MEMS IMPEDANCE BIOSENSOR FOR ACCURATE AND RAPID DETECTION OF E.COLI 0157:H7

116 Pages·2016·4.26 MB·English

MEMS IMPEDANCE BIOSENSOR FOR ACCURATE AND RAPID DETECTION OF
E.COLI 0157:H7
_______________________________________
A Dissertation
Presented to
The Faculty of the Graduate School
At the University of Missouri
_______________________________________________________
In Partial Fulfillment
of the Requirements for the Degree
Doctor of Philosophy
_____________________________________________________
by
SHIBAJYOTI GHOSH DASTIDER
Dr. Mahmoud Almasri, Dissertation Supervisor
JULY 2016
The undersigned, appointed by the dean of the Graduate School,
have examined the dissertation entitled

MEMS IMPEDANCE BIOSENSOR FOR ACCURATE AND RAPID DETECTION OF
E.COLI 0157:H7
Presented by Shibajyoti Ghosh Dastider
A candidate for the degree of
Doctor of Philosophy
And hereby certify that, in their opinion, it is worthy of acceptance.

Mahmoud Almasri

Kevin Gillis

Satish Nair

Naz Islam

Majed El-Dweik
ACKNOWLEDGEMENT
I’d like to thank my advisor, Dr. Mahmoud Almasri, for giving me the opportunity
to work in his research group, and for providing me with the guidance, constructive
feedback, and encouragement to complete this dissertation. I am grateful to my colleagues
at Microsystem Research Lab and would like to extend my thanks to them. I would
specially like to thank Dr. Syed Barrizuddin and Dr. Baskar Balakrishnan for their help
and support during the experimentation.
I take this opportunity to thank my committee members Dr. Mahmoud Almasri, Dr.
Kevin Gillis, Dr. Satish Nair, Dr. Naz Islam and Dr. Majed El-Dweik for their time and
consideration in reading my dissertation and for their help in defining and completing my
research.
I specially want to thank my family, especially wife Navneet, my sister and my
parents for their encouragement, love and support throughout my doctorate program.

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TABLE OF CONTENTS
ACKNOWLEDGEMENT ................................................................................................ ii
LIST OF FIGURES ......................................................................................................... vi
LIST OF TABLES ......................................................................................................... xiii
ABSTRACT .................................................................................................................... xiv
Chapter 1 INTRODUCTION AND MOTIVATION .................................................... 1
1.1 Introduction to Microelectromechanical systems (MEMS) ...................................... 1
1.2 MEMS biosensors ..................................................................................................... 3
Chapter 2 THEORETICAL BACKGROUND ............................................................ 20
2.1 Equivalent circuit models ....................................................................................... 20
2.1.1 Equivalent circuit model of a cell .................................................................... 20
2.1.2 Equivalent circuit model of a cell suspending in electrolyte between two
electrodes .................................................................................................................. 21
2.2 Dielectrophoretic focusing ...................................................................................... 23
Chapter 3 DESIGN AND FABRICATION .................................................................. 29
3.1 Design I ................................................................................................................... 29
3.2 Design II.................................................................................................................. 32
3.3 Fabrication .............................................................................................................. 35
3.3.1 Fabrication techniques ..................................................................................... 36
3.3.2 Materials used .................................................................................................. 45
3.3.3 Fabrication Process of Design I MEMS impedance biosensors ...................... 47
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3.3.4 Fabrication Process of Design II MEMS impedance biosensors ..................... 55
3.4 Simulation ............................................................................................................... 62
Chapter 4 RESULTS AND DISCUSSIONS ................................................................. 66
4.1 Design I: Culture preparation of bacteria ................................................................ 66
4.1.1 E.coli broth preparation ................................................................................... 66
4.1.2 Agar preparation .............................................................................................. 66
4.1.3 Antibody preparation ....................................................................................... 67
4.1.4 Surface functionalization and antigen immobilization .................................... 67
4.1.5 Fluorescent labeling ........................................................................................ 69
4.2 Test setup ................................................................................................................ 69
4.3 Time response ......................................................................................................... 71
4.4 Design I: Focusing testing ...................................................................................... 72
4.5 Design I: Electrical testing and Impedance measurement ...................................... 74
4.5.1 Impedance response without focusing effect ................................................... 74
4.5.2 Impedance response with focusing effect ........................................................ 75
4.5.3 Impedance response with focusing effect and secondary DEP ....................... 76
4.6 Design: Bacterial sample preparation and enumeration ......................................... 78
4.7 Design II: Antibody Immobilization and Antigen Capturing ................................. 79
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4.8 Design II: Focusing testing ..................................................................................... 79
4.9 Design II: Electrical testing and Impedance measurement ..................................... 81
4.10 Analysis of impedance spectrum .......................................................................... 85
4.11 Specificity Testing ................................................................................................ 87
Chapter 5 CONCLUSION ............................................................................................. 89
REFERENCES ................................................................................................................ 91
VITA .............................................................................................................................. 100

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LIST OF FIGURES
Figure 1.1 Interdigitated array microelectrode immunosensor. ......................................... 5
Figure 1.2 Microimpedance biosensor for bacterial detection. .......................................... 6
Figure 1.3 Double interdigitated microelectrode based impedance biosensor. ................. 7
Figure 1.4 Flow cell design with interdigitated electrodes. ............................................... 8
Figure 1.5 Interdigitated Microelectrode Array chip with gold microelectrodes and
PDMS microchannel. .......................................................................................................... 9
Figure 1.6 Lab on a Chip packaged biosensor and 3D schematic. .................................. 10
Figure 1.7. Interdigitated microelectrode sensor with polystyrene micro chamber for
monitoring growth of E.coli in urine. ............................................................................... 10
Figure 1.8 Impedance based infectivity sensor. ............................................................... 11
Figure 1.9 Schematic illustrates nanoliter-sized plug, with a few are occupied by a single
bacterium and the experimental procedure of detection of bacteria incubated in plugs. .. 12
Figure 1.10 (a) Optical image of the integrated chip, the sensing area and the excitation
circuits. (b) Sensor surface profile and cross section diagram. (c) Schematic diagram of a
sensing pixel and the dimensions of each pixel. ............................................................... 13
Figure 1.11 Schematic and optical image of the GMI sensor, gold film surface
modification and test setup. .............................................................................................. 14
Figure 1.12 Layout of the interdigitated microelectrode array based biosensor. ............ 15
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Figure 1.13 Schematic and optical images of the µECS biosensor. ................................ 16
Figure 1.14 Schematic of the PDMS microfluidic immunosensor with antibody coated
alumina nanoporous membrane. ....................................................................................... 17
Figure 1.15 Schematic and optical images of the microelectromechanical biosensor. .... 18
Figure 1.16 Schematic of immunomagnetics seperation of polyaniline-labeled
Escherichia coli O157:H7. ................................................................................................ 19
Figure 2.1 An equivalent circuit model of a single bacteria cell. .................................... 20
Figure 2.2 Equivalent circuit of a single cell suspended in the microchannel. ................ 21
Figure 2.3 Electric double layer between electrode and electrolyte. ............................... 22
Figure 2.4. Equivalent circuit model of electric double layer........................................... 23
Figure 2.5 (a) No DEP force in uniform electric field, (b) DEP force in non-uniform
electric field. ..................................................................................................................... 24
Figure 2.6 Schematic diagram showing the positive and negative dielectrophoretic forces
on particles subjected to non-uniform electric fields. ....................................................... 26
Figure 3.1. Schematic of impedance biosensor featuring (a) SU-8 microchannel with
focusing and measuring electrodes, (b) magnified view of focusing and sensing region,
(c) complete device with PDMS cover and connectors. ................................................... 30
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Figure 3.2. 2-D schematic of the a) microelectrode mask, b) magnified view of the
microelectrode arrays, c) magnified view of the individual IDE, d) microchannel mask
and e) overlay of the two masks. ...................................................................................... 32
Figure 3.3. Schematic of impedance biosensor featuring (a) SU-8 microchannel with
focusing and measuring electrodes, (b) magnified view of focusing and sensing region,
(c) complete device with PDMS cover and connectors. ................................................... 33
Figure 3.4. 2-D schematic of the a) microelectrode mask, b) electroplating mask, c)
microchannel mask, d) magnified view of the focusing electrode and e) sensing IDE
arrays. ................................................................................................................................ 35
Figure 3.5 Schematics showing the photolithography process of positive and negative
photoresist. ........................................................................................................................ 38
Figure 3.6 Capacitive coupled oxygen plasma generated by RF power supply in a
vacuum chamber. .............................................................................................................. 39
Figure 3.7 Sputtering deposition. ..................................................................................... 41
Figure 3.8 RF magnetron sputtering target. ..................................................................... 42
Figure 3.9 Electroplating setup. ....................................................................................... 43
Figure 3.10 Schematics of a general electroplating process showing a) deposition of
metal seed layer, b) patterned photoresist electroplating mold, c) electroplated metal and
d) electroplated structure post photoresist striping. .......................................................... 43
Figure 3.11 Isotropic wet etching. ................................................................................... 44
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Figure 3.12 Layouts of masks by L-Edit.......................................................................... 47
Figure 3.13 Graphical illustration of Step 1 to 3 of design I’s process flow. .................. 48
Figure 3.14 Optical images of a) focusing electrode array, b) sensing electrode array on
the substrate after Cr/ Au etching. .................................................................................... 49
Figure 3.15 Magnified views of the substrate with photoresist mold for electroplating. 50
Figure 3.16 Graphical illustration of Step 4 and 5 of design I’s process flow. ................ 50
Figure 3.17 Optical images of a) fluidic inlet, b) focusing electrode array with
microchannel, c) microfluidic junction near focusing region, d) microfluidic junction near
sensing region, e) sensing electrode array with microfluidic channel and f) fluidic outlet .
........................................................................................................................................... 51
Figure 3.18 Optical image of the biosensor after patterning the microchannel. ............. 52
Figure 3.19 SEM micrographs of a) microchannel near the focusing array, b)
microchannel showing a fluidic junction, c) focusing and d) sensing electrode arrays
embedded the microchannel. ............................................................................................ 52
Figure 3.20 The optical images show the completed biosensor wire bonded to PCB for
external electrical connections. ......................................................................................... 54
Figure 3.21 Layouts of masks designed using L-Edit. ..................................................... 55
Figure 3.22 Graphical illustration of Step 1 to 3 of design II’s process flow. ................. 56
Figure 3.23 Magnified optical image of the focusing electrode post Au etch. ................ 57
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