Table Of ContentMolecular Engineering of Selective Recognition Elements as Coatings for Sensor
Platforms
by
Justyn Wayne Jaworski
B.S. (Boston University) 2004
A dissertation submitted in partial satisfaction of the
requirements for the degree of
Joint Doctor of Philosophy
with the University of California, San Francisco
in
Bioengineering
in the
GRADUATE DIVISION
of the
UNIVERSITY OF CALIFORNIA, BERKELEY
Committee in charge:
Professor Seung-Wuk Lee, co-Chair
Professor Arun Majumdar, co-Chair
Professor Tejal Desai
Professor Ting Xu
Spring 2009
The dissertation of Justyn Wayne Jaworski is approved:
Prof. Seung-Wuk Lee, co-Chair Date
Prof. Arun Majumdar, co-Chair Date
Prof. Tejal Desai Date
Prof. Ting Xu Date
University of California, Berkeley
Spring 2009
Molecular Engineering of Selective Recognition Elements as Coatings for Sensor
Platforms
Copyright 2009
by
Justyn Wayne Jaworski
Abstract
Molecular Engineering of Selective Recognition Elements as Coatings for Sensor
Platforms
by
Justyn Wayne Jaworski
Joint Doctor of Philosophy
with the University of California, San Francisco
in
Bioengineering
in the
GRADUATE DIVISION
of the
UNIVERSITY OF CALIFORNIA, BERKELEY
Professor Seung-Wuk Lee, co-Chair
Professor Arun Majumdar, co-Chair
This dissertation focuses on the aspects of selectivity in chemical sensing systems. While
a number of sensing platforms exist that are capable of highly sensitive detection, the
common factor of poor selectivity continues to limit their widespread use. In this work,
we explore the use of sequence specific biopolymers identified through combinatorial
screening approaches for the creation of molecular recognition elements for chemical
sensor coatings. Particularly, a library of bacteriophage was screened to identify which
1
of the unique peptide sequences present on their protein coat could provide the highest
affinity binding to a target chemical. We specifically targeted small molecules including
trinitrotoluene (TNT) and dinitrotoluene (DNT). From phage display experiments, we
identify consensus peptide motifs, and we analyzed their binding efficacy based on
affinity and specificity. Additionally, we demonstrate that the standalone receptor for
TNT could be incorporated into a polymeric coating while retaining its functionality. In
doing so, a peptide based sensor coating was developed and implemented onto a common
Quartz Crystal Microbalance sensing platform. Liquid phase experiments demonstrated
the sensing ability of this system selectivity respond to TNT while remaining relatively
inert to the analogue DNT molecule. Furthermore, a polymeric based sensing system
was developed with the TNT receptive motif to create a widely deployable sensing
system. Integration was simply a matter of coupling a chromic responsive polymer at the
final step of receptor synthesis. In doing so, a modular sensing system was created which
demonstrated target binding to small molecules, such as TNT, or large cells, such as
fibroblasts, depending on the surface receptor motif. Finally, we show that the
fabrication approach could be optimized to enhance the sensitivity of the system to small
molecule targets.
Our results demonstrate that short amino acid sequences can be identified through phage
screening for small molecule binding and further developed into a sensor coating. The
receptors may be implemented onto a common QCM based sensor or onto a newly
develop chromic responsive system, thus demonstrating the broad sensor integration
capabilities of these receptive motifs. We anticipate this approach may lead to furthering
2
the development of molecular recognition elements by utilizing the biological toolkit of
evolutionary screening for selective receptors. In the future, we hope such approaches
will be used to gain a mechanistic understanding of molecular recognition which would
have a profound impact on the chemical sensing community.
_________________________
Co-Chair, Dissertation Committee
_________________________
Co-Chair, Dissertation Committee
3
Dedicated to Sonja Jaworski and Hilda Todd
i
Table of Contents
List of Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iv
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiv
Chapter 1: Introduction
1.1 Challenges and Current Approaches in Chemical Sensing . . . . . . . . . . . . . . . . . . . . . 1
1.2 Molecular Recognition. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
1.3 Approaches to Achieving Molecular Recognition. . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
1.4 Challenges and Future Trends in Chemical Sensor Coatings. . . . . . . . . . . . . . . . . . .19
Chapter 2: Phage Screening of Small Molecule Targets for Selectivity Motifs
2.1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .20
2.2 Phage Display for Selection of TNT and DNT Binding Peptide Motifs . . . . . . . . . .21
2.3 Phage Display for Selection of Methyl Parathion Binding Peptide Motifs. . . . . . . . 27
2.4 Phage Display for Selection of Eugenol Binding Peptide Motifs. . . . . . . . . . . . . . . 30
2.5 Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Chapter 3: Analysis Techniques for Identifying Selective Binding Motifs
3.1 Selective Binding. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
3.2 Synthesis of Standalone Receptors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .45
3.3 Mutational Analysis Binding Assay. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .46
3.4 Isothermal Titration Calorimetry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
3.5 Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
ii
Chapter 4: Selective Coatings for Chemical Sensing
4.1 Introduction to Chemical Sensing. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .53
4.2 Overview of a Peptide Based Sensor Coatings. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
4.3 Coating Design for Surface Stress Based Sensors. . . . . . . . . . . . . . . . . . . . . . . . . . . 54
4.4 Coating Design for Quartz Crystal Microbalance Sensing Platform. . . . . . . . . . . . . 62
4.5 Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
Chapter 5: Development and Optimization of a Polymeric Sensing Vesicle
5.1 Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .85
5.2 Overview of Polydiacetylene as a Sensing Platform. . . . . . . . . . . . . . . . . . . . . . . . . 86
5.3 Experimental Section. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
5.4 Results and Discussion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .96
5.5 Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 108
Chapter 6: Summary and Outlook
6.1 Molecular Recognition Elements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
6.2 Sensor Coatings. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112
6.3 Sensing Platforms. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .114
Bibliography. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
iii
List of Figures
Figure-1: a) Typical synthetic polymers consist of a monomer repeated multiple times.
Target analyte molecules generally have one binding site with such polymers. b)
Sequence-specific polymers have different residues strung together in a single chain.
Depending on the sequence, target molecules can form multiple binding sites with the
polymer, which leads to higher free energy of binding and thereby higher selectivity
against background interfering molecules.
Figure-2: Schematic of molecular imprinting approach to creating target selective
recognition motifs.
Figure-3: Overview of phage display screening process against molecular crystal TNT.
Figure-4: Schematic of SELEX screening process for identifying RNA or DNA based
aptamers for target specific binding.
Figure-5: Schematic diagram showing our biomimetic approach to develop selective
coatings for gas-phase explosive molecules. Identified molecular recognition elements
from the directed evolution process of phage display are used for their multivalent
recognition of explosive targets in liquid-phase screening. Through chemical
modification, the peptide receptors are linked with oligo(ethylene glycol) and
immobilized as coatings capable of binding explosive targets in air.
iv
