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[资源] PARYLENE AS A NEW MEMBRANE MATERIAL FOR BIOMEMS APPLICATIONS

PARYLENE AS A NEW MEMBRANE MATERIAL FOR BIOMEMS APPLICATIONS
Thesis by
Bo Lu
In Partial Fulfillment of the Requirements for the Degree of
Doctor of Philosophy
California Institute of Technology
Pasadena, California
2012
(Defended April 26th, 2012)
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© 2012
Bo Lu
All Rights Reserved

Abstract
Parylene as a New Membrane Material for BioMEMS Applications
Thesis by
Bo Lu
Doctor of Philosophy in Electrical Engineering
California Institute of Technology
The work in this thesis aims to use MEMS and microfabrication technologies to develop two types of parylene membrane devices for biomedical applications. The first device is the parylene membrane filter for cancer detection. The presence of circulating tumor cells (CTC) in patient blood is an important sign of cancer metastasis. However, currently there are two big challenges for CTC detection. First, CTCs are extremely rare, especially at the early stage of cancer metastasis. Secondly, CTCs are very fragile, and are very likely to be damaged during the capturing process. By using size-based membrane filtration through the specially designed parylene filters, together with a constant-pressure filtration system, we are able to capture the CTCs from patient blood
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with high capture efficiency, high viability, moderate enrichment, and high throughput. Both immunofluorescence enumeration and telomerase activity detection have been used to detect and differentiate the captured CTCs. The feasibility of further cell culture of the captured CTCs has also been demonstrated, which could be a useful way to increase the number of CTCs for future studies. Models of the time-dependent cell membrane damage are developed to predict and prevent CTC damage during this detection process. The results of clinical trials further demonstrate that the parylene membrane filter is a promising device for cancer detection.
The second device is the parylene artificial Bruch’s membrane for age-related macular degeneration (AMD). AMD is usually characterized by an impaired Bruch’s membrane with much lowered permeability, which impedes the transportation of nutrients from choroid vessels to nourish the retinal pigment epithelial (RPE) cells and photoreceptors. Parylene is selected as a substitute material because of its good mechanical properties, transparency, biocompatibility, and machinability. More importantly, it is found that the permeability of submicron parylene is very similar to that of healthy human Bruch’s membrane. A mesh-supported submicron parylene membrane structure has been designed and its feasibility as an artificial Bruch’s membrane has been demonstrated by diffusion experiments, cell perfusion culture, and pressure deflection tests. RPE cells are able to adhere, proliferate and develop into normal in vivo-like morphology and functions. Currently this artificial membrane is under clinical trials.

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Table of Contents
Chapter 1: Introduction ............................................................................................. 1
1.1 Parylene: An Ideal Material for BioMEMS ......................................................... 1
1.2 Latest Achievements in Parylene BioMEMS ....................................................... 2
1.3 Parylene Membrane Devices ................................................................................ 4
1.4 Parylene Processing Technologies ....................................................................... 7
1.4.1 Parylene dry etching........................................................................... 8
1.4.2 Parylene adhesion consideration ........................................................ 9
1.4.3 Parylene molding technique ............................................................. 11
1.4.4 Parylene channel formation ............................................................. 12
1.4.5 Parylene fill-in technique ................................................................. 15
1.5 Layout of the Dissertation .................................................................................. 16
1.6 References .......................................................................................................... 16
Chapter 2: Characterizations of Parylene Membranes ........................................ 19
2.1 Overview ............................................................................................................ 19
2.2 Mechanical Characterization .............................................................................. 20
2.2.1 A comparison of normal and ultrathin membranes .......................... 20
2.2.2 Strategy to enhance the strength of ultrathin membrane.................. 21
2.3 Semipermeability of Submicron Parylene Membranes ...................................... 22
2.3.1 Measurements of the diffusion coefficients ..................................... 23
2.3.2 Molecular weight/radius exclusion limits ........................................ 26
2.4 Parylene Autofluorescence ................................................................................. 27
2.4.1 Motivation ........................................................................................ 27
2.4.2 Comparisons of parylene with other polymers/plastics ................... 29
2.4.3 Autofluorescence behaviors during UV illumination ...................... 32
2.4.4 The mechanism of autofluorescence in parylene-C/D/N films ........ 36
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2.4.5 Parylene-HT: A better choice for autofluorescence concerns .......... 41
2.4.6 Autofluorescence induced in microfabrication process ................... 43
2.5 Hydrophilic and Hydrophobic Parylene Membranes ......................................... 45
2.5.1 The importance of surface hydrophilicity/hydrophobicity .............. 45
2.5.2 Plasma treatments and their effects on parylene surfaces ................ 46
2.5.3 Superhydrophobic parylene membrane ........................................... 48
2.6 Summary ............................................................................................................ 54
2.7 References .......................................................................................................... 54
Chapter 3: Parylene Membrane Filters for Cancer Detection ............................. 59
3.1 Overview ............................................................................................................ 59
3.2 Current Technologies for CTC Detection .......................................................... 61
3.2.1 Enriching methods ........................................................................... 61
3.2.2 Detection methods ........................................................................... 64
3.3 Parylene Membrane Filters ................................................................................ 65
3.3.1 Parylene: An ideal membrane filter material ................................... 65
3.3.2 1st generation: 2D pore filter ............................................................ 66
3.3.3 2nd generation: 3D pore filter ........................................................... 68
3.3.4 3rd generation: 3D gap filter ............................................................. 72
3.3.5 4th generation: 2D slot filter ............................................................. 77
3.4 Experiment Results and Clinical Trials .............................................................. 81
3.4.1 Immunofluorescence method ........................................................... 81
3.4.2 Telomerase activity detection ........................................................... 84
3.5 Discussion: Further Optimizations ..................................................................... 88
3.5.1 Enrichment improvement ................................................................. 88
3.5.2 Sensitivity improvement .................................................................. 89
3.5.3 Viability improvement ..................................................................... 90
3.5.4 Throughput improvement ................................................................ 91
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3.6 CTC Culture After Capture ................................................................................ 91
3.7 A Biomechanical Study of Cell Membrane Damage ......................................... 97
3.7.1 Motivation ........................................................................................ 97
3.7.2 Experiment approach ....................................................................... 98
3.7.3 Cell modeling ................................................................................... 99
3.7.4 Simulation ...................................................................................... 100
3.7.5 Time-dependent viability drop ....................................................... 101
3.7.6 Molecular membrane failure model ............................................... 103
3.7.7 Griffith’s membrane failure model ................................................ 105
3.7.8 Comparison of models ................................................................... 107
3.7.9 Prediction of the safe “golden zone” .............................................. 108
3.8 Summary .......................................................................................................... 109
3.9 References ........................................................................................................ 110
Chapter 4: Parylene Artificial Bruch’s Membrane .............................................. 115
4.1 Overview .......................................................................................................... 115
4.2 Existing Therapies for AMD ............................................................................ 117
4.3 Parylene Artificial Bruch’s Membrane ............................................................. 118
4.3.1 Submicron parylene: A potential candidate ................................... 118
4.3.2 Perfusion cell-culture experiments ................................................. 120
4.3.3 Mesh-supported submicron parylene ............................................. 122
4.3.4 Mechanical optimization ................................................................ 124
4.3.5 RPE cell culture on the MSPM ...................................................... 126
4.3.6 Comparison of MSPM and porous membrane ............................... 128
4.4 Animal Trials .................................................................................................... 130
4.4.1 The “lollipop” design ..................................................................... 130
4.4.2 Mechanical implantation platform ................................................. 131
4.4.3 Microfluidic implantation tool ....................................................... 133
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4.4.4 Post-implantation staining and imaging ......................................... 136
4.5 RPE Cage ......................................................................................................... 137
4.5.1 Motivation ...................................................................................... 137
4.5.2 Cage design and fabrication ........................................................... 138
4.5.3 Cage assembling ............................................................................ 141
4.5.4 Preliminary results ......................................................................... 142
4.6 Summary .......................................................................................................... 143
4.7 References ........................................................................................................ 144
Chapter 5: Conclusions .......................................................................................... 147
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List of Figures
Figure 1-1: Chemical structures of major members in the parylene family ................ 2
Figure 1-2: Gold coated parylene membrane with gratings for SPR application ....... 5
Figure 1-3: The concept and operation method of the parylene-based cell origami ... 6
Figure 1-4: The concept and operation procedure of the selective patterning of cells or proteins using parylene membranes as peeling masks ............................................ 7
Figure 1-5: Parylene molding technique used in forming a parylene membrane with gratings for SPR application ...................................................................................... 11
Figure 1-6: Parylene molding technique for superhydrophobic films ...................... 12
Figure 1-7: Fabrication process flow of surface-micromachined parylene channels with sacrificial photoresist ......................................................................................... 13
Figure 1-8: The fabrication process of an embedded parylene channel .................... 14
Figure 1-9: Illustration of the parylene fill-in process .............................................. 15
Figure 2-1: Stress-strain curves of parylene films with different thicknesses, measured by DMA at room temperature ................................................................... 20
Figure 2-2: (a) The membrane deflection test setup; (b) Uniform submicron parylene is broken at a low pressure load; (c) The composited membrane is broken at a much higher pressure load. .................................................................................................. 22
Figure 2-3: Schematic of the diffusivity measurement with blind-well chambers ... 24
Figure 2-4: Diffusion coefficients of dextran molecules in submicron parylene-C membranes with different thicknesses ....................................................................... 25
Figure 2-5: Molecular weight exclusion limits of submicron parylene-C ................ 26
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Figure 2-6: Strong autofluorescence in parylene-C based dual-layer membrane CTC
microfilter .................................................................................................................. 28
Figure 2-7: Structure of parylene-C, -D, -N, -HT films ............................................ 29
Figure 2-8: Comparisons of relative initial autofluorescence intensities of parylene-C
with other polymers and plastics ............................................................................... 31
Figure 2-9: Enhanced blue, green, and red autofluorescence of parylene-C film after
2 minutes short-time UV illumination ....................................................................... 33
Figure 2-10: Quantitative fluorescence intensity variations of parylene films during
continuous short-time UV illumination ..................................................................... 34
Figure 2-11: Quantitative blue fluorescence intensity variations of parylene films
during continuous long-time UV illumination .......................................................... 36
Figure 2-12: Fluorescence spectra of parylene-C film, under 280 nm excitation,
measured by fluorimeter ............................................................................................ 38
Figure 2-13: Infrared spectra of parylene-C film ...................................................... 38
Figure 2-14: Fluorescence spectra of parylene-HT dimer and film .......................... 42
Figure 2-15: Comparisons of autofluorescence of unpatterned parylene-C film and
parylene-C based devices .......................................................................................... 44
Figure 2-16: The effects of fluorine plasma treatment .............................................. 47
Figure 2-17: AFM evaluation of the rms surface roughness ..................................... 48
Figure 2-18: Fabrication process of two types of superhydrophobic films ............... 50
Figure 2-19: SEM images of the superhydrophobic films ........................................ 50
Figure 2-20: Water droplet on a superhydrophobic film (lotus leaf) ......................... 51
Figure 2-21: Water droplet on a superhydrophobic film (rose petal) ........................ 52
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Figure 2-22: Advantages of parylene superhydrophobic film ................................... 53
Figure 3-1: SEM images of 2D pore filters. .............................................................. 67
Figure 3-2: Cells were partially damaged or totally lysed after filtration ................. 68
Figure 3-3: 3D pore filter design ............................................................................... 69
Figure 3-4: Fabrication process flow of the 3D pore filter ........................................ 69
Figure 3-5: Photos of the fabricated 3D microfilter .................................................. 70
Figure 3-6: Device is assembled inside a housing cassette ....................................... 70
Figure 3-7: Comparison of 2D and 3D filters with unfixed MCF-7 cells ................. 71
Figure 3-8: SEM image of a MCF-7 cell captured on the 3D pore filter .................. 72
Figure 3-9: Comparison of “pore capture” and “gap capture” mechanisms ............. 73
Figure 3-10: Fabrication process flow of the 3D gap filter ....................................... 73
Figure 3-11: Photos of the 3D gap filter. ................................................................... 74
Figure 3-12: Filtration setup using constant pressure driving ................................... 74
Figure 3-13: Fluorescent image of a model system testing ....................................... 75
Figure 3-14: Release of trapped tumor cells from filter using a brush ...................... 76
Figure 3-15: The low enrichment of the 3D pore filter ............................................. 77
Figure 3-16: Constant-pressure-driven filtration system, the filter assembly, and the
SEM image of fabricated slot filter ........................................................................... 78
Figure 3-17: 2D slot filter characterization ............................................................... 80
Figure 3-18: Cancer cells captured on the slot filter ................................................. 80
Figure 3-19: On-filter immunofluorescence staining of captured CTCs .................. 83
Figure 3-20: Histogram demonstrating performance comparison of membrane
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microfilter vs. CellSearch® assay in clinical samples .............................................. 84
Figure 3-21: Detection of telomerase activity from live cancer cells captured on a
slot microfilter ........................................................................................................... 85
Figure 3-22: Single-cell telomerase measurement .................................................... 87
Figure 3-23: CTC filtration and enumeration experiments with parylene-C and
parylene-HT membrane filters .................................................................................. 89
Figure 3-24: On-filter (a) and off-filter (b) cell culture of captured PC-3 cells from
human blood after 3 days and 6 days in RPMI complete medium ............................ 92
Figure 3-25: Parylene surface treatments and their influences on cell adherence .... 93
Figure 3-26: Parylene-C/HT filter fabrication process .............................................. 94
Figure 3-27: Operation procedure of 3D on-filter culture ......................................... 95
Figure 3-28: 3D on-filter cultured tumor growth rate and viability .......................... 96
Figure 3-29: Immuno-staining of cultured 3D tumor ................................................ 96
Figure 3-30: Cortical-shell/liquid-core model of a cell captured on a slot ............... 99
Figure 3-31: COMSOL simulation results .............................................................. 101
Figure 3-32: Time-dependent viability drop at different drive pressures ................ 102
Figure 3-33: Schematic of nanopore formation in lipid bilayer membrane ............ 104
Figure 3-34: Relations between the mean cell lysis time τ and the membrane tension
σ for (a) the molecular membrane failure model and (b) the Griffith’s membrane
failure model ............................................................................................................ 106
Figure 3-35: The safe “golden zones” predicted by (a) the molecular membrane
failure model and (b) the Griffith’s membrane failure model ................................. 109
Figure 4-1: Schematics of the Bruch’s membrane and RPE cells ........................... 116
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Figure 4-2: Comparisons of the permeability of submicron parylene, human Bruch’s
membranes, lens capsule and collagen film in terms of the diffusion flux ............. 119
Figure 4-3: Perfusion cell viability tests .................................................................. 121
Figure 4-4: Fabrication process of the MSPM ........................................................ 122
Figure 4-5: SEM images of (a) the front side, (b) the back side, and (c) the cross
section of a MSPM with 0.30 μm ultrathin parylene .............................................. 123
Figure 4-6: Membrane deflection experiment ......................................................... 124
Figure 4-7: A wrinkled MSPM (a) and a broken MSPM (b) ................................... 125
Figure 4-8: Yielding pressures and breakdown pressures for different MSPMs ..... 125
Figure 4-9: H9-RPE cell culture on the MSPM ...................................................... 127
Figure 4-10: Porous parylene substrate with through holes (1 μm in diameter) ..... 129
Figure 4-11: Comparisons of morphologies of RPE cells on different substrates .. 129
Figure 4-12: Current designs of rat’s and pig’s lollipops and their dimensions ...... 131
Figure 4-13: Diagrammatic sketch of the implantation tool ................................... 132
Figure 4-14: Fabrication process flow of the microfluidic implantation tool ......... 134
Figure 4-15: The microfluidic implantation tool ..................................................... 134
Figure 4-16: Implantation of a lollipop device into a rat’s eye ............................... 135
Figure 4-17: Hematoxylin-eosin staining one week after implantation .................. 136
Figure 4-18: Immunofluorescence staining of the of RPE cells ............................. 136
Figure 4-19: The concept of the RPE cage .............................................................. 138
Figure 4-20: Cell migration experiments. The inset shows the transwell setup ...... 139
Figure 4-21: Fabrication process flow of the cage’s top cover ............................... 140
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Figure 4-22: SEM image of the cover and a close-up view of the opening ............ 140
Figure 4-23: The assembling and operation procedure of the cage for in vitro cell
culture. ..................................................................................................................... 141
Figure 4-24: RPE cell culture experiments inside the 3D cage ............................... 142
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List of Tables
Table 1-1: Percentage of papers related to parylene in recent conferences ................. 3
Table 2-1: Comparison of the mechanical strength between the uniform submicron parylene membrane and the composited membrane ................................................. 22
Table 2-2: Molecular radius exclusion limits of submicron parylene-C membranes 27
Table 3-1: Comparisons of various CTC enriching technologies ............................. 64
Table 3-2: Capture efficiency with different gap sizes .............................................. 76
Table 3-3: Comparison of 2D slot filters with different open factors ....................... 88
Table 3-4: Fitting parameters of experimental data in our work on cancer cells by filtration, Rand’s work on RBCs by micropipette aspiration, and Taupin’s work on RBCs by osmotic expansion .................................................................................... 108

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