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A Parylene Real Time PCR Microdevice
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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 2009 (Defended December 4th, 2009) ii © 2009 Quoc (Brandon) Quach All Rights Reserved iii To Cuong Quach and Nga Huynh iv It never ceases to amuse me that I was once a 7year old weekend factory worker, a 10year old dry cleaner, and now a Caltech PhD graduate all within a 10 mile radius. Cheers to the American Dream! 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 is 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 new 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. ix Table of Contents 1 PCR and Real Time PCR ...................................1 1.1 Introduction to the Polymerase Chain Reaction ................................................. 1 1.1.1 Components ................................................................................................2 1.1.2 Procedure ....................................................................................................8 1.1.3 Molecular Level Theory ........................................................................... 12 1.1.4 Equipment .................................................................................................12 1.1.5 Gel Electrophoresis...................................................................................13 1.1.6 Applications ..............................................................................................14 1.2 Real Time PCR ................................................................................................. 15 1.2.1 Theory.......................................................................................................15 1.2.2 Fluorescent Indicators...............................................................................16 1.2.3 Calibration Curves ....................................................................................19 1.2.4 Equipment .................................................................................................22 1.2.5 Applications ..............................................................................................23 1.3 Chapter Summary .............................................................................................26 2 Parylene Microfluidics......................................27 2.1 MEMS Background ..........................................................................................27 2.2 General Microfluidics Technology ................................................................... 27 2.3 MEMS Technologies for PCR Microdevices ................................................... 31 2.3.1 Bulk Micromachining ...............................................................................31 x 2.3.2 Soft Lithography .......................................................................................35 2.3.3 Surface Micromachining...........................................................................37 2.4 Parylene MEMS Technology............................................................................ 38 2.4.1 Why Use Parylene?................................................................................... 38 2.4.2 Parylene Chemical Structure..................................................................... 40 2.4.3 Physical Properties....................................................................................41 2.4.4 Chemical Vapor Deposition Method ........................................................ 42 2.4.5 Patterning ..................................................................................................43 2.4.6 Biocompatibility of Parylene as a Real Time PCR Material .................... 53 2.5 Chapter Summary .............................................................................................61 3 RTPCR Microdevice, Air Gap Version...........62 3.1 Fabrication ........................................................................................................62 3.2 Fluidic Channel Design..................................................................................... 74 3.3 Device Thermal Engineering ............................................................................ 76 3.3.1 Heat Transfer Background........................................................................ 76 3.3.2 Device Thermal Design ............................................................................ 81 3.3.3 Thermal Performance Results................................................................... 87 3.4 Interface with Housing...................................................................................... 91 3.5 Device Performance..........................................................................................93 3.5.1 Real Time Polymerase Chain Reaction Components ............................... 93 3.5.2 Thermal Cycling Protocol (94, 72, 55; 30 s each) .................................... 96 3.5.3 Optical Detection Protocol........................................................................ 97 3.5.4 Results.....................................................................................................101 xi 3.6 Chapter Summary ........................................................................................... 103 4 RTPCR Microdevice, Free Standing Version 104 4.1 Fabrication ...................................................................................................... 104 4.2 Fluidic Channel Design................................................................................... 112 4.3 Device Thermal Engineering .......................................................................... 112 4.3.1 Heat Transfer Background...................................................................... 112 4.3.2 Device Thermal Design .......................................................................... 112 4.3.3 Thermal Performance Results................................................................. 116 4.4 Interface with Housing.................................................................................... 119 4.5 Device Performance........................................................................................ 122 4.5.1 Real Time Polymerase Chain Reaction Components ............................. 122 4.5.2 Thermal Cycling Protocol....................................................................... 122 4.5.3 Optical Detection Protocol...................................................................... 122 4.5.4 Results and Discussion ...........................................................................123 4.6 Chapter Summary ........................................................................................... 124 5 Conclusion........................................................125 References .............................................................126 xii List of Figures Figure 1-1: Basic concept of PCR amplification ................................................................ 1 Figure 1-2: Chemical structure of nucleoside triphosphates............................................... 3 Figure 1-3: Typical thermal recipe for PCR ...................................................................... 8 Figure 1-4: PCR schematic illustrating selective amplification of the target region between the primer pairs................................................................................................... 11 Figure 1-5: MJ Thermal Cycler from Bio-RAD .............................................................. 13 Figure 1-6: Chemical Structure of SYBR Green I............................................................ 16 Figure 1-7: Fluorescence spectrum of SYBR Green I ..................................................... 17 Figure 1-8: Schematic of TaqMan probes ........................................................................ 18 Figure 1-9: Amplification plots for a calibration curve. Replaces 16–20 are 10-fold serial dilutions............................................................................................................................. 20 Figure 1-10: Calibration curve for an M13 virus DNA sample........................................ 21 Figure 1-11: Strategene MX3005P benchtop RTPCR system.......................................... 23 Figure 1-12: Serial dilutions to determine sensitivity of assay......................................... 24 Figure 1-13: Assay specificity .......................................................................................... 25 Figure 2-1: Basic schematic of photolithography ........................................................... 28 Figure 2-2: Photolithography using a stepper.................................................................. 31 Figure 2-3: Example of bulk micromachining.................................................................. 32 Figure 2-4: PDMS micromolding ..................................................................................... 36 Figure 2-5: Surface micromachining ................................................................................ 38 Figure 2-6: Chemical Structure of Parylene ..................................................................... 40 Figure 2-7: Schematic of parylene CVD deposition......................................................... 42 xiii Figure 2-8: Chemical structure of di-p-xylylene, the dimer precursor to parylene N .... 42 Figure 2-9: Surface micromachined parylene channel ..................................................... 46 Figure 2-10: Embedded channel technology .................................................................... 49 Figure 2-11: Microfluidic components fabricated using parylene technology................. 51 Figure 2-12: Thermal isolation by parylene “stitches” ..................................................... 52 Figure 2-13: Integrated HPLC system .............................................................................. 53 Figure 2-14: QPCR on low volumes in parylene coated tubes........................................ 53 Figure 2-15: Amplification of 0.5 μl QPCR solution ...................................................... 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: QPCR with glass added into reaction tubes ................................................. 60 Figure 3-1: Overall process flow ..................................................................................... 62 Figure 3-2: Silicon chip ................................................................................................... 63 Figure 3-3: Patterned oxidation layer .............................................................................. 64 Figure 3-4: Metal deposition and patterning. Oxide layer (purple) underneath the metal (orange) acts as in electrical insulator............................................................................... 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. ..................................................... 66 Figure 3-6: First parylene deposition............................................................................... 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 is etched. ............................................................................................................................... 68 Figure 3-8: Etching of first parylene layer (light blue) and XeF2 etching of underlying silicon................................................................................................................................ 69 Figure 3-9: Inlet outlet hole ............................................................................................. 69 Figure 3-10: Second parylene patterning. Underlying oxide is once again the top layer.71 Figure 3-11: Air gap formation......................................................................................... 72 Figure 3-12: Zoom showing the parylene “stitches” used to connect the island to the main body................................................................................................................................... 72 Figure 3-13: Wire bonding on the completed chip. The wire bonds provide electrical continuity across the parylene-stitched air gap................................................................. 73 Figure 3-14: Bubble trapped in reaction chamber from early chip designs..................... 74 Figure 3-15: Channel layout ............................................................................................. 75 Figure 3-16: Channel cross section................................................................................... 76 Figure 3-17: EE analogue for thermal characterization................................................... 80 Figure 3-18: Thermally isolated island ............................................................................. 82 Figure 3-19: Parylene stiches............................................................................................ 83 Figure 3-20: Extended RC Model..................................................................................... 83 Figure 3-21 Temperature sensor calibration ..................................................................... 85 Figure 3-22: Temperature control hardware arrangement ............................................... 87 Figure 3-23: Steady state temperature ............................................................................. 88 Figure 3-24: Heating with step function applied power ................................................... 89 xv Figure 3-25: Temperature cooling dynamic with zero applied power.............................. 90 Figure 3-26: Chip housing assembly ................................................................................ 91 Figure 3-27: Chip housing components............................................................................ 92 Figure 3-28: Chip housing with external valves .............................................................. 93 Figure 3-29: Structure of the M13 virus ........................................................................... 94 Figure 3-30: Genome of the M13 virus ............................................................................ 95 Figure 3-31: Temperature recipes..................................................................................... 96 Figure 3-32: Filter block for SYBR Green I detection ..................................................... 97 Figure 3-33: SYBR Green fluorescence in microchannel .............................................. 100 Figure 3-34: Detection of M13 virus. Data normalization described above................... 101 Figure 3-35: Air gap chip versus conventional QPCR machine.................................... 102 Figure 4-1: Overall device fabrication steps.................................................................. 104 Figure 4-2: Bare silicon chip.......................................................................................... 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 transparent....................................................................................................................... 105 Figure 4-4: First parylene layer with representative holes. The holes are actually present throughout the outlined channel region. ......................................................................... 106 Figure 4-5: Channels etched into silicon. Bottom right is i/o hole. .............................. 107 Figure 4-6: Second parylene layer deposited................................................................. 108 Figure 4-7: Platinum pattern .......................................................................................... 109 Figure 4-8: Back side finishing. View from back side. Left: After DRIE. Right: After XeF2 ................................................................................................................................ 110 xvi Figure 4-9: Finished chip front and back....................................................................... 111 Figure 4-10: Platinum traces directly on parylene. Left: contact pads. Right: heaters113 Figure 4-11: Metal layout .............................................................................................. 113 Figure 4-12: Temperature sensor calibration.................................................................. 115 Figure 4-13: Simple circuit analogy ............................................................................... 116 Figure 4-14: Thermal resistance to heat transfer into environment............................... 118 Figure 4-15: Cooling experiments used to determine thermal time constant and capacitance...................................................................................................................... 120 Figure 4-16: Heating experiments .................................................................................. 120 Figure 4-17: Chip housing assembly. O-rings and pins not shown .............................. 121 Figure 4-18: Chip housing components......................................................................... 121 Figure 4-19: Detection of M13 virus on chip ................................................................. 123 Figure 4-20 Comparison of chip versus conventional machine. Sample volumes and surface-area-to-volume ratios of parylene were comparable.......................................... 123 xvii List of Tables Table 1-1: Technical specifications for the Bio-Rad MJ Mini PCR Machine................. 13 Table 2-1: Methods for etching silicon............................................................................ 32 Table 2-2: Physical values of parylene (Unless otherwise stated, values are from ref 17) ........................................................................................................................................... 41 Table 3-1: Thermal conductivity of selected materials.................................................... 77 Table 3-2: Values for calculation of Rayleigh number for air......................................... 79 Table 3-3: Nusselt numbers. All correlations from CRC Handbook47 ........................... 80 Table 3-4: Power specifications for the air gap version .................................................. 86 Table 3-5: Parameters used for thermal model ................................................................ 90 Table 4-1: Parameters for heater.................................................................................... 116[ Last edited by yipddicorp on 2014-2-20 at 09:52 ] |
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