Towards Single Molecule Studies by Ahmed Al Balushi B.Eng., University of Bath, 2005

192 Pages·2016·1.98 MB·English

Double Nanohole Aperture Optical Tweezers:
Towards Single Molecule Studies
by
Ahmed Al Balushi
B.Eng., University of Bath, 2005
M.Sc., University of Bath, 2007
A Dissertation Submitted in Partial Fulfillment of the
Requirements for the Degree of
DOCTOR OF PHILOSOPHY
in the Department of Electrical and Computer Engineering
(cid:13)c Ahmed A. Al Balushi, 2016
University of Victoria
All rights reserved. This dissertation may not be reproduced in whole or in part, by
photocopying or other means, without the permission of the author.
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Double Nanohole Aperture Optical Tweezers:
Towards Single Molecule Studies
by
Ahmed Al Balushi
B.Eng., University of Bath, 2005
M.Sc., University of Bath, 2007
Supervisory Committee
Dr. Reuven Gordon, Supervisor
(Department of Electrical and Computer Engineering)
Dr. Poman So, Departmental Member
(Department of Electrical and Computer Engineering)
Dr. Stephanie Willerth, Outside Member
(Department of Mechanical Engineering)
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Supervisory Committee
Dr. Reuven Gordon, Supervisor
(Department of Electrical and Computer Engineering)
Dr. Poman So, Departmental Member
(Department of Electrical and Computer Engineering)
Dr. Stephanie Willerth, Outside Member
(Department of Mechanical Engineering)
ABSTRACT
Nanoaperture optical tweezers are emerging as useful tools for the detection and
identification of biological molecules and their interactions at the single molecule
level. Nanoaperture optical tweezers provide a low-cost, scalable, straight-forward,
high-speed platform for single molecule studies without the need to use tethers or
labeling. This thesis gives a general description of conventional optical tweezers and
how they are limited in terms of their capability to trapping biological molecules.
It also looks at nanoaperture-based optical tweezers which have been suggested to
overcome the limitations of conventional optical tweezers. The thesis then focuses
on the double nanohole optical tweezer as a tool for trapping biological molecules
and studying their behaviour and interactions with other molecules. The double
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nanohole aperture trap integrated with microfluidic channels has been used to detect
single protein binding. In that experiment a double-syringe pump was used to deliver
biotin-coated polystyrene particles to the double nanohole trapping site. Once stable
trapping of biotin-coated polystyrene particle was achieved, the double-syringe pump
wasusedtoflowinstreptavidinsolutiontothetrappingsiteandbindingwasdetected
by measuring the transmission through the double nanohole aperture. In addition,
the double nanohole optical tweezer has been used to observe the real-time dynamic
variations in protein-small molecule interaction (PSMI) with the primary focus on the
effect of single and multiple binding events on the dynamics of the protein in the trap.
Time traces of the bare form of the streptavidin showed slower timescale dynamics as
compared to the biotinylated forms of the protein. Furthermore, the double nanohole
aperture tweezer has been used to study the real-time binding kinetics of PSMIs and
to determine their disassociation constants. The interaction of blood protein human
serum albumin (HSA) with tolbutamide and phenytoin was considered in that study.
The dissociation constants of the interaction of HSA with tolbutamide and phenytoin
obtained using our technique were in good agreement with the values reported in
the literature. These results would open up new windows for studying real-time
binding kinetics of protein-small molecule interactions in a label-free, free-solution
environment, which will be of interest to future studies including drug discovery.
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Contents
Supervisory Committee ii
Abstract iii
Table of Contents v
List of Tables xi
List of Figures xii
Acknowledgements xxix
Dedication xxx
1 Introduction 1
1.1 Optical Tweezers: at a Glance . . . . . . . . . . . . . . . . . . . . . . 1
1.2 Thesis Outline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
1.3 Author’s Contributions . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2 Nanoapertures and Optical Trapping 9
2.1 Single Beam Optical Tweezers and their Limitations . . . . . . . . . . 10
2.2 Nanoaperture-based Optical Tweezers . . . . . . . . . . . . . . . . . . 12
2.3 Self-Induced Back-Action Optical Trapping . . . . . . . . . . . . . . . 15
2.3.1 Motivation for Self-Induced Back-Action Trapping . . . . . . . 15
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2.3.2 SIBA Trapping Regimes . . . . . . . . . . . . . . . . . . . . . 17
2.3.3 Advantages of SIBA Trapping . . . . . . . . . . . . . . . . . . 18
2.3.4 Nanoaperture Trapping Geometries . . . . . . . . . . . . . . . 20
2.4 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
3 Double Nanohole Optical Tweezer System 23
3.1 Motivation for Double Nanohole Apertures . . . . . . . . . . . . . . . 23
3.2 Resonances of the Double Nanohole Aperture . . . . . . . . . . . . . 25
3.3 Double Nanohole Aperture for Optical Trapping . . . . . . . . . . . . 27
3.3.1 Experimental Setup . . . . . . . . . . . . . . . . . . . . . . . . 27
3.3.2 Trapping Detection . . . . . . . . . . . . . . . . . . . . . . . . 27
3.3.3 Fabrication of the Double Nanohole Aperture . . . . . . . . . 29
3.3.4 Double Nanohole Aperture Chip Assembly . . . . . . . . . . . 31
3.4 Single Particle Spectroscopy of Trapped Particles . . . . . . . . . . . 33
3.4.1 Raman Spectroscopy . . . . . . . . . . . . . . . . . . . . . . . 33
3.4.2 Acoustic Raman . . . . . . . . . . . . . . . . . . . . . . . . . 33
3.5 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
4 Double Nanohole Optical Tweezers for Single Protein Studies 36
4.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
4.2 Advantages of Using the Double Nanohole Tweezer System for Protein
Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
4.3 Observing Single Protein Binding by Optical Transmission Through a
Double Nanohole Aperture in a Metal Film . . . . . . . . . . . . . . . 39
4.3.1 Experiment and Results . . . . . . . . . . . . . . . . . . . . . 40
4.3.2 Control Experiments . . . . . . . . . . . . . . . . . . . . . . . 42
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4.4 Label-free Free solution Single-Molecule Protein-Small Molecule Inter-
action Observed by Double Nanohole Trapping . . . . . . . . . . . . . 43
4.4.1 Experiment and Results . . . . . . . . . . . . . . . . . . . . . 44
4.4.2 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
4.5 ALabel-FreeUntetheredApproachtoSingle-MoleculeProteinBinding
Kinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
4.5.1 Experiment and Results . . . . . . . . . . . . . . . . . . . . . 48
4.5.2 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50
4.6 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
5 Conclusions and Future Work 54
5.1 Thesis Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
5.2 Future Work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
Bibliography 57
A Observing single protein binding by optical transmission through
a double nanohole aperture in a metal film 78
A.1 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
A.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79
A.3 Experimental Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
A.4 Protein Binding Experiments . . . . . . . . . . . . . . . . . . . . . . 83
A.5 Control Experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . 84
A.5.1 Saturated streptavidin . . . . . . . . . . . . . . . . . . . . . . 85
A.5.2 Non-functionalized PS particles . . . . . . . . . . . . . . . . . 85
A.6 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
A.6.1 DNH trap for single protein binding detection . . . . . . . . . 86
A.6.2 Future directions . . . . . . . . . . . . . . . . . . . . . . . . . 89
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A.7 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 90
B Label-free free solution single-molecule protein-small molecule in-
teraction observed by double nanohole plasmonic trapping 91
B.1 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
B.2 Paper Content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
B.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
B.3.1 Fabrication of DNH . . . . . . . . . . . . . . . . . . . . . . . . 101
B.3.2 Gold sample preparation . . . . . . . . . . . . . . . . . . . . . 101
C A label-free untethered approach to single-molecule protein bind-
ing kinetics 102
C.1 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
C.2 Paper Content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103
C.3 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
C.3.1 Fabrication of DNH . . . . . . . . . . . . . . . . . . . . . . . . 113
C.3.2 Gold sample preparation . . . . . . . . . . . . . . . . . . . . . 113
C.4 Supporting Information . . . . . . . . . . . . . . . . . . . . . . . . . . 114
C.4.1 Time Traces of a Bare HSA in the DNH . . . . . . . . . . . . 114
C.4.2 Time Traces of HSA Interaction with Phenytoin . . . . . . . . 114
C.4.3 Transmission spectrum of the DNH aperture . . . . . . . . . . 115
C.4.4 Varying Threshold Level Tolerance . . . . . . . . . . . . . . . 116
C.4.5 Goodness of fit . . . . . . . . . . . . . . . . . . . . . . . . . . 117
C.4.6 Effect of Noise on Decay Rate Constants . . . . . . . . . . . . 118
D Raman Spectroscopy of Single Nanoparticles in a double-nanohole
optical tweezer system 120
D.1 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120
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D.2 Paper Content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
E Nanoscalevolumeconfinementandfluorescenceenhancementwith
double nanohole aperture 129
E.1 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129
E.2 Paper Content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
E.2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
E.2.2 Results: zeptoliter volume with 100-fold fluorescence enhance-
ment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
E.2.3 Fluorescence photodynamics acceleration and LDOS enhance-
ment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138
E.2.4 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 144
F Double nanohole optical trapping: dynamics and protein-antibody
co-trapping 148
F.1 Abstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
F.2 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
F.3 Microfluidic integration of double nanohole trap . . . . . . . . . . . . 150
F.3.1 Setup . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 150
F.3.2 Trapping nanoparticles . . . . . . . . . . . . . . . . . . . . . . 150
F.4 Dynamics of the double nanohole trap . . . . . . . . . . . . . . . . . 152
F.4.1 Roll-off frequency . . . . . . . . . . . . . . . . . . . . . . . . . 152
F.4.2 Skewness distribution . . . . . . . . . . . . . . . . . . . . . . 153
F.5 Cotrapping of protein-antibody . . . . . . . . . . . . . . . . . . . . . 153
F.6 Numerical simulations of DNH optical trap . . . . . . . . . . . . . . . 156
F.7 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
F.7.1 Kramers hopping explanation of low frequency roll-off . . . . . 158
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F.7.2 Co-trapping towards protein-protein interaction . . . . . . . . 161
F.8 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162