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[资源] A Parylene Real Time PCR Microdevice

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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