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«Thesis by Bo Lu In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy California Institute of Technology Pasadena, ...»

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Thesis by

Bo Lu

In Partial Fulfillment of the Requirements

for the Degree of

Doctor of Philosophy

California Institute of Technology

Pasadena, California

(Defended April 26th, 2012)


© 2012

Bo Lu

All Rights Reserved



All the work in this thesis would definitely not be possible without the remarkable guidance from my Ph.D. research advisor, Dr. Yu-Chong Tai. Frequently, I feel I have been so lucky to be able to work with him in the past five years at Caltech. He has taught me how to think and behave like a Ph.D. What I have learned from him is not only limited to research, but also includes many interpersonal skills. Whenever I got inspired and proposed new ideas, he gave me encouragement and strong support. Whenever I met difficulties, he helped me analyze the problem and find the solutions. Whenever I made a mistake, he was always very tolerant, pointing out my mistake first and then helping me correct it, patiently. I ought to give most credit to him for my research achievements so far, and for possibly more in the future.

I will never forget the tremendous help from all the previous and current members at Dr. Tai’s Caltech MEMS Group, including the previous members Dr. Siyang Zheng, Dr.

Changlin Pang, Dr. Wen Li, Dr. Po-Jui (PJ) Chen, Dr. Nick Lo, Dr. Jason Shih, Dr. Mike Liu, Dr. Quoc (Brandon) Quach, Dr. Ray Huang, Dr. Luca Giacchino, Dr. Jeffrey Chun-Hui Lin, Ms. Yingying Wang, and Mr. Zhao Liu, and the current group members Mandheerej (Monty) Nandra, Justin Young-Hyun Kim, Wendian Shi, Penvipha (Yok) Satsanarukkit, Jay Han-Chieh Chang, Yu Zhao, Charles DeBoer, Dongyang Kang, Yang Liu, Nick Scianmarello, and Shell Zhang. They are not only my colleagues but also my friends. I also appreciate our lab manager Trevor Roper and group assistant Christine iv Garske. Without Trevor, the cleanroom would be down; without Christine, our group would be unorganized.

The multidisciplinary nature of my research enabled me to gain plenty of biological, medical, and surgical knowledge from my collaborators at the Norris Cancer Center and the Doheny Eye Institute of the University of Southern California. I would like to thank Dr. Mark S. Humayun, Dr. David Hinton, Dr. Biju Thomas, Dr. Amir Goldkorn, Dr. Tong Xu, Dr. Danhong Zhu, Ms. Laura Liu, Dr. Yuntao Hu, Dr. Jing Xu, and Mr. Kaijie He for their helps and suggestions on my research. I would also like to thank my friend, Mr.

Kuang Shen from the Chemistry Department of Caltech, who helped me alot on the material studies, as well as Mr. Guoan Zheng from Electrical Engineering of Caltech, who helped me a lot with the optics.

Moreover, I would like to thank my Ph.D. candidacy and defense committee members, including Dr. Changhuei Yang, Dr. Hyuck Choo, Dr. Joel W. Burdick, and Dr.

Chin-Lin Guo.

I am deeply grateful to my parents, grandma, and uncle. They have always stood firmly with me and backed me up throughout my life. Last, but the most important, special thanks are due to my fiancée, Jie Zhu. Her love and support helped me overcome

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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

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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

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Chapter 1: Introduction

1.1 Parylene: An Ideal Material for BioMEMS

1.2 Latest Achievements in Parylene BioMEMS

1.3 Parylene Membrane Devices

1.4 Parylene Processing Technologies

1.4.1 Parylene dry etching

1.4.2 Parylene adhesion consideration

1.4.3 Parylene molding technique

1.4.4 Parylene channel formation

1.4.5 Parylene fill-in technique

1.5 Layout of the Dissertation

1.6 References

Chapter 2: Characterizations of Parylene Membranes

2.1 Overview

2.2 Mechanical Characterization

2.2.1 A comparison of normal and ultrathin membranes

2.2.2 Strategy to enhance the strength of ultrathin membrane.................. 21

2.3 Semipermeability of Submicron Parylene Membranes

2.3.1 Measurements of the diffusion coefficients

2.3.2 Molecular weight/radius exclusion limits

2.4 Parylene Autofluorescence

2.4.1 Motivation

2.4.2 Comparisons of parylene with other polymers/plastics

2.4.3 Autofluorescence behaviors during UV illumination

2.4.4 The mechanism of autofluorescence in parylene-C/D/N films........ 36 viii 2.4.5 Parylene-HT: A better choice for autofluorescence concerns.......... 41 2.4.6 Autofluorescence induced in microfabrication process

2.5 Hydrophilic and Hydrophobic Parylene Membranes

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

2.6 Summary

2.7 References

Chapter 3: Parylene Membrane Filters for Cancer Detection

3.1 Overview

3.2 Current Technologies for CTC Detection

3.2.1 Enriching methods

3.2.2 Detection methods

3.3 Parylene Membrane Filters

3.3.1 Parylene: An ideal membrane filter material

1st generation: 2D pore filter

3.3.2 2nd generation: 3D pore filter

3.3.3 3rd generation: 3D gap filter

3.3.4 4th generation: 2D slot filter


3.4 Experiment Results and Clinical Trials

3.4.1 Immunofluorescence method

3.4.2 Telomerase activity detection

3.5 Discussion: Further Optimizations

3.5.1 Enrichment improvement

3.5.2 Sensitivity improvement

3.5.3 Viability improvement

3.5.4 Throughput improvement


3.6 CTC Culture After Capture

3.7 A Biomechanical Study of Cell Membrane Damage

3.7.1 Motivation

3.7.2 Experiment approach

3.7.3 Cell modeling

3.7.4 Simulation

3.7.5 Time-dependent viability drop

3.7.6 Molecular membrane failure model

3.7.7 Griffith’s membrane failure model

3.7.8 Comparison of models

3.7.9 Prediction of the safe “golden zone”

3.8 Summary

3.9 References

Chapter 4: Parylene Artificial Bruch’s Membrane

4.1 Overview

4.2 Existing Therapies for AMD

4.3 Parylene Artificial Bruch’s Membrane

4.3.1 Submicron parylene: A potential candidate

4.3.2 Perfusion cell-culture experiments

4.3.3 Mesh-supported submicron parylene

4.3.4 Mechanical optimization

4.3.5 RPE cell culture on the MSPM

4.3.6 Comparison of MSPM and porous membrane

4.4 Animal Trials

4.4.1 The “lollipop” design

4.4.2 Mechanical implantation platform

4.4.3 Microfluidic implantation tool

x 4.4.4 Post-implantation staining and imaging

4.5 RPE Cage

4.5.1 Motivation

4.5.2 Cage design and fabrication

4.5.3 Cage assembling

4.5.4 Preliminary results

4.6 Summary

4.7 References

Chapter 5: Conclusions


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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

Figure 1-5: Parylene molding technique used in forming a parylene membrane with gratings for SPR application

Figure 1-6: Parylene molding technique for superhydrophobic films

Figure 1-7: Fabrication process flow of surface-micromachined parylene channels with sacrificial photoresist

Figure 1-8: The fabrication process of an embedded parylene channel

Figure 1-9: Illustration of the parylene fill-in process

Figure 2-1: Stress-strain curves of parylene films with different thicknesses, measured by DMA at room temperature

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

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

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Figure 2-6: Strong autofluorescence in parylene-C based dual-layer membrane CTC microfilter

Figure 2-7: Structure of parylene-C, -D, -N, -HT films

Figure 2-8: Comparisons of relative initial autofluorescence intensities of parylene-C with other polymers and plastics

Figure 2-9: Enhanced blue, green, and red autofluorescence of parylene-C film after 2 minutes short-time UV illumination

Figure 2-10: Quantitative fluorescence intensity variations of parylene films during continuous short-time UV illumination

Figure 2-11: Quantitative blue fluorescence intensity variations of parylene films during continuous long-time UV illumination

Figure 2-12: Fluorescence spectra of parylene-C film, under 280 nm excitation, measured by fluorimeter

Figure 2-13: Infrared spectra of parylene-C film

Figure 2-14: Fluorescence spectra of parylene-HT dimer and film

Figure 2-15: Comparisons of autofluorescence of unpatterned parylene-C film and parylene-C based devices

Figure 2-16: The effects of fluorine plasma treatment

Figure 2-17: AFM evaluation of the rms surface roughness

Figure 2-18: Fabrication process of two types of superhydrophobic films............... 50 Figure 2-19: SEM images of the superhydrophobic films

Figure 2-20: Water droplet on a superhydrophobic film (lotus leaf)

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Figure 2-22: Advantages of parylene superhydrophobic film

Figure 3-1: SEM images of 2D pore filters.

Figure 3-2: Cells were partially damaged or totally lysed after filtration................. 68 Figure 3-3: 3D pore filter design

Figure 3-4: Fabrication process flow of the 3D pore filter

Figure 3-5: Photos of the fabricated 3D microfilter

Figure 3-6: Device is assembled inside a housing cassette

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

Figure 3-11: Photos of the 3D gap filter.

Figure 3-12: Filtration setup using constant pressure driving

Figure 3-13: Fluorescent image of a model system testing

Figure 3-14: Release of trapped tumor cells from filter using a brush

Figure 3-15: The low enrichment of the 3D pore filter

Figure 3-16: Constant-pressure-driven filtration system, the filter assembly, and the SEM image of fabricated slot filter

Figure 3-17: 2D slot filter characterization

Figure 3-18: Cancer cells captured on the slot filter

Figure 3-19: On-filter immunofluorescence staining of captured CTCs.................. 83

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microfilter vs. CellSearch® assay in clinical samples

Figure 3-21: Detection of telomerase activity from live cancer cells captured on a slot microfilter

Figure 3-22: Single-cell telomerase measurement

Figure 3-23: CTC filtration and enumeration experiments with parylene-C and parylene-HT membrane filters

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

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