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Design, Fabrication, and Characterization of Polymer-Based DESIGN, FABRICATION, AND CHARACTERIZATION OF POLYMER-BASED CANTILEVER PROBES FOR ATOMIC FORCE MICROSCOPY OF LIVE MAMMALIAN CELLS IN LIQUID By FANGZHOU YU A thesis submitted to the Graduate school-New Brunswick Rutgers, the State University of New Jersey In partial fulfillment of the requirements For the degree of Master of Science Graduate Program in Electrical and Computer Engineering Written under the direction of Jaeseok Jeon And approved by New Brunswick, New Jersey October 2016 ABSTRACT OF THE THESIS Design, Fabrication, and Characterization of Polymer-Based Cantilever Probes for Atomic Force Microscopy of Live Mammalian Cells in Liquid by FANGZHOU YU Thesis Director: Jaeseok Jeon This thesis presents the design, fabrication, and characterization of polymer-based cantilever probes for atomic force microscopes (AFMs), in order to enable biological research requiring non-destructive high-speed high-resolution topographical imaging and nanomechanical characterizations of sub-cellular and cellular samples. A reliable low-cost surface- micromachining process is developed for the rapid prototyping of bio-compatible polymer-based V-shaped AFM probes. The physical properties of fabricated prototypes, such as effective spring constant, resonant frequency, and quality factor, are determined experimentally via thermal noise method and analytically via finite element and parallel-beam approximation methods. Using a prototype, AFM nanoindentation measurements are performed on live mammalian cells— human cervical epithelial cancer cells (called “HeLa”) in a liquid culture medium. Experimental results are compared to those obtained using a commercial Si-based probe; when the prototype probe is used, the deformation and/or distortion of the cell membrane are reduced significantly albeit repeated indentations on the cell surface. For further AFM-based biological studies, the ii design and fabrication process of the prototype probe are fine-tuned; a reasonably straight cantilever with a strain gradient as low as 10-4 μm-1 is achieved via corrugating the optical reflection coating or confining it to the tip region, and a sharp tip with a radius of curvature as small as ~40 nm, which is comparable to that of a Si-based probe, is achieved via sequential depositions of low- and high-viscosity acrylic polymers. iii Acknowledgments First, I would like to express the deepest appreciation to my advisor Prof. Jaeseok Jeon for his support, patience, and guidance in my research towards completing my master’s thesis. Without his guidance and persistent help, this dissertation would not be possible. I would like to thank my committee members, Prof. Zoran Gajic, Prof. Qingze Zou, and Prof. Mehdi Javanmard for their evaluation on my research work. Without their direction, I would not have been able to complete my master’s thesis. It is my pleasure to work with selfless and professional people in this project. I would like to thank Yanbiao Pan and Jiangbo Liu for helping me with design and testing. Their countless inputs make this project come true. I would like to thank Zhengyu Yang and Nabeela Khan for simulation section. I would also like to thank Ai-Lian Lin and Shiyan Yu for their assistance in device fabrication and testing. I am also indebted to all the staff and co-workers in Microelectronics Research Laboratory (MERL) of Rutgers University and Center for Functional Nanomaterials (CFN) of Brookhaven National Lab. Specially thanks to Robert Lorber, Pavel Reyes, Ming Lu, Wen-Chiang Hong, and Rui Li for their training and valuable advice on my project. Finally, I would like to thank my parents and my fiancée for their support and encouragement to pursue my research. iv Table of Contents Abstract ................................................................................................................................................... ii Acknowledgment .................................................................................................................................... iv List of Tables ......................................................................................................................................... vii List of Figures ................................................................................................................................... viii 1. INTRODUCTION .............................................................................................................................. 1 1.1. Silicon-based AFM cantilever probe ................................................................................... 1 1.2. Polymer-based AFM cantilever probe ................................................................................. 2 1.3. Objective and Approach ...................................................................................................... 2 1.4. Organization of Thesis ......................................................................................................... 4 2. PROPOSED POLYMER-BASED AFM CANTILEVER PROBE ................................................. 5 2.1. Design and structure ............................................................................................................ 5 2.2. Fabrication Process ............................................................................................................. 6 2.3. Characterization .................................................................................................................. 9 2.3.1. Thermal Noise Method ...................................................................................... 9 2.3.2. Finite Element Method ...................................................................................... 11 2.3.3. Parallel Beam Approximation Method .............................................................. 11 3. EXPERIMENTAL EVALUATION WITH NANOINDENTATION ........................................... 18 3.1. Preparation of Biological Samples ..................................................................................... 18 3.2. Experimental Setup and Method ........................................................................................ 19 3.3. Experimental Setup and Method ........................................................................................ 19 4. FINE-TUNED PMMA-BASED CANTILEVER PROBE .............................................................. 23 4.1. Design and Fabrication ....................................................................................................... 23 4.2. Implementation ................................................................................................................... 25 4.2.1. Sharpness of Probe ............................................................................................ 25 4.2.2. Strain Gradient of Cantilever ............................................................................. 27 v 4.3. Characterization ................................................................................................................. 30 5. CONCLUSION ................................................................................................................................. 33 vi List of Tables 4.1. Adhesion force Fad for a spherical tip on a flat surface. ….......................................................... 26 4.2. Viscosity values of various PMMA compounds …..................................................................... 27 vii List of Figures 2.1 Isometric, Design parameters and values of polymer-based cantilever probes. …........................ 5 2.2 A low-cost three-mask surface-micromachining process. …......................................................... 7 2.3 Plan-view micrographs and SEMs of fabricated cantilever probes. ….......................................... 8 2.4 Simplified schematic of the AFM system setup used for this work. …......................................... 9 2.5 Measured thermal noise frequency spectrum. …........................................................................... 10 2.6 Rectangular AFM cantilever. …..................................................................................................... 12 2.7 Cross-sectional view of V-shaped AFM cantilever. ….................................................................. 12 2.8 Customized parallel beam approximation. …................................................................................. 14 2.9 Measured, FEM, and analytical keff values of probe prototype. …................................................. 17 3.1 Phase-contrast microscopy of HeLa cells. ….................................................................................. 18 3.2 Experimental setup for AFM nanoindentation on live cells in liquid. …....................................... 19 3.3 Measured force-probe interaction forces Fint with Vpp = 3 V and fload = 1 Hz load input. ….......... 21 3.3 Measured force-probe interaction forces Fint with Vpp = 3 V and fload = 8 Hz load input. ….......... 21 3.5 Measured time to half Fint vs. force load rate. …............................................................................ 22 3.6 Measured time to half Fint vs. peak-to-peak input voltage, Vpp. ….................................................. 22 4.1 Isometric, Design parameters and values of fine-tuned polymer-based cantilever probes. …....... 23 4.2 Surface-micromachining process flow used to fabricate the AFM probes and SEMs. ….............. 25 4.3 Measured radii of curvature of
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