorescent dyes Optical measurements are performed on the back side of the device so that the platinum layer serves as a reflector along with its normal roles as heater and temperature sensor for increased optical efficiency 112 4 2 Fluidic Channel Design Requirements for a PCR chamber were discussed in the previous chapter Like the previous version of the device this design features a constant cross section channel for easy removal of bubbles by flushing with high velocity liquid should they appear The effective radius of the channel is 75 um with a total length such that the total volume of liquid contained within the amplification region 1s about 550 nL 4 3 Device Thermal Engineering 4 3 1 Heat Transfer Background Please refer to the previous chapter for a theoretical introduction to the heat transfer principles used in this work 4 3 2 Device Thermal Design Since the theoretical framework for thermal design was provided in the previous chapter only quick explanations pertaining to differences between this version and the previous are discussed here Unlike the previous version which had a silicon support piece this version has free standing channels with no support This m
53. e uses the same setup as the previous version so please refer to the previous chapter for details 4 3 3 Thermal Performance Results The simple RC circuit analogue described in the previous chapter is used to describe the thermal results Figure 4 13 Simple circuit analogy 117 U t Ue hc T JR I e RC Equation 4 2 l hA Equation 4 3 C pC V where h heat transfer coefficient A heat transfer area p density of the solid C heat capacity V volume of the solid Chip characterization can be performed by analyzing some special cases At steady state too Equation 4 2 reduces to U t 2oo JR Equation 4 4 Data from this case can be analyzed to obtain the thermal resistance R Another interesting case occurs when applied power is zero J 0 which reduces the equation to DRE e RC Equation 4 5 With this equation one can deduce the thermal capacitance C by analyzing the rate of cooling Both these analyses will be discussed below 4 3 3 1 Steady State Temperature Resistance The resistance to heat transfer into the environment in this case 1s the slope of the line in Figure 4 14 R 1 hA 0 923 C mW Thus for an area of 25 mm 12 5 mm on each side the overall including top and bottom heat transfer coefficient h 43 5 W m C 118 This is in good agreements with the calculated heat transfer coefficient of h 46 W m C based on empirical correlations 95 4 85 y 0 92
54. ectrical and computational devices has inspired an analogous effort in the mechanical and biological realms using similar technology Since MEMS was born from microelectronics both fields share similarities in materials and patterning technology Recently however with increasing interest in biological assays in MEMS new materials and methods are being introduced that are more biocompatible 2 2 General Microfluidics Technology At the core of microfluidics technology is photolithography using an energy beam to pattern thin photosensitive films called photoresists The energy beam can be composed of photons light UV electrons X ray photons or even ions In this work 28 only UV photons are used The thin films used depend on the energy source but their general principle 1s shown in Figure 2 1 Light Sut Photoresist ae Substrate Film deposition Photoresist application Exposure DUM p E AGL Development Etching Resist removal Figure 2 1 Basic schematic of photolithography E e Film deposition A thin film to be patterned is deposited onto a substrate Because MEMS originated from microelectronics silicon is still commonly used as a substrate The thin film can be silicon oxide silicon nitride metal poly silicon or a polymer Often it 1s the silicon itself that 1s to be patterned in which case no thin film is deposited e Photoresist application To pattern a thin film deposited on a substr
55. eeees 27 MEMS Technologies for PCR Microdevices sss 3l Bulk NIicronmacbintlD uoo o etie tt IL as tie nae ub eae 3l 2 9 2 10 Hel ICHO EODD ere een me A oobis redet euet td Eee 35 2 9 9 S race Micromachining sooo oes op DR OA qa Dd eia esi teu e d ee iUt 37 24 Parylene MEMS TechnoloBVsaasacnedns vt teintes v oo E En Pene cute use iue ten a 38 2 4 NAP USePdtylefie ae en ee Ee debel oit do inh ubt eee beide 38 2 4 2 Parydene Chemical StP C DEG seasea teat eb OR ERE M RE ATLA 40 2 4 3 Physical Proprie Sie aeo at bon bere ae Rad abr tutela 4 2 4 4 Chemical Vapor Deposition Method seen 42 2 4 5 LEES TR S DI SPD RN EN RE RR RI I RE RENI RO RI E ERO ERE FORERO ER 43 2 4 6 Biocompatibility of Parylene as a Real Time PCR Material 23 2 5 CHaDIGE SUNMA neern S O rales n tst case 61 3 RTPCR Microdevice Air Gap Version 62 3 1 FAD PIC AO Meares oct HDte d nien ci LEID iD COMI D HI ELSE IS iC MD DEMANDE 62 3 2 hndic Channel D68191l sete eoa eise rte rr tec te eua Misa rte emm eee lines 74 3 3 Device Thermal BEtig te Siti coe dee o petis aid edid hum dee io epe eh ade ae ies 76 3 951 Heat Frans ter Back oround ox s dnt den atn teen epe mates 76 21914 Device WS ria SS pct side oe ete tigate A nee asta neat sca 81 3 3 3 Thermal Performance Results eene 87 3o J i ridce WAC ETOUSIHTO cocco mato REESE HEN Eo E NES Ee inet dina as 9 3 5 Device Pertobm
56. emical Analysis and Optimization of Inhibition and Adsorption Phenomena in Glass Silicon PCR Chips Sensors and Actuators B 96 685 692 2003 G M Whitesides E Ostuni S Takayama X Jiang and D E Ingber Soft Lithography in biology and biochemistry Annual Revue of Biomedical Engineering 3 335 373 2001 6 J Liu M Enzelberger and S Quake A Nanoliter Rotary Device for Polymerase Chain Reaction Electrophoresis 23 1531 1536 2002 J S Marcus W F Anderson S R Quake Parallel Picoliter RT PCR Assays Using Microfluidics Analytical Chemistry 78 956 958 2006 Q Xiang B Xu R Fu and D Li Real Time PCR on Disposable PDMS Chip with a Miniatuizede Thermal Cycler Biomedical Microdevices 7 4 273 279 2005 J Shih PAD Thesis 2008 3 J C Salamone Concise Polymeric Materials Encyclopedia pp 1363 1365 1999 3l V amp P Scientific Inc Website www vp scientific com 128 C Y Shih T A Harder and Y C Tai Yield Strength of Thin Film Parylene C Microsystem Technologies 10 407 411 2004 33 X Q Wang Q L Lin and Y C Tai A Parylene Micro Check Valve Proceeds from The Twelfth IEEE International Conference on MicroElectroMechanical Systems 1999 MEMS 99 Orlando Florida pp 177 182 1999 B Lu S Zhang and Y C Tai Parylene Background Fluorescence Study for BioMEMS Applications Proceedings of the 15 International Conferences on Solid State Sensors and Actuators Transducers 2
57. entually shorted after processing to allow larger bonding pads for interfacing with the chip housing More on this will be discussed in the section dealing with chip housing 4 3 2 1 Temperature Sensor The 2000 A platinum trace resulted in a resistance of about 2 9 kOhms at room temperature The temperature calibration curve showed a highly linear relationship see Figure 4 12 The TCR temperature coefficient of resistance value was about 2 0 x 10 C which is consistent with the previous version 115 3300 y 6 2117x 2749 3200 R 0 9999 3200 3150 E an 3100 Cc 3050 3000 2950 20 40 60 80 100 Temperature C Figure 4 12 Temperature sensor calibration 4 3 2 2 Heater The thin film metal trace heater based on joule heating provides a sufficient and simple heat source The power dissipated by such a metal line is given by Joule s First Law V 5 P VI R R Equation 4 1 In designing such a heater it is important to choose a resistance value such that the available power sources can supply the required voltages and current Below are the relevant parameters for the heater 116 Table 4 1 Parameters for heater Maximum Power Required 100 mW Heater resistance 2900 ohms Maximum voltage required for 100 mW 20 Volts Maximum current required for 100 mW 5 mA These values are within the capabilities of the available power supply 4 3 2 3 Equipment Setup This devic
58. es used vary from reaction to reaction For a given reaction a series of conditions must be tested to find an optimal set Here are some guidelines for choosing a thermal recipe 10 o Denaturation step Increasing temperature causes faster breakdown of Taq polymerase while lowering the temperature below the melting point of the target DNA would halt replication o Annealing step This temperature varies the most as it has the most effect on PCR Too high temperature will result in reduced products since a smaller fraction of primers can overcome the thermal energy required to remain bonded Too low temperature is also detrimental as it allows primers to bind nonspecifically resulting in multiple products seen as multiple bands on gel electrophoresis Low temperatures promote secondary structures on DNA o Extension step Excessively high or low temperatures will result in non optimal performance by Taq polymerase zi d 5 5 3 5 3 o 5 C 5 d 5 d n 5 5 i LL Mi 3 5 5 5 gt D Figure 1 4 PCR schematic illustrating selective amplification of the target region between the primer pairs 12 1 1 3 Molecular Level Theory In Figure 1 4 step 1 refers to the denaturalization of the template forming two single stranded pieces of DNA Step 2 shows the annealing step where the primers bind Step 3 is the elongation step Notice that although elongation starts at
59. essing can be performed in a standard laboratory environment with relatively few pieces of equipment Furthermore PDMS is cheap and easily obtained Inlet and outlet fluidic connections become simple a needle poked hole is formed which acts as both a gasket and stabilizer for the capillary tubes Flexibility allows structures such as pumps and valves to be integrated with channels This simplicity has allowed a burst of attention and innovation to this microfluidics paradigm Components such as pumps valves and mixers have been fabricated for applications in biochemistry and cell culture Liu et al demonstrated a novel rotary device in which a plug of PCR solution was passed through a circular channel containing 3 heated regions at 94 C 55 C and 72 C The heaters were fabricated using metal lift off on the supporting glass slide with tungsten as the 37 heating component with aluminum leads This design allowed usage of only 12 nL of solution The fluid was pushed by peristaltic style pumps fabricated on a separate PDMS layer control layer on top of a layer of PDMS with the fluidic channels fluidic layer This control scheme provides the actuation for valves pumps and fluidic metering for large scale integration The same group later developed the technology to enable real time PCR on the picoliter scale A much simpler design for a PDMS QPCR device which does not require any photolithography was presented by Q Xiang et al
60. g into an oven or hotplate to further drive away remaining development solution casting solvent or moisture resulting in a hardened film with increased resistance to etching environments A mild oxygen treatment referred to as de scumming may also be executed here to etch away any photoresist residue that may not have been developed away Etching With the photoresist in place the wafer can be placed into an etching environment such as a plasma or acidic metal etching solution The photoresist 30 protects the layers that are underneath it from the etchant such that the thin film s pattern matches the resist pattern e Resist removal After etching the resist has served its purpose and can now be removed A photoresist stripper solution can be purchased from the resist vendor Some resists easily dissolve in common organic solvents such as acetone A key feature of the photolithography process is that features can be mass produced on wafers As seen in Figure 2 2 the pattern from one mask can be projected onto a wafer repeatedly to make tens to hundreds of devices simultaneously This projector can also reduce the size of mask features for example by a factor of 10 The optical limit for feature sizes is given by Rayleigh s criteria Won A Equation 2 1 NA Where k is a constant related to the contrast of the photoresist typically 0 75 NA is the numerical aperture of the projection system about 0 6 and is the wave
61. h as shown in Figure 1 10 21 Calibration Curve i o 2 E Z o gt O o o o H 0 1 1 Concentration ug ml Figure 1 10 Calibration curve for an M13 virus DNA sample From this plot the concentration of an unknown sample assuming identical experimental conditions can be estimated by observing its threshold cycle number Using linear regression a relationship for this curve yields C 3 423 Log T 11 33 E 95 9 Equation 1 4 where C is the number of cycle required to reach the threshold fluorescence To 1s the initial concentration of template DNA molecules E 1s the efficiency of the reaction 22 A mathematical derivation of Equation 1 4 is now given By rearranging Equation 1 3 the relationship between starting DNA copy number To and cycle number c can be derived using simple algebra as C log E 80 ET Equation 1 5 To find a one to one correlation the parameters T and E must be kept constant Fora constant value of target molecules T we choose a set value for the threshold fluorescence this assumes a linear relationship between fluorescence reading and number of target molecules which is generally a good assumption and call it T To obtain a constant efficiency E we narrow the range of threshold fluorescence values to only when the reaction is in the exponential phase or the linear phase in a log plot When T T the observed c is the threshold cycle num
62. his move is considered by many as the most important development towards the wide usage of PCR Taq polymerase was originally isolated from Thermus aquaticus bacteria that live in hot springs and thus have polymerase that can withstand high temperatures The enzyme has optimal activity around 70 80 C and a half life of about 10 minutes at 97 C Not only did this lead to survivability at the denaturization step it allowed higher annealing temperatures which lead to increased primer binding specificity and reduction in secondary structures in the template and target strands At the optimal temperature Taq polymerase has an estimated extension speed of about 60 nucleotides per second For the 120 bp target region in this study this would mean a minimum of 2 seconds is required for the DNA synthesis step however the processivity average number of nucleotides incorporated until the polymerase dissociates of the enzyme is only about 60 nucleotides thus a full 15 seconds is used The enzyme also features a 5 3 exonuclease activity meaning it can destroy a pre existing strand of DNA that is in front of it when synthesizing the new strand This feature 1s useful for FRET based real time PCR discussed in Section 1 2 2 2 Taq polymerase used in modern reactions are recombinant 1 e have an altered protein sequence and packaged with molecules that disable activity at room temperature but restore full activity upon heating to 95 C hot st
63. ic acid to form a solution that oxidizes silicon caused by the nitric acid then etches the oxide caused by the hydrofluoric acid subsequently oxidizing the freshly exposed silicon caused by the nitric acid Acetic acid is used as a diluent instead of water because it prevents dissociation of the nitric acid since oxidation requires undissociated NHO5 The overall reaction is 33 Si HNO 6HF gt H gt 2SiFs HNO H20 H gt Reaction 2 1 KOH etching utilizes potassium hydroxide which anisotropically etches the 111 planes of silicon 30 100x slower than the 100 planes These solutions are usually kept at high pH values gt 12 and high temperatures 70 C as they are considerably slower than the isotropic etching due to the slow chemistry at the surface One proposed overall reaction mechanism is Si 20H 2H 0 gt SiO OH 2H Reaction 2 2 In addition to KOH other oxides used are sodium hydroxide NaOH tetremethyl ammonium hydroxide TMAH ammonium hydroxide NH4OH and many more For dry isotropic etching XeF2 gas is used The overall reaction is 2 XeF Si gt 2Xe SiF Reaction 2 3 This reaction does not require ion bombardment heat or other external energy sources Since it is dry and chemical in nature many masking materials can be used including aluminum silicon dioxide silicon nitride photoresist and parylene These properties make it useful in post processing CMOS integrated circu
64. ich are down in Figure 2 6 Cl CH A on Parylene C CH Jch Parylene N CI CH o CI Parylene D Figure 2 6 Chemical Structure of Parylene Variants that have halogens placed in the aliphatic carbons are also available These simple chemical changes result in differences in chemical and physical properties as well as deposition kinetics In this work the word parylene will imply Parylene C unless otherwise stated 41 2 4 3 Physical Properties Below 1s a list of properties for Parylene C Table 2 2 Physical values of parylene Unless otherwise stated values are from ref 17 Property Value Comparison Tensile strength Mpa 69 Yield strength MPa 59 Elongation at break 200 Young s Modulus GPa 2 8 0 027 times the value for silicon Density g cm 1 289 Index of refraction 1 639 Melting temperature C 290 Thermal Conductivity W m k 0 082 3 2 times the value for air Aside from these quantifiable properties parylene has the distinguished advantage of being classified as Class VI by the United States Pharmacopeia USP a classification given to materials and methods which are used to produce them with superior biocompatibility suitable for long term implantation in humans It is also transparent to light above 300 nm 2 4 3 1 Parylene fluorescence Unprocessed Parylene C fluoresces weakly at 515 555 nm upon excitation with 465 495 nm light from a fluorescence microscope Processing of Paryle
65. ight heaters113 Feur Aad Ts Moara you ea T E 113 Figure 4 12 Temperature sensor calibration seen 115 Figure 4 13 Simple circuit analogy cccccccccccccccenssssseseeseeseeececeecccccessecenaaaassseseees 116 Figure 4 14 Thermal resistance to heat transfer into environment 118 Figure 4 15 Cooling experiments used to determine thermal time constant and AU IN ENTE TT berate teas 120 Figure 4 16 Heatine expertme ls iioi em e deiode etiaai o a be eie o vaio 120 Figure 4 17 Chip housing assembly O rings and pins not shown 121 Fig r 4 15 Chip housime Components o oe eret e E e Hr aerate 121 Figure 4 19 Detection of M13 virus on chip eeeessesesseeeeeeeeeeeeereeeereeererereereerererererereeees 123 Figure 4 20 Comparison of chip versus conventional machine Sample volumes and surface area to volume ratios of parylene were comparable sssuss 125 Table 1 1 Table 2 1 Table 2 2 Table 3 1 Table 3 2 Table 3 3 Table 3 4 Table 3 5 Table 4 1 xvii List of Tables Technical specifications for the Bio Rad MJ Mini PCR Machine 13 Methods for Gichin amp SIICOTOG o s oo aH aea UU e oa ad d tee 32 Physical values of parylene Unless otherwise stated values are from ref 17 EE A A EPOR 4 Thermal conductivity of selected materials cccccccccccccceeeeeeeeeeeeeee
66. inimizes excess mass which reduces heat capacity which in turn reduces the characteristic time constant t allowing faster heating and cooling rates Once again platinum was used as the heater and temperature sensor This time however no metal adhesion layer was necessary as platinum was deposited directly onto the parylene channels see Figure 4 10 and the materials have good adhesion to each other 113 ITI b e be ee D 72 o ET LE e a OCD i e 2 2 20 T 3 lt DL d p 3 gt D nay _ i D _ Figure 4 10 Platinum traces directly on parylene Left contact pads Right heaters Heater 1 Heater 2 Pad 1 Heater 3 Pad 2 Pad 3 Pad 4 Pad 5 Pad 6 Pad 7 Pad 8 Figure 4 11 Metal layout 114 The metal layout is diagrammed in Figure 4 11 There are three heaters labeled 1 2 and 3 Heater 2 1s the main heater and the one used for all the experiments here Heaters 1 and 3 were placed for possible use in future experiments including measuring the temperature at those distant positions It should be clear from Figure 4 11 that pads 1 2 5 and 6 serve heaters 1 and 3 Pads 3 and 4 and also pads 7 and 8 connect near the bottom of heater 2 The original intent was for them to be used in a four wire resistance measurement configuration however they were ev
67. ip capable of many functions Such a system has been realized in a fully integrated HPLC system comprised of the column mixer electrolysis pumps composition sensor mixer filter and electrospray nozzle This promising example proves the feasibility of a fully integrated RTPCR chip with possibly integrated solution storage units mixers pumps valves and even gel electrophoresis chamber 53 Column Mixer 4 2 cm x 80 um x 0 8 cm x 30 um x 25 um LxWxH Fig HOT ff 25 um LxWxH Filter 2 Composition 73 um r ES 7 am B n Sensor 80 um x particles P 25 um WxH Flow Sensor Electrospray 80 um x 3 um WxH Nozzle A EF LENIN mn om n 2 n E Figure 2 13 Integrated HPLC system 2 4 6 Biocompatibility of Parylene as a Real Time PCR Material 2 4 6 1 Parylene C Coated Reaction Tubes with Low Volume Solutions T nl T z ma a m LL Figure 2 14 QPCR on low volumes in parylene coated tubes 54 Amplification Plots 700 600 e 9 ee X wee Tuy e c 500 ee s D 400 E 2 3o Ka Pewee Lory LL E AA g A al B A N 200 t m a pT A O A z inia TTT Te 11 21 100 Cycles e 0 5uL d10 0 5uLd10O0 A 0 5uL dik O buLdiknp gm 0 5uLd10k 0 5uL d10knp Figure 2 15 Amplification of 0 5 ul QPCR solution As show in Figure 2 14 and Figure 2 15 using a conventional
68. its Etch rates are in the 1 um min order of magnitude with actual rate highly dependent on silicon load and feature sizes 34 Slight anisotropic etching 2 1 aspect ratio can be achieved using a plasma based on SF sulfer hexafluoride and will etch silicon to form the gaseous product SiF4 which diffuses back into the plasma creating a new surface to be etched In the reactive ion etching RIE configuration a parallel plate chamber is formed producing a biasing electric field that drives ions towards the substrate Since the pressure is in the 300 mTorr range compared to 2 5 Torr for XeF2 the mean free path is low enough for slightly directional in the z direction etching with a smaller component of lateral etching caused by interactions between etching molecules If true anisotropic etching is required deep reactive ion etching DRIE can be used In this modification of the RIE configuration a SFs plasma is still used to etch silicon but the plasma is either alternated with a C4Fg passivation layer for the side walls Bosch process or the substrate is chilled to 110 C cryogenic process to minimize chemical etching rates while preserving the 10n bombardment mechanism still present in the upward facing surfaces Another advantage of the ICP configuration is the separation of the plasma power source and the substrate bias voltage power source This allows independent control of plasma density and kinetic energy at which ions
69. l Cycler Figure 1 5Error Reference source not found Some specifications are provided in Table 1 1 below Table 1 1 Technical specifications for the Bio Rad MJ Mini PCR Machine Figure 1 5 MJ Thermal Cycler from Bio RAD Currently PCR machines are relatively large heavy slow and require hundreds of watts power The use of a bulky thermal block causes slow temperature ramp rates These issues can be addressed using MEMS technology 1 1 5 Gel Electrophoresis Following PCR gel electrophoresis is used to determine if the anticipated DNA target was amplified Using this technique a DNA ladder mixture of fragments of DNA of known size is run alongside the PCR sample to obtain an estimate of the size of the product sometimes referred to as the amplicon If only one product is formed that is of the anticipated length one can be reasonably assured that the amplicon 1s in fact that 14 identical to the target DNA If further reassurance is required one can sequence parts of the amplicon or bind it to a probe strand of known sequence 1 1 6 Applications Since PCR is a DNA based analysis it is versatile due to the fact that all organisms have DNA or RNA which can be converted to DNA The usage of DNA primers takes advantage of the naturally evolved base pairing phenomenon that provides excellent specificity The assay is also practical because tests can be performed in about one hour using a few pieces of equipment that a
70. length of light used 365 nm In this example the minimum feature size under the optical limit would be about 0 5 um As a result of these basic fabrication features MEMS devices are smaller lighter and possibly cheaper In some cases performance is improved and potential integration of many functions onto one chip exists 3l Ear i Lens I l l 4 Mask or Reticle t s Lens A Scan directions Figure 2 2 Photolithography using a stepper 2 3 MEMS Technologies for PCR Microdevices The general outline of photolithography given above is now extended to include MEMS techniques that are of particular interest in making PCR microdevices 2 3 1 Bulk Micromachining Bulk micromachining refers to the fabrication schemes that form a fluidic channel by etching into the substrate glass or silicon then bonding to a cover unit also glass or silicon 32 Glass Glass Anodic i Bonding Silicon Silicon Figure 2 3 Example of bulk micromachining Silicon is often used as a substrate because of the wide variety of etching methods already developed for it and the potential of starting fabrication with a pre fabricated CMOS chip Both isotropic and anisotropic wet and dry etching technologies are available see Table 2 1 Table 2 1 Methods for etching silicon mE Wet Etching Dry Etching HNA etching is an isotropic mixture of hydrofluoric acid nitric acid and acet
71. mated volume of 500 nL and SA vol of 50 mm Figure 4 20 shows the chip compared to cycling done with a conventional machine where the sample volume was 500 nL and SA vol ratios of 6 mm and 66 mm were used The 66 mm sa vol sample was obtained by adding the appropriate surface area of parylene sheets into the reaction vessel The chip performs fairly well in the comparison 4 6 Chapter Summary An improved design based on the previous version was fabricated and tested Results were similar to the previous version but with improved thermal efficiency a more elegant structure and easier fabrication 125 5 Conclusion Recent advances in PCR have lead to real time PCR where the reaction product is quantified during the reaction via fluorescent indicators This allows quantification of starting material and faster reaction times since the reaction can be stopped once the amount of fluorescence reaches an acceptable level Recent advances in MEMS have lead to parylene based bio MEMS ranging from implantable microdevices to lab on a chip Since this material is more bio compatible while fitting in nicely with current microfabrication techniques its usage is becoming more common By combining advances in both these fields this thesis presented a real time PCR device based on parylene MEMS technology Compared to current devices the chip uses less fluidic reagents and less power while providing more efficient heating can cooling Future
72. monochromatic mode and all signal processing noise reduction auto contrast etc was turned off Manual settings ensured consistent shutter speeds The image analysis software was the free software Image J from the NIH Measurements were made in units of intensity related to the 16 bit images Lowest intensity was given a value of 1 whereas the saturation intensities had value 65536 2 16 Due to fluctuations in the mercury arc lamp intensity normalization was performed for every image 3 5 3 3 Normalization Fluorescence emission intensity I 1s related to the excitation intensity X and quantum yield by I X Equation 3 13 99 When SYBR Green binds to DNA it is of the DNA in solution that changes Thus in quantitative PCR we are actually interested changes in with respect to time which we try to extract by measuring I at a region of interest region A in Figure 3 33 D 4x Ar APA L h A Equation 3 14 A 9 I Xo dy XS where I Fluorescence intensity at time t Io Fluorescence intensity at time 0 first cycle of PCR X Excitation intensity at time t Xo Excitation intensity at time 0 x quantum yield at time t o quantum yield at time 0 100 The problem is that X is also changing with time due to the nature of the halogen light source and thus must also be measured Fortunately one only needs the relative change in time of excitation intensity Xo X not its absolute value
73. nd thus not be destroyed by DNA polymerase Also if the target region is present but the primer pairs fail to function correctly no fluorophore is emitted The specificity of the Taqman system limits its scope in usage Custom probes must be synthesized for each target increasing costs for research 1 2 3 Calibration Curves The key that allows real time PCR to be quantitative is the relationship between the staring number of template DNA molecules and the number of cycles required to amplify it to a set amount If there are more starting molecules fewer cycles are required Below is an example of how a calibration curve is obtained followed by an analysis of the process e Obtain a sample of known template DNA concentration e Prepare 10 fold serial dilutions to generate multiple data points for the curve e Perform RTPCR on these samples 20 The data from these steps results in a graph such as the one below oO a iD LE a LL Cycles Figure 1 9 Amplification plots for a calibration curve Replaces 16 20 are 10 fold serial dilutions e From the above data choose a threshold fluorescence value blue solid line that crosses the sample curve where they are all linear in a log fluorescence versus linear cycle number plot e Note the number of cycles required by each known concentration to reach the threshold fluorescence call it c and plot it against the concentration These steps yield a curve suc
74. ne C using standard MEMS fabrication techniques such as plasma etching and electron beam metal deposition are suspected to increase fluorescence however further studies need to be done and conclusive evidence is not yet available Parylene HT has been shown to PE 34 fluoresce less in this range 42 2 4 4 Chemical Vapor Deposition Method Parylene coats substrates at room temperature conformally because of its chemical vapor deposition process outlined in Figure 2 7 THE PAARYLENE PROCESS P PCi y D nce pra ENE PARA WYLENE l FK T PARYLENE IN MONOMER Di PARPA XYLENE DINE FA OE PARYLENE C PARYLENE D eee Fd F WAPORIZATION 17m c F YPEX IBYA IN Seo C E 0 DEPOSITION COLD TRAF WALL as C TU PLMP Figure 2 7 Schematic of parylene CVD deposition 2 Figure 2 8 Chemical structure of di p xylylene the dimer precursor to parylene N e The desired parylene precursor in the form of a dimer is weighed and loaded into the vaporization chamber There is a direct correlation between grams of dimer and microns of parylene deposited e The system is pumped down to a few millitorr base pressure 43 e As the vaporization chamber gradually heats from room temperature to about 175 C molecules of dimer sublime and flow due to the constant pumping by the vacuum pump through the pyrolysis chamber which is at 680 C e Inthe pyrolysis chamber the dimer is converted into reactive monomer units e These u
75. ne layers a layer of conductive epoxy epoxy made conductive by incorporating silver particles into the matrix was placed over the contact pads This had the unfortunate consequence of shorting the narrowly pitched pads that were originally designed to provide a 4 wire resistance measurement configuration Good electrical function was still achieved however using a 2 wire configuration 120 Cooling Dynamics Celcius or mW 20 0 BEHENEHNHBEBEEEHNHEBHENHEEHEHEBEEHENHEBEHNH 4 0 1 2 3 4 5 Time s Temperature m Applied Power Model Temperature Figure 4 15 Cooling experiments used to determine thermal time constant and capacitance Heating Dynamics 80 me epee BBBeBeeeee EEEH EE Celcius or mW 1 0 1 2 3 4 5 6 Time s Temperature Applied Power Model Temperature Figure 4 16 Heating experiments 121 Figure 4 17 Chip housing assembly O rings and pins not shown Clamping screw x4 Acrylic top cover Chip Electrical O rings x2 contact pin x2 Fluidic port Ultem base Figure 4 18 Chip housing components 122 4 5 Device Performance 4 5 1 Real Time Polymerase Chain Reaction Components The same RTPCR mixture was used as in the previous version Please see chapter 3 for more details 4 5 2 Thermal Cycling Protocol The same thermal recipe was used as in the previous version Please see chapter 3 for
76. ng the parameters in the model described by Figure 3 20 These can be isolated by studying special limiting cases as described below 3 3 3 1 Steady State Temperature Thermal Resistances In order to determine the rate of heat transfer to the surrounding air and through the air gap steady state power experiments were performed 88 95 y 0 339x 23 5 85 B 1 R 0 9974 75 4 65 55 Steady State Temperature C 45 B e e s e E d4 3 0 50 100 150 200 250 Applied Power mW Figure 3 23 Steady state temperature From Figure 3 20 at infinite time the capacitors become open circuit elements leaving an effective resistance of R Rk R RAUS FR Equation 3 11 RK k R Rss R R de R Here Rss is simply the slope of Figure 3 23 Furthermore R and R3 are scaled by area since they share the same mechanism of heat transfer A R R TE Equation 3 12 2 Where A and A are the heat transfer areas of the island and body respectively Thus by calculated R using heat transfer correlations Rg can be determined using Rss Caution should be used when using the empirical heat transfer correlations however as they are mainly determined for macro scale millimeter or larger features and may not 89 be as accurate at these small length scales where viscous forces are much higher relative to buoyancy forces Values used in the model are shown in Ta
77. ning contaminants and produce a more hydrophobic surface Also before deposition a masking material such as removable tape can be applied to the back side of the wafer to prevent parylene deposition there if necessary Excessive area of both parylene layers are etched in oxygen plasma Depending upon design the excessive parylene may remain intact or be removed in later steps If wafer dicing is performed on the front side of the wafer removal of excess parylene is recommended as the water jet from the dicing saw can flow underneath this parylene and lift off the entire structure if adhesion is poor With a trimmed channel the water can be stopped by application of a dummy non patterned layer of photoresist Even if dicing is performed on the back side of the wafer excess thick photoresist can impede smooth dice separation as the parylene layer needs to be manually ripped This can leave jagged edges on the parylene edges 48 that can interfere with any chip housing In general removal of excess parylene is recommended e For the inlet and outlet holes a section of the second layer can be patterned and etched using an oxygen plasma The sacrificial photoresist underneath acts as a safety buffer should over etching occur e The sacrificial photoresist is dissolved using an organic solvent such as acetone For long channels 1 cm this step at room temperature can take weeks as it 1s diffusion limited If the photoresist has been excessivel
78. nits flow into the room temperature deposition chamber maintained at a steady 22 mTorr by feedback from a pressure sensor during deposition For increased uniformity the monomers flow past a baffle and the substrates are placed in the center both in the horizontal plane and in height of a rotating stage e Monomers that do not bind flow past the deposition chamber where they adsorb to the walls of the cold trap The standard temperature of the cold trap is 70 C although a simpler option is to use a liquid nitrogen filled cylinder as a cold trap resulting in a temperature approaching 196 C This lower cold trap temperature has no effect on the deposition process e A vacuum pump continually runs during the process to power the flow of molecules This room temperature plasma free deposition process allows the use of organic adhesion promoters such as silanes One commonly used promoter is A 174 gamma methacryloxy propyltrimethoxysilane which bonds to silicon oxide and presents an organic surface to promote adhesion of parylene 2 4 5 Patterning Parylene is chemically inert to most liquids at room temperature Although dissolution in some aromatic solvents such as chloronaphthalene has been reported to occur at temperatures about 150 C this process is not compatible with many photo resist 44 based MEMS fabrication steps The most effective method for etching it is using an oxygen plasma Typically etching rates range from 0 0
79. ntification of amounts of a particular mRNA gives insight into a cell s natural function and reaction to stimuli such as drugs or signaling molecules from other cells and is of great importance in the field of 26 biology Although there are various methods in recent years RT QPCR has emerged as the method of choice for this analysis 1 3 Chapter Summary Requiring simple affordable machinery and components real time PCR is easy to implement for extension of PCR that provides a new dimension to PCR analysis By monitoring the amount of DNA present via fluorescence quantification of the initial amount of DNA or RNA in the sample can be achieved Zi 2 Parylene Microfluidics This chapter serves as a short introduction to microfluidics including some background reasons for using microfluidics and basic technologies Parylene as a microfluidics material is discussed in the middle of the chapter and microfluidics as applied to real time PCR 1s discussed towards the end 2 1 MEMS Background The microelectronic industry has greatly matured since the discovery of the transistor effect in semiconductors in the 1940s Whereas early computers filled entire rooms and were only accessible to a few users through terminals today portable smart cell phones have far superior computational power packed into a hand held device These devices are small light and affordable Such a dramatic change in capabilities and portability in el
80. on steps are presented in Figure 4 1 Details of each step are presented in the text body Grow pattern oxide Back side DRIE Deposit pattern 1 parylene Front side DRIE XeF2 Deposit 2 parylene Deposit pattern metal Back side DRIE XeF s I silicon EM 2 parylene md oxide mH m etal a 1 parylene Figure 4 1 Overall device fabrication steps 105 The starting substrate is a 100 mm diameter 500 um thick one side polished silicon wafer For diagrammatic purposes only one cm x 1 cm chip is used to represent the entire wafer Figure 4 2 Bare silicon chip Following piranha cleaning submersion in a 120 C bath of HySO and H202 a 1 um thermal oxide layer is grown onto the wafer using an oxidation furnace at 1050 C Front Back Grow pattern oxide Back side DRIE Figure 4 3 Oxide layers Notice the back side shows silicon etched by the DRIE Back side also shows the legs of the front side oxide pattern for clarity Actual silicon is not transparent 106 The oxide layer is then patterned using 1 um of AZ 1518 photoresist as mask for 8 minutes of etching in buffered hydrofluoric acid The buffered acid is used instead of diluted hydrofluoric acid for a more consistent etching rate The oxide layer is preserved everywhere except the channels and back side window Figure 4 3 Initial back side etching of about 350 um of silicon is performed at this step to reduce the amount of
81. ooth flat surface the fluidic port was drilled using a 7 64 inch end mill instead of a drill bit The acrylic top cover contained four holes for the clamping screws and four holes for electrical contact pins although only two were used 93 Figure 3 28 Chip housing with external valves 3 5 Device Performance 3 5 1 Real Time Polymerase Chain Reaction Components The components of the PCR reaction will be detailed including the substrate the M13 virus 3 5 1 1 PCR Solution The protocol for preparation of the PCR solution is given below Details about each component are discussed in Chapter 1 e Start with 22 ul PCR Mix Platinum PCR Supermix from Invitrogen o Mix contains 22 U ml DNA polymerase 22 mM Tris HCl pH 8 4 55 mM KCl 1 65 mM MgCh 220 uM dGTP dATP dTTP dCTP and stabilizers e Add 2 40 DMSO 2 2 o DMSO disrupts base pairing to reduce results of secondary DNA structure of the targets and reduce non specific primer dimer formation e Add 0 6ul of 40X SYBR Green I 94 o Fluorescent dye for more details see Chapter 1 e Add 1 ul of each primers right then left o Final concentration is 0 01 nmoles of each primer e Add lul virus sample or its pure DNA o See below for more details 3 5 1 2 The Sample M13 Virus The sample used in this study 1s the M13 bacteriophage a model DNA virus The template was thus its 6 4 kilobases long circular single stranded DNA genome The phage is about 900 nm long and
82. or qPCR surface binding dominates evidence suggests the surface binding occurs at the minor groove of dsDNA The stock stain solution comes dissolved in DMSO dimethylsulfoxide and stored frozen 20 C until use After a 10 000X dilution for RTPCR analysis the working concentration 1s about 2 uM Figure 1 6 Chemical Structure of SYBR Green I 17 Its double stranded DNA bound fluorescence spectrum is available in Figure 1 7 100 90 excitation emission x 6 80 i tf TU e o 60 z o a 90 4 40 E 30 G 5 20 10 0 300 350 400 450 500 660 600 660 700 wavelength nm Figure 1 7 Fluorescence spectrum of SYBR Green I Since SYBR Green I binds to all double stranded DNA at the working concentrations it can be used irrespective of target DNA sequence making it versatile As the number of double stranded DNA molecules increases so does the fluorescence This feature 1s also its biggest limitation as non specific non target DNA sequences that are unintentionally amplified also produce a fluorescence signal This limitation is partially addressed by the generation of melting curves and gel electrophoresis to check for purity and length of the product Removal of SYBR Green from DNA can be achieved by ethanol precipitation Add ethanol to cause the DNA to precipitate then centrifuge the pellet wash it again in ethanol allow to dry then resuspend in buffer solution 18 1 2 2 2 TaqMan An alternative
83. other resistor and capacitor in parallel representing the body of the chip and a resistor that represents the resistance to leaking heat through the parylene stitches and air gap Figure 3 20 Figure 3 20 Extended RC Model Where R Thermal resistance from island to the environment C Heat capacity of the island R2 Thermal resistance from chip body to environment C5 Heat capacity of chip body R Thermal resistance from island to chip body 84 An analytical solution for temperature as a function of time exists however it is a long expression that does not offer any insight into the system Instead the solutions will be plotted in the thermal characterization sections for various special cases 3 3 2 1 Temperature Sensor A resistance temperature detector RTD configuration was used because of its simplicity Such a sensor takes advantage of the relationship between temperature and resistance of a metal given by the Callendar Van Dusen equation R T R 1 AT BT Equation 3 8 Where R is the resistance at temperature T in Celsius Ro is the resistance at 0 C and A and B are coefficients that are characteristic of the material For the temperature range of interest for PCR 0 C 100 C the coefficient B becomes negligible Furthermore instead of creating a 0C environment for measuring Ro it is common practice to replace the Callendar Van Dusen equation with an equation of the form tU 1
84. own in Figure 2 15 a 10 fold serial dilution showed that a minimum of 1 63E7 DNA template molecules in 0 5 ul showed amplification in this setup This suggests on chip QPCR would be possible for these low volumes The combination of the parylene surface and low volume effects is shown in Figure 2 17 For this 0 5 ul sample cycled in the conventional machine a surface area to volume ratio of 60 mm is noticeably attenuated whereas a ratio of 80 mm is indistinguishable from the no primers control Thus a parylene based chip should not have a ratio greater than 60 mm 5 i13 iii ee eo 9 e e9e 1 09 Pa E 1 07 m A i e O 0np y T A 60 mm np S e 80 mnvnp 3 N T 4 0np 80np 1 03 A O 1 01 0 99 0 97 0 5 10 15 20 25 30 Cycles Figure 2 17 High SA vol ratios of Parylene on a 0 5uL RTPCR sample In an attempt to quantitate the extent to which parylene or any material in general inhibits RTPCR comparisons were made between samples with parylene added to those with small volume It was observed that adding parylene sheets into the reaction tube had an effect similar to that of using small volumes Thus by adding sheets of parylene the sample acts as if its volume was decreased This lost volume was then plotted against the surface area added to yield Figure 2 18 Fitting a straight line resulted in a reasonable linear fit and a slope of 0 0
85. p and thus allow for fluidic access to the channels Deposit 2 parylene Figure 4 6 Second parylene layer deposited A second parylene layer 20 um is then deposited Figure 4 6 This layer covers the silicon sidewalls with parylene and also fills the holes completing the channel 109 Deposit pattern metal Figure 4 7 Platinum pattern A 2000 A platinum layer is then deposited on the second parylene It is patterned using the metal lift off method No adhesion layer 1s necessary because parylene and platinum have good adhesion to each other The engineering reasons behind the metal layout are discussed in the section about thermal engineering below Despite the 1 2 um dimples on the surface due to the filled holes the metal is still a continuous film Figure 4 7 110 Back side DRIE XeF2 Figure 4 8 Back side finishing View from back side Left After DRIE Right After XeF After metallization the back side of the wafer is etched using DRIE with the oxide layer asa mask This etches the silicon down to the lower surface of the channels Figure 4 8 upper left Further plasma etching at this point can damage the parylene channels To etch the remaining silicon XeF2 is used The gas based chemical reacting mechanism has excellent selectivity to silicon and leaves the parylene intact Following this step all the silicon in the back side window is removed making the platinum layer on the front side of the
86. phoshate dATP deoxyguanosine triphosphate dGTP deoxythymidine triphosphate dTTP and deoxycytidine triphosphate dCTP They are composed of a ribose sugar three phosphate groups and a base Figure 1 2 The base component determines the type of nucleotide it is RNA has an OH group at the 2 of the ribose sugar while DNA has only a hydrogen atom there For normal PCR operation equal ratios of the four standard dNTPs are added to the reaction mix Deviation from the standard recipe can be useful for special applications For example to study random mutations to the target region one of the dNTPs can be added in excess to increase the chance of erroneously incorporating that nucleotide into the amplicons For other applications slight variants of the standard nucleotides can be used Deoxyuridine triphosphate dUTP can be substituted along with biotin or fluorescently labeled variants of dUTP These modified nucleotides provide useful functions such as binding to streptavidin and detection by fluorescence two properties that are heavily exploited in biochemistry and biotechnology NH Purines 0 N N H fjs 5 4M a EN Ies Os Base d B 3 G a 2 Q 4 Boe 3 ih H H H NH o g o 3 glycosidic bond R R HO OH Rhose Adenine Guanine H Deoxyrib ose Pyrimidines Pent ento se 5 ide l Nucleoside _H HAC _H 4 Nucleoside monophosphate J E i He 1 i Nucleoside diphosphate j 0 N 0 Nucleoside
87. rature cooling dynamic with zero applied power The same parameters were used as in the heating model The only fitted parameter here was the initial body temperature which was given a value of 56 C It should be noted that the time varying portion of the model begins to lose accuracy at time greater than a few seconds possibly due to non linearity in the actual system thermal leakage to 9 the body housing and non uniform temperature distributions Nevertheless these experiments and model describe the time and temperature regions that are most important to an RTPCR chip 3 4 Interface with Housing To interface the chip to the macro world a custom built housing was designed using solid works and fabricated in house using a computer numerical controlled CNC milling machine acrylic spring loaded pins qPCR chip Fluidic coupling Figure 3 26 Chip housing assembly 92 Electrical Clamping contact pin x2 screw x4 Acrylic top cover Chip O rings x2 Ultem base Fluidic port oet screw hole Figure 3 27 Chip housing components The base was machined from Ultem polyetherimide a material that can be supplied as USP Class VI compliant implying good biocompatibility It contains two grooves for positioning the O rings a set screw hole to receive the set screw four through holes to receive the clamping screws and 2 fluidic ports machined to accept a 6 40 fluidic coupling nut A achieve a sm
88. re occurs when two regions of the primer compliment each other causing the DNA strand to curve and bind to itself much like tape sticking to itself This shields the primer DNA from accessing the template DNA Furthermore primers should not be complementary to each other to avoid primer dimer formation Partial dimer formation will result in amplification of the primers themselves o In some cases mispriming is specifically designed into the primers Point mutations a difference of one nucleotide between the mutant and original DNA strand can be introduced into DNA sequences by first introducing them into primers Larger sets of nucleotides such as a restriction endonuclease recognition site can be added to the 5 ends of primers to allow the amplicons to be inserted into a cloning vector and expressed in bacterial cells These techniques are part of the field of molecular biology enabled by PCR molecular cloning DNA Polymerase 1s an enzyme that continues synthesis of a complementary strand of DNA In the early implementations of PCR E Coli DNA polymerase in particular the Klenow fragment was destroyed at the melting DNA temperatures 95 C and thus replaced after every cycle Furthermore the ideal temperature for synthesis using this polymerase was 37 C however this allowed primers to bind to noncomplementary regions thus reducing specificity Biologists eventually moved to Taq Polymerase I because it is thermally stable at 95 C T
89. re standard in modern biology laboratories and using cheap easily obtainable reagents PCR can supply large amounts of specific DNA for further analysis and can be used downstream from other assays This is particularly useful when only small amounts of the original template DNA is present such as in forensic analysis It can be used to isolate a specific region of DNA for purposes such as bacterial transformation and is a central part of the Sanger sequencing method for determining the sequence of DNA fragments Perhaps the most well known application of PCR is DNA fingerprinting used in criminal trials DNA fingerprinting can also be used in paternity testing and even determine evolutionary relationships between organisms By designing primers that amplify a DNA sequence that is unique to virus one can identify its presence in a sample PCR can also be used to diagnose diseases such as cancer 15 1 2 Real Time PCR The real time polymerase chain reaction RTPCR or quantitative polymerase chain reaction qPCR is a procedure based on PCR where a piece of target DNA 1s both amplified and quantified using fluorescence simultaneously 1 2 4 Theory From the background of PCR it is seen that the number of DNA target molecules doubles after every cycle Mathematically this can be written as c I 2 Equation 1 2 where T current number of target molecules To initial number of target molecules c number of cycles F
90. right and with a chuckle hesitantly acknowledge that might work I would like to thank my colleagues from the Caltech Micromachining Lab Justin Boland for introducing me to machines and the stock market Matt Liger for that night we tried to use the CMP machine it has yet to be touched again to this day Damien Rodger for being very niche and introducing me to the lab Scott Miserendino for being a cool officemate Victor Shih for helping to improve my processes Siyang Zheng for being my student mentor when I first joined the lab Qing He for helping me with fluidic couplings Nick Lo for being my contact person for any EE theory Wen Li for our discussions about everybody else while in the cleanroom and for helping me keep the Ebeam running PJ Chen for lending me the pressure regulators Jason Shih for being the master of fluidics Mike Liu for being from Berkeley too Luca Giacchino for taking over for Matt Liger as the representative of all of Europe Ray Huang for continuing the consulting club Jeffrey Lin for the late night dinners which you still owe me one for helping you with the SEM vil Monty Nandra for being the master of EE and optics Justin Young Hyun Kim for always saying hi Bo Lu for putting my name on that paper Yu Zhao for being a fun officemate Penvipha Satsanarukkit for being a hard working mentee Bo Lu for inspiring me with enthusiasm for your lab results Wendian Shi for all your answers to my photography q
91. rmal isolation technology for microfluidic system on chip applications Sensors and Actuators A Physical 126 1 pp 270 276 2005 5 J Shih PhD Thesis 2008 A Gonzalez R Grimes E J Walsh T Dalton and M Davies Interaction of Quantitative PCR Components with Polymeric Surfaces Biomedical Microdevices 9 261 266 2007 Y Chen and Y Momose Reaction of Argon Plasma treated Teflon PFA with Aminopropyltriethoxysilane in its n Hexane Solution Surface and Interface Analysis 27 1073 1083 1999 C Zhang and D Xing Survey and Summary Miniaturized PCR chips for nucleic acid amplification and analysis latest advances and future trends Nucleic Acids Research 35 13 pp 4223 4237 2007 F Kreith Editor in Chief The CRC Handbook of Mechanical Engineering Section 4 16 1998 3 C Y Shih Y Chen J Xie Q He and Y C Tai On Chip Temperature Gradient Liquid Chromotography Technical Digest of the 18th IEEE International Conference on MicroElectroMechanical Systems MEMS 2005 Miami Florida pp 539 542 2005 E Meng MEMS Technology and devices for micro fluid dosing system PhD Thesis p 123 2003 C Y Shih Temperature Controlled Microchip Liquid Chromatogrphy System PhD Thesis p 86 2006 l J S Kim B K Park and J S Lee Natural Convection Heat Transfer Around Microfin Array Experimental Heat Transfer 21 55 72 2008 52 G Frackman G Kobs D Simpson and D Storts Bet
92. s have not been done Poor primer binding or sluggish extension steps can result from altered salt concentrations or reactants becoming denatured from interactions with the parylene chamber walls comparison of the air gap QPCR chip versus a conventional thermal cycler The chip 1 18 102 Conventional Verus Chip m e Conventional Machine e oc 114 Conventional Machine a No Primers ee ad e 1 12 m Air Gap Chip e a ti e 1 08 d i e m 1 06 E e j 1 04 7 e 1 02 M a T3 5 e 14 e 9 te ES Sade ee eee a E 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 Cycles Figure 3 35 Air gap chip versus conventional QPCR machine performs fairly although exponential amplification 1s muted The amplification curve appears Figure 3 35 shows a 103 3 6 Chapter Summary The first version of the device was designed fabricated and tested Further improvements can be made by simplifying the fabrication process removing the passive silicon on the thermal island and varying the biochemical parameters during the PCR step 104 4 RTPCR Microdevice Free Standing Version 4 1 Fabrication Here an improved version of the RTPCR chip is presented featuring a free standing chamber with the heater temperature sensor placed on top This new design allows for even more efficient heat transfer by eliminating the passive silicon underneath the channel The overall fabricati
93. s on the silicon The glass sealing also allows optical access to the solution while thermal cycling for usage in real time PCR The glass and silicon surface of these devices however inhibit the PCR reaction by adsorbing components such as Taq polymerase to an extent that surface treatments such as the addition of BSA must always be used Furthermore this scheme uses two rigid materials complicating the integration of moving parts such as valves and pumps 2 3 2 Soft Lithography Soft lithography generally refers to the use of elastomers such as polydimethylsiloxane PDMS in conjunction with molds to form channels First a master mold is formed by bulk methods such as DRIE of silicon or surface methods such 36 as SU 8 photoresist on top of silicon A fluid mix of pre polymer is then poured onto the master and allowed to cure at 80 100 C The PDMS can then be peeled from the master mold forming a negative replica Similar to bulk micromachining a second surface is required to complete the channel Common glass slides work well as they bond well with PDMS Surface treatment such as oxygen plasma surface cleaning or application of a thin adhesion layer of PDMS on the glass slide 1s often used to enhance bonding PDMS PDMS i Il II E fT Figure 2 4 PDMS micromolding The main advantage of soft lithography is its ease of use Formation of the master mold is the only step that requires a clean room environment Further proc
94. se of the superior properties of the material and its method of deposition This technology has matured enough for the fabrication of advanced HPLC devices Based on initial studies of low volume QPCR in a parylene environment building and designing a parylene based RTPCR device should be possible and is the first step towards a fully integrated bio analysis device capable of performing multiple bio assays 62 3 RTPCR Microdevice Air Gap Version 3 1 Fabrication Thermal oxidation and patterning not shown Metal deposition and patterning DRIE trenches for sidewalls and stitches a ease Ces es First parylene deposition Backside DRIE for inlet outlet formation E First layer parylene etch eF etching of exposed silicon 1 TT od second parylene deposited and patterned Air gap formation Dicing and Wire bonding Silicon ES 1 parylene m 2 parylene ms Metal Figure 3 1 Overall process flow 63 The process flow diagram is provided in this section with explanations for each step This is a detailed explanation describing how to build this device Familiarity with semiconductor or MEMS processing procedures may be required to fully understand some steps The overall process flow diagram is shown followed by illustrated step by step elaborations Figure 3 2 Silicon chip All photoresist exposure was performed using a 10X reduction stepper at 436 nm g line wavelength The substr
95. seeeees 27 Values for calculation of Rayleigh number for air sssssssss 79 Nusselt numbers All correlations from CRC Handbook 80 Power specifications for the air gap version ccccceceececeeeeeeeeaeeeeseeseeeeees 86 Parameters used for thermal model ccc ccccccccseeceeeceeeeeeeeeeeeeeeeeeeeeeeeeeeees 90 Parameters TOL Bedl et pina etes talent aes alike tak abelian ce a bitty 116 1 PCR and Real Time PCR 1 1 Introduction to the Polymerase Chain Reaction The polymerase chain reaction PCR is an in vitro molecular biology technique used to amplify deoxyribosenucleic acids DNA Developed primarily by Kary Mullis Nobel Prize Chemistry 1993 while at Cetus Corporation in the 1980s PCR is now a standard technique used in nearly all biology labs in the world Using the reaction scientists can amplify a target region of DNA located within the template DNA Figure 1 1 The product is called the amplicon This technique greatly simplifies DNA amplification which before the development of PCR required DNA to be reproduced in vivo in bacteria using cloning techniques The amplification of a specific target region makes PCR an exceptional tool for a wide variety of applications including paternity tests genotyping and pathogen detection Template DNA Figure 1 1 Basic concept of PCR amplification To explain PCR in detail a list of components will
96. supplies a hydrophobic surface for improved adhesion between parylene and the substrate These filled trenches function as either vertical etch stops for silicon etching or stitches that hold the air gap to the main chip body A simple layer of tape was placed on the back side of the wafers to prevent parylene deposition there The resulting top layer was flat enough 2 um features for further processing steps such as application of photoresist by spinning Back side DRIE for inlet outlet formation Overlap Figure 3 7 Inlet outlet formation Notice the back side etching shaded in brown overlaps the channel etching region ensuring a continuous path when the channel is etched 69 Taking advantage of dual side alignment marks AZ 9260 photoresist was deposited and patterned on the back side of the wafer to serve as a mask for 800 loops of a modified Bosch process to form the inlet outlet holes The depths and diameter of these holes were designed such that when the channels are formed there 1s a continuous path from the back of the chip to the channel First parylene patterning and XeF gt etching of silicon Figure 3 8 Etching of first parylene layer light blue and XeF etching of underlying silicon Figure 3 9 Inlet outlet hole 70 At this stage the parylene layer was etched in an oxygen plasma using an RF parallel plate reactive 10n etcher RIE AZ 9260 was used as an etching mask for etching in 350 mTorr
97. t 16 bases The upper limit 1s driven by specificity as well An extremely long primer will base pair despite a single base mismatch because the other correct pairings provide enough thermodynamic driving force to sustain one mismatch The most common primer lengths used are about 20 bases long Within these 20 bases the 3 end of the primer is most important since Taq DNA Polymerase will extend this end when assembling the new strand even if the 5 end is slightly mismatched and not binding efficiently In fact the 5 end can be modified to carry additional sequences such as restriction sites that are not complementary to the template The melting temperature Tm of primers should be about 52 58 C This is the temperature above which the DNA separates from its compliment to become two single strands of DNA T that is too low will require a low annealing temperature that allows nonspecific primer binding while temperatures should not be higher than the elongation temperature as DNA synthesis will prematurely start The annealing temperature is often set at 5 C lower than the lower Tm of the two primers to start then further optimized using trial and error A simplified correlation for estimating the melting temperature for primers 1s T C 2 A G C 2 A T Equation 1 1 Where G C A T represent the number of instances of the respective bases Primers should not contain sequences that result in secondary structure This structu
98. tandard A thermocouple coupled to a signal processor that converts degrees to voltage was used without checking its certificate of calibration thus this inaccuracy 1s likely to be above the precision of the system 86 3 3 2 2 Heater The thin film metal trace heater based on joule heating provides a sufficient and simple heat source The power dissipated by such a metal line is given by Joule s First Law 2 P VI LL I R Equation 3 10 In designing such a heater it 1s 1mportant to choose a resistance value such that the available power sources can supply the required voltages and current Below are the relevant parameters for the heater Table 3 4 Power specifications for the air gap version Maximum Power Required 300 mW Heater resistance 2300 ohms Maximum voltage required for 300 mW 30 Volts Maximum current required for 300 mW 10 mA These values are within the capabilities of the available power supply 3 3 2 3 Equipment Setup The off chip hardware and control software complete the temperature control system 87 R on chip Heater and temperature sensor Figure 3 22 Temperature control hardware arrangement The temperature control hardware consisted of the chip power source multimeter set to ammeter mode and computer The hardware was connecting via GPIB connections and controlled using LabView 3 3 3 Thermal Performance Results Thermal performance can be characterized by establishi
99. th a 5 color filter wheel This design eliminates non uniformity in the fluorescence detection a problem faced by the CCD image capture approach Figure 1 11 Strategene MX3005P benchtop RTPCR system 1 2 5 Applications Two applications of real time PCR are described below pathogen detection and mRNA expression profiling 24 1 2 5 1 Pathogen Detection Pathogen detection is a popular application of real time PCR The versatility of this technique is demonstrated by Zeng et al as they detect the airborne mold Cladosporium an allergen They determined the presence of 10 spores m in two locations a countryside house that houses firewood and a paper factory A higher level at 10 spores m was detected in a cow barn These levels exceed the medically recommended maximum exposure of 3000 spores m Prolonged exposure can weaken the immune system and cause severe asthma In past studies detection of Cladosporium was based on slower methods including cell culture in which spores were grown in an incubation chamber before microscopic identification by eyes a time consuming and labor intensive approach To test the sensitivity of their array they performed a serial dilution test Figure 1 12 The most dilute sample detected labeled 6 corresponded to only 2 spores This sensitivity of down to 1 genome copy is not uncommon in RTPCR assays 10000 1000 PCR Base Line Subtracted CF RFU m i j 100 O 2 4 6 8 10 12 14 16
100. the overhanging parylene layer thus creating a channel Excess parylene on the wafer was then eliminated by etching in the RIE system exposing the underlying oxide and gold contact pads see Figure 3 10 72 Air Gap Formation Figure 3 11 Air gap formation Figure 3 12 Zoom showing the parylene stitches used to connect the island to the main body The air gap was formed by front and back side DRIE Both sides use thick AZ 9260 photoresist as the masking material The parylene stitches then became visible as a stack of parylene sheets bridging the gap between the air gap and main body see Figure 3 13 At this point light from the back side was able to shine through the air gap a good test to check if the gap has been completely formed 73 Dicing and wire bonding Figure 3 13 Wire bonding on the completed chip The wire bonds provide electrical continuity across the parylene stitched air gap A dicing saw was used to cut the wafer into 1 cm x 1 em dies After device separation a wedge wire bonder was used to electrically join the main chip body with the air gap This provides a simple elegant way to join the pads with the heaters on the island Since both sections of the heaters were joined in series they form one heater and can be controlled as one resistor 74 3 2 Fluidic Channel Design There are many design requirements for a quantitative PCR chamber From a materials standpoint the surface
101. tic thin walled reaction tube and placed in a thermal cycler for 30 40 cycles Denature DNA eI Extend Primers Anneal Primers Time OQ 1 2 3 4 minutes Figure 1 3 Typical thermal recipe for PCR A typical thermal recipe is 1 94 C for 2 minutes This initialization step activates specially modified Taq polymerases which are designed for minimal activity at room temperature Also during this step the template DNA and primers fully dissociated This step occurs only once in the reaction 2 94 C for 15 30 seconds This denaturation step dissociates the DNA targets also called amplicons produced during previous cycles by disrupting the hydrogen bonds between complementary bases This exposes the bases and allows the primers bind to them in the next step 3 55 C for 15 30 seconds During this annealing step the primers bind to their complementary sequences in the target DNA 4 72 C for 15 30 seconds This is the extension step when the DNA polymerase extends the DNA strand starting from the primers assembling from the 5 to 3 end of the new DNA strand by adding the complementary dNTP to the elongating strand 5 Repeat steps 2 4 for about 30 40 cycles After each cycle the number of DNA molecules is theoretically doubled if 100 efficient 6 Final Elongation This step is often performed at 72 C for 5 minutes to ensure any remaining single stranded DNA 1s fully extended The exact temperatures and tim
102. uestions and Charles DeBoer for keeping me company during lunch The friendly administrative staff of Tanya Owen Christine Garske and Agnes Tong were highly efficient while the lab engineer Trevor Roper kept the lab and machines running smoothly Finally I would like to thank the Caltech Glee Club and Caltech Consulting Club for making my last years at Caltech memorable and music filled viil Abstract The polymerase chain reaction PCR is a powerful biochemical assay that is used in virtually all biochemical labs By specifically amplifying a small sample of DNA this technique 1s useful in the fields of paternity testing forensics and virus detection just to name a few A useful advancement of PCR involves monitoring the fluorescence generated by an increase in DNA during the amplification This so called real time RT PCR allows quantification of the initial sample amount and allows for shorter assay times by stopping the reaction when enough fluorescence has been detected Technology in the field of micro electro mechanical systems MEMS has advanced from the academic laboratory level to a handful of commercially successful devices Work on adapting MEMS to biochemical applications however is still at the laboratory research stage Recent breakthroughs in the use of more biocompatible materials in MEMS devices have helped to advance bio MEMS In particular the polymer Parylene has superior properties that present a promising ne
103. urthermore if one allows for a nonidealistic efficiency the value 2 can be substituted with 1 E where E 0 E I 1s the average efficiency after c cycles Thus Equation 1 2 becomes f T l iE E Equation 1 3 since we choose a constant value for E here Equation 1 3 is only valid for the early cycles before the efficiency becomes unpredictable due to a variety of factors such as degradation of Taq polymerase after repeated thermal cycling These early cycles are referred to as the exponential phase and their termination can be identified by the 16 deviation in exponential shape of a T versus c curve or deviation of linearity in a log T versus C Curve 1 2 2 Fluorescent Indicators 1 2 2 1 SYBR Green SYBR Green I SG is a nucleic acid stain with many uses including double stranded DNA dsDNA quantification in real time PCR and gel electrophoresis For the latter it is generally considered a safer alternative to ethidium bromide with 25X better sensitivity Upon binding to double stranded DNA its fluorescence intensity becomes 1 000 times that of its unbound state with a quantum yield of 0 8 This large gain in fluorescence contributes to a good signal to noise ratio as non bound SG in the solution and walls of the container contribute minimal noise Its chemical structure 1s shown in Figure 1 6 At low concentrations the dye binds to DNA by intercalation however at the higher working concentrations f
104. w platform for this field This work presents the design fabrication and testing of a parylene based MEMS RTPCR device By combining advancements in both biology and MEMS engineering this work demonstrates the feasibility of such a device along with quantitative analysis and data that serve as a guide for its future development Table of Contents 1 PCR and Real Time PCR L 1 1 1 2 1 2 1 1 2 2 1 2 3 1 2 4 1 2 5 1 3 Introduction to the Polymerase Chain Reaction ccccceseseeeesseeeeeeeeeeseeeeeaeees l CODON A aana a E 2 POCO a a er ae 8 Molecular Level DBeOEy veieren A acdsee 12 I UTES ATS DIU oes EN AE E EA E E AAE E A asec 12 CTCL IC CH OPMORSS 1S ein a E A ES 13 ZXDDIICalloliS iee E E A 14 Real inte PO Raani a N A E SE 15 iki T ae AIEN A AN PO IE T EE eg A E 15 Fluorescent Indicatots a2 entis E E foe po Da Dee eE 16 Calbrattonc C UPVE Suse estia t end CO ES Ops ee cutis i ape uo Seo baut OE c 19 TE QUIDIHODID secrete emet o ate Unten vette etui an EATE omen ce etas 22 UNO DUNC AUTO DIS raa Ro etospe etu snos uo AA eee 29 CHIPET SUM Maly o309 Aes yeast adres rales tae as eden ee etum oat eee 26 2 Parylene Microfluidics 277 ZA 2 2 2 3 2 3 MEMS Bae ke OrO Und eoe eee ied 10e epe ridotto oot io onto eil tete iae eiusd 27 General Microfluidics Technology ccccccccccccccececeeeeeeceaeseeesssssseeeeeeeeee
105. work on this device should focus on improving the fluorescent signal by adjusting the biochemical parameters and focusing on optical engineering In the longer term highly parallel arrays of these devices should be fabricated to allow simultaneous reactions including a calibration set for quantifying the initial DNA amount in the sample 126 References http en wikipedia org wiki Nucleotide www idtdna com http frodo wi mit edu primer3 input htm M McPherson and S Moller PCR Second Edition p 5 2006 F C Lawyer S Stoffel R K Saiki S Y Chang P A Landre R D Abramson and D H Gelfand High level expression purification and enzymatic characterization of full length Thermus aquaticus DNA polymerase PCR Methods and Applications 2 275 287 1993 User s manual for Platinum PCR Supermix part no 11306 pps Invitrogen Revised 01 July 2003 http www juliantrubin com encyclopedia biochemistry pcr html Bio Rad website www bio rad com Product Manual for SYBR Green I MP07567 Invitrogen Revised 16 March 2006 I H Zipper et al nvestigations on DNA intercalation and surface binding by SYBR Green I its structure determination and methodological implications Nucleic Acids Research 32 e103 2004 11 Invitrogen company website www invitrogen com Q Y Zeng S O Westermark A Rasmuson Lestander and X R Wang Detection and quantification of Cladosporium in aerosols by real
106. y baked by a long several days and hot 140 C or higher hard bake dissolution can take longer More aggressive conditions such as higher temperature soaking and other solvents may speed up this step however this increases the likelihood that solvent can penetrate the parylene parylene interface or parylene substrate interface to delaminate the channel For soak times in the time scale of months solvent penetration through the parylene is a concern as solvent molecules can remain in the parylene interfaces A high performance liquid chromatography HPLC chip based on this scheme has been fabricated Alternatively fluidic channels can also be made using embedded channel technology 49 b Figure 2 10 Embedded channel technology An outline of the steps shown in Figure 2 10 is provided below An overhang material black such as silicon oxide is deposited and patterned over a substrate dark grey such as silicon This overhang material must be able to withstand the following substrate etching step and still be a free standing structure This limits the extent to which the silicon can be undercut the width of the channel For microfluidics it is highly preferred that this material 1s transparent to allow optical monitoring of fluid in the channel The silicon is then isotropically etched causing an undercut that forms the overhang structure A suitable method is silicon etching by XeF gas especially if wide channels

A Parylene Real Time PCR Microdevice

Contents

Download Pdf Manuals

Related Search

Related Contents