Bottom-up Surface Engineering of DNA Macro-assemblies for 
Nanoelectronics and Optical Biosensors 
 
by 
 
Tony Jefferson Gnanaprakasa 
 
 
 
A thesis submitted to the Graduate Faculty of 
Auburn University 
in partial fulfillment of the 
requirements for the Degree of 
Master of Science 
 
Auburn, Alabama 
May 09, 2011 
 
 
 
 
Keywords: Optical DNA biosensors, SECM, 
charge transfer kinetics, nanoelectronics 
 
 
Copyright 2011 by Tony Jefferson Gnanaprakasa 
 
 
 
Approved by 
 
Aleksandr L. Simonian, Chair, Professor of Materials Engineering 
Dong-Joo Kim, Associate Professor of Materials Engineering 
ZhongYang Cheng, Associate Professor of Materials Engineering 
 
ii 
 
Abstract 
 
 
 Nanotechnology has promised an out of the ordinary impact on mankind. From the most 
mundane of consumables to the indispensible advancements in health care and medicine that it 
has potential in; make it an exciting revolution in science and engineering. Design and 
implementation of nanomaterials for the applications mentioned afore is the next big challenge. 
Despite the prodigious efforts carried out in unraveling the complications encountered, it is still 
problematical to engineer nanomaterials because of their physiochemical properties that make 
them complex systems to manifest, yet enthralling to work with. Standing out from this category 
of fascinating materials, single-walled carbon nanotubes (SWNTs) have been of great interest for 
their outstanding physical and chemical properties and their potential in the future of 
nanoelectronics and nanomedicine. In recent years, three dimensional nanoarchitectures have 
been found to be the apt candidates for the development of nanoelectronic devices and novel 
sonde diagnostic platforms. But lack of the proper chemical functionalities and biocompatibility 
has impeded their impact on nanomedicine and clinical diagnostics. Deoxyribonucleic acid 
(DNA) is a bio-nanomaterial from nature?s tool box and forms the central icon of modern 
medicine and has been greatly admired for its versatility and complex design. 
As a result, DNA was used as a template to design a layer-by-layer assembled SWNT 
based macromolecular surfaces for nanoelectronics with great control. These 3D 
nanoarchitectures provide a promising step in developing novel biomimetic antimicrobial thin
iii 
 
films. The electron transfer properties and conductivity of these materials was then studied using 
scanning electrochemical microscopy (SECM) as a non-destructive method. It was verified that 
the charge injection through the multilayers were solely due to electron hoping across the DNA-
SWNT adducts, as a result of an increase in feedback response from the SECM.  
 Subsequently, portable and sensitive optical biosensors were designed for the detection of 
the virulent disease causing gene in foodborne pathogens. In this case, the virulent hipO gene 
from Campylobacter jejuni was detected using a DNA self-assembled monolayer developed over 
gold surface on a surface plasmon resonance (SPREETA) platform and avidinated polystyrene 
surface on the diffraction optics technology (DOT) platform in real-time with high specificity. 
Hence the research presented here elaborates the use of DNA as a molecular tool to develop 
novel biomimetic nanoelectronic and medical diagnostic sensor platforms. 
 
 
 
 
 
 
 
 
 
 
 
 
iv 
 
Acknowledgements 
 
 
 In the first place, I would like to express my deepest and sincere gratitude to my mentor 
Dr. Aleksandr L. Simonian for providing me with his diligent support and guidance. His 
enduring motivation has fostered in me, a never-ending opportunity to excel during the course of 
this research and will be a pillar of support throughout my professional career. 
 I am grateful to my committee members, Dr. Dong-Joo Kim and Dr. ZhongYang Cheng 
for providing their time and invaluable review of this manuscript. My sincere thanks to Dr. 
Virginia A. Davis for her collaboration and insightful discussions during this research. I am also 
thankful to Dr. Omar A. Oyarzabal and Dr. Eric V. Olsen for their critical and intuitive reviews 
and their collaboration. This work wouldn?t have been possible without the financial support 
from NSF, AUDFS, USAF and NATO. I extend my sincere gratitude to Dr. Minseo Park for the 
collaborative research. I am always thankful to Mr. Roy Howard, Ms. Alison Mitchell and Mr. 
Steven Moore for all their help. My professors at the Central Electrochemical Research Institute 
(CECRI, India), Dr. N. G. Renganathan, Dr. K. R. Murali and Dr. S. Mohan have been 
responsible for setting up the foundation for my research career.  
 I wouldn?t have initiated and enjoyed my research career without the constant support 
from my colleague and friend Shankar. Furthermore, I would like to extend my thanks to my 
colleagues Valber, Saroja, Shawn, Rigved, Sheetal, Madhumati, Siddharth, Nitil and Jeff for all 
their help and making the research lab a fun place to work.  
v 
 
 I would like to pay tribute to all my friends at Auburn, who have been more than a 
friendly ear or an encouraging word. Though I am not able to accommodate everyone here, my 
special gratitude to Anand, Karthik, Anty, Borat, Nono, Cheechu, Rahul, Shyam, Kausar, Jola, 
Sonal, Kristen, Bala and Raja for making my three and a half year lodge at Auburn a long lasting 
memory. I would also like to thank all my friends back at home for staying in touch and for 
being there when I needed them.  
 Finally, I would like to thank my parents Grace Mary and Paul Arasu, and loving sister 
Steffi for being extra supportive and determined towards my education since I was a child. They 
have been the source of encouragement and unconditional love and I dedicate my work to their 
indomitable spirit. In the end, I would like to thank the immaculate mother of god for guiding me 
all the way through. Thank you guys!!! 
 
 
 
 
 
 
 
 
 
 
 
vi 
 
The following peer-reviewed publication and conference presentations were a product of this 
thesis: 
Peer-reviewed publication: 
 
? Valber A. Pedrosa#, Tony Gnanaprakasa#, Shankar Balasubramanian, Eric V. Olsen, 
Virginia A. Davis, Aleksandr L. Simonian, ?Electrochemical properties of interface 
formed by interlaced layers of DNA- and Lysozyme-coated single-walled carbon 
nanotubes?, Electrochemistry Communications, 2009, 11, 1401-1404 (# Authors 
contributed equally in this work) 
 
? Tony J. Gnanaprakasa#, Omar A. Oyarzabal#, Eric V. Olsen, Valber A. Pedrosa, 
Aleksandr L. Simonian, ?Tethered DNA scaffolds on optical sensor platforms for 
detection of hipO gene from Campylobacter jejuni?, Sensors and Actuators B: Chemical, 
(Submitted) (# Authors contributed equally in this work) 
 
? Siddharth Alur, Tony J. Gnanaprakasa, Yaqi Wang, Yogesh Sharma, Jing Dai, Jong W. 
Hong, Aleksandr L. Simonian, Michael Bozack, Claude Ahyi, Minseo Park, 
?AlGaN/GaN HEMT Based Biosensor?, ECS Transactions, 2010, 28, 61-64 
 
Conference Presentations: 
? Gnanaprakasa, T.J.; Simonian, A.L.; ?Electrochemical DNA nano-biosensors based on 
MWCNT-AuNP nanocomposites?, 218th meeting of The Electrochemical Society, 
October 10-15, 2010, Las Vegas, NV  
 
? Gnanaprakasa, T.J.; Davis, V.A., Simonian, A.L.; ?Development of MWCNT/AuNP 
Nanostructures for Label-Free Localized SPR Based Biosensing?, AIChE Annual 
Meeting, November 7-12, 2010, Salt lake City, UT  
 
? Gnanaprakasa, T.J.; Pedrosa, V.A.; Olsen, E.V.; Oyarzabal, O.A.; Chin, B.A.; Simonian, 
A.L.; ?DNA  biosensors  for  the  detection  of  the  hippuricase  gene  of  Campylobacter  
jejuni  based  on SPREETA?   and   dotLab?   platforms?, Biosensors  2010, 20th 
Anniversary   World   Congress   on Biosensors, May 26-28, 2010, Glasgow, United 
Kingdom 
vii 
 
? Gnanaprakasa, T.J.; Pedrosa, V.A.; Olsen, E.V.; Oyarzabal, O.A.; Simonian, A.L.; 
?Electrochemical Impedance Spectroscopy Studies of hippuricase Gene 
Hybridization for Campylobacter  jejuni  Specific  Detection?,  217th meeting of The 
Electrochemical Society, April 25-30, 2010, Vancouver, Canada 
 
? Gnanaprakasa, T.J.; Pedrosa, V.A.; Olsen, E.V.; Oyarzabal, O.A.; Simonian, A.L.; 
?DNA  biosensors  for  the  detection  of  Campylobacter  jejuni  nucleic  acids  based  on  
SPR  and  laser diffraction platforms?, AIChE Annual Meeting, November 8-13, 2009, 
Nashville, TN 
 
? Pedrosa, V.A.; Gnanaprakasa, T.J.; Olsen, E.V.; Davis, V.A.; Simonian, A.L.; 
?Electrochemical properties of interface formed by interlaced layers of DNA and 
lysozyme-coated single-walled carbon nanotubes?, 216th meeting of The 
Electrochemical Society, October 4-9,2009, Vienna, Austria 
 
? Gnanaprakasa, T.J.; Pedrosa, V.A.; Olsen, E.V.; Davis, V.A.; Simonian, A.L.; ?Probing 
conductivity and lateral charge transfer across LSZ-SWNT/DNA-SWNT nanocomposites 
using scanning electrochemical microscopy?, 215th meeting of The Electrochemical 
Society, May 24-29, 2009, San Francisco, CA 
 
? Alur, S.; Gnanaprakasa, T.J.; Xu, H.; Wang, Y.; Simonian, A.L.; Oyarzabal, O.A.; Park, 
M.; ?A Biosensor Based on GaN Field Effect Transistor?, International Conference on 
Compound Semiconductor Technology, May 18-21, 2009, Miami, FL 
 
? Pedrosa, V.A.; Gnanaprakasa, T.J., Davis, V.A.; Simonian, A.L.; ?Lateral conductivity 
of single-walled carbon nanotube reinforced with biopolymers by scanning 
electrochemical microscopy?, Mini Symposium, ECS Local Section and Atlanta Student 
Chapter at the Georgia Institute of Technology, February 18, 2009, Atlanta, GA (invited) 
 
? Balasubramanian, S.; Nepal, D.; Gnanaprakasa, T.J.; Davis, V.A.; Simonian, A.L.; 
?Electrochemical characteristics of SWNT-biopolymer nanocomposites?, 213th meeting 
of The Electrochemical Society, May 18-23, 2008, Phoenix, AZ 
 
 
 
 
viii 
 
Table of Contents 
 
Abstract ........................................................................................................................................... ii 
Acknowledgements ........................................................................................................................ iv 
List of Figures .............................................................................................................................. xiii 
List of Tables ............................................................................................................................... xvi 
List of Abbreviations, Acronyms and Symbols .......................................................................... xvii 
1. Introduction ................................................................................................................................ 1 
1.1. References ........................................................................................................................ 7 
2. Literature Review ..................................................................................................................... 10 
2.1. Agenda ........................................................................................................................... 10 
2.2. DNA as dispersant for SWNTs ...................................................................................... 11 
2.3. Layer-by-Layer assembly (LbL) .................................................................................... 12 
2.4. Charge transfer across DNA based carbon nanotube hybrid materials ......................... 15 
2.5. DNA templated diagnostic platforms ............................................................................ 17 
2.6. Campylobacter jejuni (C. jejuni) ................................................................................... 19 
2.7. Pathogenesis of C. jejuni diseases ................................................................................. 20 
2.8. Biosensors ...................................................................................................................... 24 
2.9. Biorecognition elements ................................................................................................ 24 
2.9.1. Enzymatic Biosensors ...................................................................................... 24 
2.9.2. Antibody-Based Biosensors ............................................................................. 25 
ix 
 
2.9.3. Aptasensors ...................................................................................................... 25 
2.9.4. Tethered DNA structures on 2D substrates ...................................................... 26 
2.10. DNA as an alternate probe for biosensors .................................................................. 28 
2.11. DNA Immobilization .................................................................................................. 29 
2.11.1. Physisorption .................................................................................................... 29 
2.11.2. Covalent Immobilization .................................................................................. 30 
2.11.3. Affinity binding ................................................................................................ 30 
2.11.4. Chemisorption .................................................................................................. 31 
2.12. Structure-Property of biological ligands as SAMs ..................................................... 33 
2.13. Commercial C. jejuni testing kits................................................................................ 34 
2.14. C. jejuni biosensors ..................................................................................................... 34 
2.15. Transducers ? Why optical transducers? .................................................................... 35 
2.16. Thesis organization ..................................................................................................... 38 
2.17. References ................................................................................................................... 39 
3. Scanning Electrochemical Microscopy ? Principle & Experimental Setup ............................. 48 
3.1. Scanning Electrochemical Microscopy (SECM) ........................................................... 48 
3.2. Ultramicroelectrodes (UME) ......................................................................................... 49 
3.3. Feedback Mode .............................................................................................................. 51 
3.4. Theoretical Predictions .................................................................................................. 52 
3.4.1. First-Order Heterogeneous Reaction for reaction between tip and substrate ... 52 
3.4.2. Steady-State Conditions considered for reaction at SWNT nanocomposites .. 55 
3.4.3. Ohmic behavior at the substrate during charge injection ................................. 58 
3.5. Theoretical fitting of conducting and insulating approach curves ................................. 60 
x 
 
3.6. SECM setup ................................................................................................................... 61 
3.6.1. Tip preparation and operation .......................................................................... 62 
3.6.2. Protocol for SECM setup ................................................................................. 64 
3.7. References ...................................................................................................................... 65 
4. Diffraction Optics Technology (DOTLAB) ............................................................................. 67 
4.1. Optical Transducers ....................................................................................................... 67 
4.2. Detection using diffraction based sensing ..................................................................... 67 
4.2.1. Diffraction measurement and ?self-referencing? .............................................. 68 
4.2.2. Sensitivity ......................................................................................................... 69 
4.3. dotLab system ................................................................................................................ 70 
4.4. Protocol for sensor setup ................................................................................................ 72 
4.5. References ...................................................................................................................... 73 
5. Nanoelectronic Properties of Single-Walled Carbon Nanotubes based Antimicrobial Films . 74 
5.1. Introduction .................................................................................................................... 74 
5.2. Coupled self-assembly of SWNT based biopolymers ................................................... 75 
5.3. Experimental Section ..................................................................................................... 77 
5.3.1. Reagents ........................................................................................................... 77 
5.3.2. Apparatus .......................................................................................................... 78 
5.3.3. Layer-by-Layer assembly ................................................................................. 78 
5.4. Results and Discussion .................................................................................................. 79 
5.4.1. Charge injection through the nano-film ........................................................... 79 
5.4.2. Nanoelectronic properties of alternate layers ................................................... 82 
5.4.3. Nano-bioelectrode ............................................................................................ 84 
xi 
 
5.4.4. Impedance Analysis ......................................................................................... 86 
5.5. Conclusion ..................................................................................................................... 86 
5.6. References ...................................................................................................................... 88 
6. Affinity Based DNA Optical Sensors for the Detection of hipO Gene from Campylobacter 
jejuni ....................................................................................................................................... 92 
6.1. Introduction .................................................................................................................... 92 
6.2. Experimental .................................................................................................................. 94 
6.2.1. Reagents ........................................................................................................... 94 
6.2.2. Sensor Platforms ............................................................................................... 95 
6.2.3. DNA Probes ..................................................................................................... 96 
6.3. Immobilization of DNA probes ..................................................................................... 97 
6.3.1. dotLab ............................................................................................................... 97 
6.3.2. SPREETA ......................................................................................................... 98 
6.4. Detection of target DNA .............................................................................................. 100 
6.4.1. dotLab ............................................................................................................. 100 
6.4.2. SPREETA ....................................................................................................... 102 
6.4.3. Electrochemical Impedance Spectroscopy (EIS) ........................................... 102 
6.5. Results and Discussion ................................................................................................ 103 
6.5.1. Immobilization of Biotinylated DNA probes (dotLab) .................................. 103 
6.5.2. Immobilization of Thiolated DNA probes (SPREETA) ................................ 104 
6.5.3. DNA detection - dotLab ................................................................................. 107 
6.5.4. DNA Detection - SPREETA .......................................................................... 108 
6.6. Conclusion ................................................................................................................... 112 
6.7. References .................................................................................................................... 112 
xii 
 
7. Overall Conclusion ................................................................................................................. 118 
8. Future Recommendation ........................................................................................................ 120 
8.1. Localized Surface Plasmon Resonance (LSPR) based biosensors .............................. 120 
8.2. References .................................................................................................................... 122 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
xiii 
 
List of Figures 
 
 
Figure 2-1 SEM Image SWNT based LbL assembly, (a) 8 layers (b) 68 layers. ......................... 14 
Figure 2-2 Number of food-borne related outbreaks and cases reported from 2002 to 2007 ....... 18 
Figure 2-3 Breakdown of foodborne outbreaks by source from 2002 to 2007 ............................. 19 
Figure 2-4 Foodborne pathogens responsible for transmitted diseases in 2007 ........................... 19 
Figure 2-5 Sample preparation procedures for different types of DNA biosensors ..................... 27 
Figure 2-6 Schematic of an ideal Self-assembled monolayer formed on a gold (111) surface .... 32 
Figure 3-1 Schematic of a cell for ultramicroelectrode voltammetry ........................................... 49 
Figure 3-2 Typical voltammogram for a 10 ?m ultramicroelectrode ........................................... 50 
Figure 3-3 Basic principles of SECM: A) Far from the substrate [Hemispherical diffusion] B) 
Near the substrate [Feedback Diffusion-conductive] and C) Near the substrate 
[Hindered diffusion-insulating] .................................................................................. 52 
Figure 3-4 Geometry of the diffusion domain and the parameters defining the diffusion problem 
in SECM...................................................................................................................... 56 
Figure 3-5 Schematic of an SECM electrochemical cell setup ..................................................... 62 
Figure 3-6 SEM image of the 10 ?m Pt UME showing the insulating sheath and the tip (RG ? 
2.2) .............................................................................................................................. 63 
Figure 4-1 Schematic representation of the binding phenomena to patterned surface of 
polystyrene sensor. The illustration shows that diffraction based sensing is 'self-
referencing' .................................................................................................................. 68 
Figure 4-2 Cross section of a polystyrene dotLab sensor showing the microfluidic setup .......... 71 
Figure 4-3 Schematic representation of a dotLab system ............................................................. 71 
Figure 5-1 Schematic showing the Layer-by-Layer assembly of SWNT nano-coating ............... 79 
xiv 
 
Figure 5-2 Schematic of the proposed mechanism of charge transfer through the nano-coating . 80 
Figure 5-3 Approach curves at identical concentrations of redox mediator to nano-composite 
films composed of different number of SWNT-LSZ/SWNT-DNA layers ................. 81 
Figure 5-4 Approach curves for 10 layers of SWNT-LSZ/SWNT-DNA at different 
concentrations of redox mediator. The concentration of the solution redox mediator 
from top to bottom is 100 ?M, 330 ?M and 1 mM ..................................................... 82 
Figure 5-5 Approach curves for alternating layers of SWNT-LSZ/SWNT-DNA assembly ........ 84 
Figure 5-6 Cyclic coltammogram at different scan rates shows that the coating behaves as a bio-
electrode ...................................................................................................................... 85 
Figure 5-7 EIS spectra for different layers of assembly ............................................................... 87 
Figure 5-8 Representative impedance responses of the different number of layers at open circuit
..................................................................................................................................... 87 
Figure 5-9 Schematic of the custom built platform to hold the glass substrates for impedance 
analysis ........................................................................................................................ 88 
Figure 6-1 Illustration of the formation of DNA scaffolds using a 50-mer long probe and 22-mer 
short probe by annealing the complimentary regions in both the strands ................... 97 
Figure 6-2 Response curve for DNA immobilization and hybridization using SPREETA sensor
..................................................................................................................................... 98 
Figure 6-3 Sensor response to probe immobilization. There was a continuous increase in net 
change, which was calculated by subtracting the subsequent PBST baseline .......... 104 
Figure 6-4 Dose-Response curve for probe binding. The net response curves were plotted by 
subtracting the response signal from the base line. ................................................... 105 
Figure 6-5 Faradaic response for immobilization of thiolated ssDNA on SPREETA. Frequency 
intervals: 10 mHz to 100 KHz and measurements carried out at 0.32 V vs. Ag/AgCl.
................................................................................................................................... 105 
Figure 6-6 Nyquist plots for the electrode in a 5.0?10?3 mol L?1 [Fe(CN)6]3?/4?+ 0.1 mol L?1 KCl 
aqueous solution. Faradaic response for detection of target ssDNA ........................ 106 
Figure 6-7 Dose-Response curve for impedance spectrum obtained during target binding ....... 106 
Figure 6-8 Representative response curves for the detection of the hippuricase gene. The target 
was serially diluted and introduced over the sensing surface, starting with the lowest 
concentration. ............................................................................................................ 108 
xv 
 
Figure 6-9 Dose-Response curve for hippuricase gene target binding. ...................................... 109 
Figure 6-10 Representative response curves for the detection of the hippuricase gene. Graded 
concentrations of ssDNA were introduced to both, sensing channel and control 
channel (both blocked with spacer thiol). ............................................................... 110 
Figure 6-11 Dose-Response curve for hippuricase gene target binding ..................................... 111 
Figure 6-12 A representative response curve for long DNA probes immobilized on gold surface. 
The concentration of probes used was 1 ?M. Inset shows calibration curve for the 
same. ........................................................................................................................ 111 
Figure 8-1 Schematic representation of localized surface plasmon resonance across a metallic 
nanoparticle ............................................................................................................... 120 
Figure 8-2 Schematic of the setup for LSPR sensing using a transmission mode UV-Vis 
Spectrophotometer .................................................................................................... 121 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
xvi 
 
List of Tables 
 
 
Table 2-1 Susceptibilities of clinical isolates of C. jejuni for different anti-microbial agents ..... 23 
Table 2-2 Different types of transducers employed in sensor development ................................. 36 
Table 6-1 Summary of method for the detection of hipO gene programmed using dotLab 
software ...................................................................................................................... 100 
 
 
 
 
 
 
 
 
 
 
 
 
xvii 
 
List of Abbreviations, Acronyms and Symbols 
 
 
2. Literature Review 
  Fraction of the surface covered by the adsorbent 
   Pressure of the adsorbent 
   Rate constant for adsorption 
   Rate constant for desorption 
      ?  
   Permittivity of free space 
  Dielectric constant of the electrolyte 
  Area of the SWNT layer 
     Distance to the outer Helmholtz plane 
    Double layer capacitance of SWNT  
 
3. Scanning Electrochemical Microscopy ? Principle & Experimental Setup 
O Oxidation species 
R Reduction species 
    Steady state diffusion controlled current 
  Number of electrons transferred per redox event 
  Faraday 
xviii 
 
  Diffusion coefficient of species O 
  Radius of the conductive disk (tip) 
   Concentration of species i (O, R) 
   Dimension sheath radius 
   Radius of insulating sheath 
   Current flowing through tip 
  Distance between tip and substrate 
   electron 
    Bulk concentration of O 
  time 
    Coordinates in the directions radial and normal to the electrode surface 
   Diffusion coefficient of species i  
   Bulk concentration of electroactive species 
  Electrode potential 
    Formal potential 
  Parameter equal to    ? ,   is the gas constant and   the temperature 
  ( ) Diffusion-limiting tip current  
  Normalized tip/substrate separation 
  ( ) Dimensionless substrate current 
  ,    Dimensionless variables equal to   (   )?  
  Diffusion flux density 
   Standard rate constant 
xix 
 
   ?  Heterogeneous rate constants for oxidation and reduction 
     ?  Dimensionless rate constant equal to      ?   ?  
  Dimensionless variable equal to     ?  
  Transfer coefficient 
      Surface diffusion coefficient 
      Solution diffusion coefficient 
   Thickness of the film 
  Electrochemical potential of electrons in the nano-film monolayer 
   Standard electrochemical potential 
  Elementary charge 
 ( ) Local electrostatic potential in the film 
    Equilibrium electrochemical potential 
  Current density 
  Conductivity  
   Avogadro?s number 
   Microelectrode radius 
 ?   Equilibrium electrochemical potential of the film with respect to the standard 
chemical potential of the redox couple 
 
 
 
 
 
 
1 
 
1. Introduction 
Interdisciplinary research in engineering, with unclouded insight and advanced 
instrumentation has brought the field of surface science into common parlance, with its inception 
from the Nobel Prize winning work by Langmuir for his studies on surface adsorption. The field 
of surface science has undergone an enormous expansion, driven by the combination of different 
techniques for the preparation of macroscopic surfaces and ultra-high vacuum environments. Yet 
many uncertainties pervade this field, ranging from self-assembly of advanced materials 
structures to the practical viabilities to invest in knowledge, on nano-molecular recognition 
processes to large-scale mesoscopic formation phenomena. Both controlled placement and 
orientation of these molecules is required for their anisotropic properties to be fully manifested in 
the macro-scale. Hence the development of new surface chemistries and nano-scale materials 
paves way for a new paradigm in developing revolutionary technologies. 
Extensive studies have been conducted on materials from microstructures to the macro 
scale. However, the challenge prevails in understanding the fundamental properties of materials 
at the nano scale. Nanomaterials exhibit significantly modified physical, chemical and biological 
properties owing to their quantum confinement. Thanks to the tremendous growth of 
nanotechnology over the past few decades, recent developments in nanoscience have been 
applied to a number of innovations in medicine and healthcare, agriculture, chemical and 
biological technologies, environmental and energy storage and materials manufacturing. 
Conventional fabrication of devices start from patterning a silicon wafer and then obtaining the 
 
2 
 
desired features on the nano scale using reliable but expensive nanofabrication methods ? simply 
called ?top-down? approach. The need for precision and reliability in the fabricated devices, 
escalate the costs of top-down routes. Preferably, nanostructures are conceived from atoms and 
molecules which are reacted chemically and ordered physically to obtain the desired features, 
which is the ?bottom-up? approach. Based upon self-organization processes and in particular 
self-assembly, bottom-up approach is the coordinated action of independent entities under local 
control of driving forces in order to achieve a group effect for macrostructures [1]. 
 The key to nanomaterials organization is not only controlled by the presence of physical 
impulses such as temperature, photonic input, electrical modulation or pH; but also changes in 
chemical impulses such as the presence of molecules which enhance stimuli between these 
materials. Apropos, nature?s tool box provides an array of nanoscale fabrication methods, 
including assembly of superstructures like lipids, organelles and microtubules. Likewise, nucleic 
acids are found to have a high affinity and specificity towards a wide range of molecules, and 
regulate the in vivo and in vitro functionalities of any biological system. Hence nucleic acids 
such as DNA can be used as templating agents for nanomaterials because their structure can be 
easily regulated by physical and chemical means, because of their rich chemical functionality. 
Recently, Belcher et al [2] reported the development of an M13 virus based scaffold for the 
development of semiconductor nano-wires; which pioneered the concept of ?bottom-up? 
nanofabrication using biological templates. Although nanofabrication using biological templates 
appears to be a long term goal, several processes such as biomineralization and nucleic acid 
mediated self-assembly appear to be a promising strategy. As a result, the extensible ruggedness 
and high functionality of DNA makes it an attractive alternative for nanofabrication and self-
 
3 
 
assembly, in developing nanoelectronic devices and functional surfaces for a wide range of 
applications.  
 DNA consists of chemical functionalities called ?chemical handles? that allow it to 
interact with a myriad of inorganic nanomaterials. The three main chemical functionalities are: 
the negatively charged phosphate backbone, metal-chelating base groups and the aromatic rings 
that form the hydrophobic core. Furthermore, Seeman et al [3] showed that DNA based 
molecular networks with four-arm junction connectivity can be used to design closed self-
assembled lattices. Therefore, DNA can be used as a versatile biological tool in developing 
mediated and template assemblies of nanomaterials.  
 More recently, single-walled carbon nanotubes (SWNTs) have been a promising 
candidate for great impact on mankind with its exceptional physical and chemical properties. 
Yet, their existence in a bundled state and the absence of chemical functionalities to minimize 
their attractive energies between each tube has hampered the development of novel building 
blocks using individually dispersed SWNTs. Nepal et al [4] reported a novel solid-state 
mechano-chemical reaction to individually disperse SWNTs using DNA as a template. These 
DNA dispersed SWNTs were then used as building blocks to develop large scale biomimetic 
antibacterial nanocomposites, which formed as a stepping stone to develop 3D nanostructures 
and devices.  
 In order for DNA to be manifested in to nanomaterials, there are several aspects that have 
to be established including its chemical and electrical properties. Much of work has been done on 
instituting the physiochemical properties of DNA-templated nanomaterials. The electrical 
properties of these hybrid materials form an inordinate factor in developing building blocks for 
nanoscale devices. Poor electrical performance of DNA based nanomaterials and the presence of 
 
4 
 
crystalline defects in these templates causes the material to suffer high resistive heating. Hence 
new schemes have to be developed in increasing their electrical properties and developing self-
assembled 3D nanostructures based on DNA templates. There are a multitude of applications that 
can be projected using DNA-templated nanofabrication. When successful, it would allow the 
large integration these materials for macromolecular electronics, medical diagnostics and gene 
therapy. 
 Away from being used as a template in nanotechnology, DNA can also be used as a 
scaffold in developing medical diagnostic platforms. Mirkin et al [5, 6] reported a gold 
nanoparticle-DNA complex for the detection of proteins and DNA sequencing. They also 
reported a novel nano-patterning technique to develop self-assembled monolayers for fast, 
responsive DNA sensing used for diagnostics and biosensing. Shionoya et al [7] were successful 
in attaching metal nano-particles to the DNA helix, which has widespread applications in photo-
electronics and biosensing. With DNA nanotechnology in the upsurge and medical science 
leaning towards developing novel diagnostics for diseases; there has been an erratic progress in 
health reforms across countries. Transmission of diseases and infections are elevated by the 
ubiquitous urbanization and globalization, thereby accelerating spread of chronic disorders and 
multiple illnesses [8]. As a result of this globalization, there is a rapid change in health systems 
which are currently not insulated from one another. Attenuated curative care, delivery of short 
term results where service is fragmented and laissez-fair approaches to health systems are a few 
of the lagging trends that need to be looked upon to reform increasing health concerns. 
Redressing these health reforms helps the commercialization of health reforms and to flourish 
with a clearer sense of purpose and direction [9].  
 
5 
 
 Consequently, there is an obvious need for the development of bioanalytical devices to 
detect these foodborne pathogens for the safety and prevention of outbreaks. There is always a 
constant demand to sense these bio-threat agents mainly for collecting genomic data, diagnosis 
of diseases and controlling of production processes. The most common bio agents range from 
bacteria to viruses [10]. Biosensing requires short sensing time, low equipment and operating 
costs, compact system size and low power consumption. Most of the modern chemical and 
biosensors have changed the way by which these foodborne pathogens are detected [11].  
A broad host range of human pathogens are an emerging threat for pathogenic diseases 
and coexist in several animal hosts and environmental reservoirs. Apparently, evidence-based 
disease control is adopted in order to identify transmission routes and sources. Isolating 
microbial samples and attributing them with different food sources has helped track microbial 
sources. Additionally, specific phage subtypes and serotypes should be found in the same host. 
This is because, the specific hosts have well adapted specific clones and secondly, a combination 
of both sero-typing and phage type provides a reliable indicator for that clone.  
This has been unsuccessful for another branch of bacterial zoonosis, most commonly - 
Campylobacter jejuni (C. jejuni), which is one of the main causes of human bacterial 
gastroenteritis. It shows marked seasonality as a result of infection in poultry products, livestock 
and most of all humans and a possibility of environmental triggering [12]. In 2010, the USDA 
reported that C. jejuni is responsible for 2.4 million illnesses and 124 deaths in the United States 
alone. A recent outbreak in the U.S. in 2009 led to 35 C. jejuni related outbreaks, because of 
consuming unpasteurized milk. Diarrheal illnesses were frequently caused by C. jejuni than 
Salmonella species, Shigella species, or Escherichia coli O157:H7. The annual cost associated 
with C. jejuni infections is estimated to be 2.4 billion dollars in the U.S. in 2000 and 
 
6 
 
approximately 136 million pounds in the United Kingdom in 1999. Besides, the emergence of 
antimicrobial-resistant Campylobacter strains has led to an increase proper clinical diagnostics 
and development of technologies for effective management of diseases [13]. 
Campylobacteriosis can occur in outbreaks, single and sporadic cases. It is associated 
with handling raw poultry, eating undercooked meat, cross-contamination from other foods, 
unpasteurized milk or contaminated water and contact with animals infected with C. jejuni, that 
show no signs of infection. The pathogenesis of C. jejuni has been proposed in four different 
ways. These include: 
1. Motility, which is the penetration of intestinal mucosa and colonization of the intestine 
leading to the production of effector molecules 
2. Chemotaxis, being the ability to sense chemical gradients that enables the organism to 
move across these gradients 
3. Adhesion and invasion 
4. Toxin production 
5. Other mechanisms including iron acquisition, oxidative stress and heat shock response 
Besides there are other factors that cause the anti-microbial resistance in C. jejuni. 
Administration of anti-microbials to farm animals either as a growth promoter or as a therapeutic 
agent enables the growth of anti-microbial resistant C. jejuni strains. Fluoroquinolone resistant 
C. jejuni strains occur in poultry animals, which renders the fluoroquinolone-susceptible C. 
jejuni drug-resistant [14]. Although, the FDA has withdrawn the use of fluoroquinolones in 
poultry animals, these organisms continue to circulate and transfer. Additionally, international 
travel is also associated with fluoroquinolone resistant C. jejuni. Millions of people travel across 
and develop fluoroquinolone-resistant Campylobacter enteric infection abroad, also known as 
 
7 
 
traveler?s diarrhea. The continuous selection pressure from the widespread use of 
fluoroquinolones has led to its outbreak [15, 16].  
Surface plasmon resonance (SPR) and Diffraction optics technology (DOT) are based on 
an optical transduction scheme and they offer, ?label-free? and ?near real-time? detection of the 
samples. Accordingly, the main objective of this work is to develop DNA-templated functional 
surfaces for sensing the virulent gene from pathogenic bacteria and to analyze charge transfer 
through self-assembled nanomaterials for nano-electronic applications. In the latter section, we 
have verified the electron transport and conductivity of DNA dispersed single-walled carbon 
nanotubes using scanning electrochemical microscopy (SECM).  Hence, this research presents 
the application of DNA as a molecular scaffold in biosensing and its significance in the 
development of nanoelectronic materials. Chapter 2 describes the necessary literature work 
followed by the experimental setup for scanning electrochemical microscopy (SECM) in Chapter 
3. Chapter 4 describes the experimental setup for the DOT system. The application of self-
assembled monolayer to develop DNA based optical sensing platforms have been discussed in 
Chapter 5. Here various challenges related to DNA assembly increasing the sensitivity have been 
discussed. The electrochemical characteristics and charge transfer kinetics of self-assembled 
single-walled carbon nanotubes and DNA based bio-nanocomposites have been discussed in 
Chapter 6.  
1.1. References 
1. Dragoman, M. and D. Dragoman, Nanoelectronics Principles and Devices. 2006, 
Norwood: Artech House, Inc. 
2. Mao, C., et al., Virus-Based Toolkit for the Directed Synthesis of Magnetic and 
Semiconducting Nanowires. Science, 2004. 303(5655): p. 213-217   
 
8 
 
3. Seeman, N.C., Nucleic acid junctions and lattices. J. Theo. Biol., 1982. 99(2): p. 237-
247. 
4. Nepal, D., et al., Supramolecular Conjugates of Carbon Nanotubes and DNA by a Solid-
State Reaction. Biomacromolecules, 2005. 6(6): p. 2919-2922. 
5. Cao, C.Y., R. Jin, and C.A. Mirkin, Nanoparticles with Raman Spectroscopic 
Fingerprints for DNA and RNA Detection. Science, 2002. 297(5586): p. 1536-1540   
6. Jin, R., et al., What Controls the Melting Properties of DNA-Linked Gold Nanoparticle 
Assemblies? J. Am. Chem. Soc., 2003. 125(6): p. 1643-1654. 
7. Tanaka, K., et al., A Discrete Self-Assembled Metal Array in Artificial DNA. Science, 
2003. 299(5610): p. 1212-1213. 
8. Van Lerberghe, W., et al., The world health report 2008 - primary health care now more 
than ever. 2008, World Health Organization. 
9. Altekruse, S.F., et al., Campylobacter jejuni?An Emerging Foodborne Pathogen Emerg 
Infect Dis, 1999. 5(1). 
10. Ding, C., H. Zhong, and S. Zhang, Ultrasensitive flow injection chemiluminescence 
detection of DNA hybridization using nanoCuS tags. Biosens. Bioelectron., 2008. 23(8): 
p. 1314-1318. 
11. Passamano, M. and M. Pighini, QCM DNA-sensor for GMOs detection. Sens. Actuators 
B, 2006. 118(1-2): p. 177-181. 
12. Galanis, E., Campylobacter and bacterial gastroenteritis. CMAJ, 2007. 177(6): p. 570-
571. 
13. Stewart-Tull, D.E.S., et al., Virulence spectra of typed strains of Campylobacter jejuni 
from different sources: a blinded in vivo study. J. Med. Microbiol., 2009. 58: p. 546-553. 
 
9 
 
14. Bradbury, W.C., et al., Investigation of a Campylobacter jejuni Outbreak by Serotyping 
and Chromosomal Restriction Endonuclease Analysis. J. Clin. Microbiol., 1984. 19(3): p. 
342-346. 
15. Engberg, J., et al., Quinolone and Macrolide Resistance in Campylobacter jejuni and C. 
coli: Resistance Mechanisms and Trends in Human Isolates. Emerg. Infect. Dis, 2001. 
7(1): p. 24-34. 
16. Pickett, C.L., et al., Prevalence of cytolethal distending toxin production in 
Campylobacter jejuni and relatedness of Campylobacter sp. cdtB gene. Infect. Immun., 
1996. 64(6): p. 2070-2078. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
10 
 
2. Literature Review 
2.1. Agenda 
 Surface chemistry combined with nanotechnology and the right placement of quantum 
confined materials is a promising field which has immense applications in the field of 
nanoelectronics and clinical diagnostics. In his avant-garde speech at Caltech, Richard Feynman 
put forth ?? the problem of manipulating and controlling things at the nanoscale? [1]. This 
marked the beginning of the manifestation of materials for the next generation of 
nanoelectronics. Today, keeping those words as the stepping stone, self-assembly of 
nanomaterials has a great impact in every possible fa?ade of science and engineering. Various 
fabrication techniques have been used as an archetype in order to arrange these materials with 
precision and harness their properties to the fullest. Although, ?top-down? approaches have been 
successful in order to develop nanofabrication techniques, and to develop smaller machines; 
equipment costs and the difficulty that arises when manipulating nanomaterials have limited the 
use of this approach. Instead, precise building of materials from the molecular level known as 
?bottom-up? approach has attracted scientists from all junctures of science exemplifying the basic 
rules of self-assembly.  
 On the contrary, naturally occurring self-assembly tools in living systems demonstrate the 
ability to produce complex molecular machines using advanced molecular engineering methods. 
Bio-nanotechnology provides a beachhead from which novel self-assembly techniques can be 
perceived and can provide a variety of tools which could lead to computational advances in 
manipulating nanomaterials. Out of these naturally occurring biomaterials, DNA has been 
11 
 
considered in the far side of the barrier that is facing conventional microelectronics.  It is a 
celebrated molecule in modern biology, especially known for its protein folding capabilities and 
chemical functionalities that make it the right successor to conventional compounds. Hence, 
DNA can be used alongside with a number of nanomaterials, presumably as a manipulator in 
advanced molecular electronics or as a powerful tool to build novel molecular detection 
technologies [2].  
2.2. DNA as dispersant for SWNTs 
 Single-walled carbon nanotubes (SWNTs) have been expected to become a potential 
source of functional materials, because of their unique combination of optical, mechanical, 
thermal and electrical properties. However, because of their strong cohesive nature, they exist in 
aggregated bundles. This is due to strong tube-tube van der Waals interactions and their axial 
geometry. Additionally, the absence of flexible group functionalities to interact with the 
surrounding medium, they are also poorly soluble in common solvents. Apparently, individual 
dispersion of SWNTs is the million dollar question. This opens opportunities for a wide range of 
applications, including nanoscale devices and novel nanostructures. Introduction of 
solubilization groups either at the ends or the side walls of SWNTs and converting them to 
amphiphilic compounds is the strategy. The SWNTs should also undergo minimum damage with 
its inherent properties intact and easily characterizable. Particularly, the method should be benign 
and should not involve harsh processing steps and functional groups. In recent years, a wide 
range of surfactants, synthetic polymers and biopolymers have been studied in order to obtain 
highly dispersed nanotubes.  
 Consequently, it has been realized that DNA can also be an apt candidate for aqueous 
solubilization and dispersion of SWNTs for potential applications in developing biochemical and 
biomedical devices. Yet challenges prevail in developing effective techniques for the large-scale 
12 
 
production of this mono-disperse solutions containing SWNTs using DNA, with reliable control 
over the interfacial chemistry. The unique double helix structure of DNA enables us to harness 
remarkable biological and non-biological applications. Other applications include decorating 
carbon nanotubes with metal nanoparticles, DNA-assisted chiral separation of SWNTs and 
sonication assisted dispersion of SWNTs in DNA. Hence, a solid-state mechano-chemical 
reaction (MCR) is adopted, in order to prepare DNA-SWNT adducts. SWNTs interact with the 
DNA as a result of the highly reactive centers generated by the mechanical energy during the 
solid phase. This resulted in a high aqueous solubility of the products with a stability of more 
than 6 months. Moreover, single-walled products were found to be 250 nm to 1 ?m in length, 
while multi-walled products were 500 nm to 3 ?m in length. This non-covalent method appears 
to be shorter in length and fully wrapped around the SWNTs and hence has been adopted for 
developing advanced 2D and 3D supramolecular nanostructures [3].  
2.3. Layer-by-Layer assembly (LbL) 
 Materials science has grown in to an interdisciplinary field encompassing organic, 
polymeric and even biological nanostructures in addition to the classic metals and inorganics. 
Hence it is of great important that materials and devices of this kind have control of molecular 
orientation and organization at the nanoscale. Consecutively, deposition of single and multi 
layered nanomaterials can also be done in a controlled fashion on 2D solid substrates. 
Consequently, a fixed relation has to be established between the nano-scopic order and its 
macroscopic orientation. Harnessing every possible application from any nanostructure requires 
knowledge of the location and orientation of every molecule with respect to each other and to the 
macroscopic coordinate (e.g. liquid crystals). The most successful method followed in 
molecularly controlled fabrication of nanostructured films is the Langmuir-Blodgett (LB) 
technique, and has dominated the field of surface science for the last 60 years, where a 
13 
 
monolayer is formed on a water surface and then transferred to a 2D support. Kuhn et al [4] were 
the first to demonstrate the development of hetero-scale nanostructures using LB technique. 
Their work gave an insight in to nano-manipulation and mechanical handling of individual layers 
with nanometer sized precision. Although the LB technique had widespread use in 
nanomolecular design, it required special apparatus and several limitations oversaw its superior 
molecular architectures and applications in thin film development.  
 Electrostatic attraction between oppositely charged nanomaterials is a good candidate in 
developing 3D nano-architectures and acts as a good driving force for multilayer buildup, 
because of the less stearic demand of all chemical bonds. A charged surface and an oppositely 
charged molecule in solution undergo strong electrostatic attraction, which has been well known 
in the field of surface adsorption. As the molecules that form an assembly have more than one 
equal charge on their surface, it regulates the adsorption of charged molecules to a single layer 
and facilitates the adsorption of one molecule over another. Cyclic repetition of both the 
adsorption steps helps developing novel 3D superstructures.  An added advantage is that it 
reduced phase segregation and makes the composite highly homogeneous, so that the 
nanostructures are well dispersed and interpenetrated [5]. Kotov et al [6] reported a 
nanostructured analog of abalone seashells using nanometer sized sheets of Na+-Montmorillonite 
clay and poly(diallyldimethylammonium chloride) (PDDA), using LbL technique. The 
nanocomposites had a tensile strength comparable with natural nacre and lamellar bones, (~100 
? 10 MPa) and a young?s modulus of 11 ? 2 GPa. LbL assembly is a highly versatile method in 
merging various functionalities in to a single composite with application-tailored mechanical 
responses. Similarly, using a ?mix and match? approach highly tunable films can be obtained on 
geometric surfaces. For instance, LbL technique has found applications in developing super-
14 
 
hydrophobic surfaces, semi-permeable membranes, drug delivery, optically active and 
responsive films, fuel cells and photovoltaic materials, biomimetic and bio-responsive coatings, 
semiconductors, catalysts and magnetic devices [7-12]. Olek et al reported a nano-composite 
using multi-walled carbon nanotubes (MWNTs) and branched polyethyleneimine (PEI) ? a 
polyelectrolyte.  
 
Figure 2-1 SEM Image SWNT based LbL assembly, (a) 8 layers (b) 68 layers. 
Recently, LbL assembly of carbon nanotubes alternated with polyelectrolytes, have 
proven to show superior electrochemical properties and have widespread applications in 
conventional electrodes for sensor applications and energy storage devices [13, 14]. 
Subramanian et al reported a novel anti-microbial nanocomposites using positively charged 
DNA-SWNT and negatively charged Lysozyme-SWNT. Precise control of both thickness and 
SWNT alignment was achieved, and the coatings robust mechanical properties [15]. Therefore, 
the use of LbL technique is used extensively to develop large scale biomimetic nanocomposites 
for widespread applications.  
 
15 
 
2.4. Charge transfer across DNA based carbon nanotube hybrid materials 
 Although integration of nanotubes in electronics files beyond the next twenty years, they 
have emerged as an outstanding class of electronics materials due to their ballistic electronic 
conduction and insensitivity to electromigration. Charge transfer and the electrical properties of 
carbon nanotubes depend on their diameter and helicity, parameterized by its roll-up vector (n, 
m). Based on this prediction, SWNTs are classified in to three types: zigzag (0, m) or (n, 0); 
Armchair (n, m), where n=m; and chiral (n, m) where n?m. Besides, SWNTs possess electrical 
characteristics comparable to the best electronic material available, which has motivated many 
researchers to develop SWNT based field-effect transistors (FETs) and interconnects [16]. 
Charge transfer across carbon nanotubes can also be analyzed using electrochemical techniques. 
A charge transfer can also occur that does not result in a chemical change, but rather in a change 
of the electrical properties of the electrical double layer at the interface. The charge distribution 
of both parts of the double layer can be altered by charge transport through the material, which is 
termed ?non-faradaic? current. On the contrary, if the electronic state of the material is changed 
which results in a new chemical entity being formed, then it is termed ?faradaic? current [17].   
 The electrochemical behavior of SWNTs is similar to that of graphite, except for the 
additional effects of the specific electronic structure of rolled grapheme. The density of 
electronics states in SWNTs exhibit singularities typical to a 1D structure. These so called van 
Hove singularities (vHSs) originate from the size-dependent quantization of the electronic wave 
function around the circumference of the SWNTs. Additionally, the energy spacing between 
corresponding vHSs is controlled by the diameter of each SWNT. SWNTs can be applied in 
electrochemical techniques as a working electrode, free-standing bucky paper or even an 
individual SWNT in contact with the electrolytic solution as a working electrode. In an 
electrochemical cell, if a potential U is applied to the electrode, then a net charge is built up 
16 
 
across the electrode, which depends on the electrical double layer formed at the electrochemical 
interface. Additionally, an iR drop is formed across the double layer because of the resistance 
value of the SWNT. As a result, the potential across the cell is defined from the 
SWNT/electrolyte interface and this system behaves like a parallel-plate capacitor. Then the 
double-layer capacitance is given by: 
         
   
 
Where,    and   are the permittivity of free space and dielectric constant of the electrolyte 
solution,   is the area of the SWNT layer and      is the distance to the outer Helmholtz plane, 
which is about 0.5 nm for SWNTs. Considering all these parameters, the double-layer 
capacitance     of SWNT is found to be 10 fF/?m of the tube length [18]. This is extremely 
difficult to evaluate if the SWNT is present as bundles as the double-layer is solely formed at the 
outer layer of the surface. Hence, it is necessary to individually disperse SWNTs in order to 
harness their electrical and charge transfer properties. Furthermore, the current flowing thorough 
a SWNT splits in to an electrostatic part over the double-layer capacitance     and a chemical 
part over the quantum capacitance,   which is estimated to be of the order to 1 fF/?m of the tube 
length.  A qualitative difference exists between a classical metal electrode and SWNT electrode. 
In the case of metal electrode, the applied potential moves the bands by      without changing 
the relative position of the Fermi level against the band edges (      ). On the other hand, the 
reaction through a SWNT is quantum-capacitance dominated. Hence, the Fermi level moves 
     but the bands remain intact. Besides,        and hence the potential dependencies vary 
according to a particular band structure of the SWNT. 
 The electrolyte is the limiting component in terms of the overall stability of the 
electrochemical system. Because the unmodified SWNTs are almost insoluble in all usual 
17 
 
solvents, electrochemical stability is the only deciding factor for any electrolyte. While ionic 
liquids are sufficiently conductive, addition of a supporting electrolyte increases the conductivity 
of the solution. As a result, the electrochemical doping of SWNTs become easier, this does not 
perturb the so called ?potential window?, and hence finds its way in a wide range of applications. 
SWNTs can be used in a different configuration of FETs, in which SWNTs is surrounded by an 
electrolyte and the system undergoes electrochemical gating, which is mainly used in analytical 
chemistry and sensors. The electrochemistry of nanotubes also finds practical applications in 
energy storage, batteries, supercapacitors, sensors and actuators and nanoelectronics. Carbon 
nanotubes can be modified using a wide range of functional groups and hence specific molecules 
can be detected via electron-transfer reactions and amperometric sensors. Most commonly used 
electrochemical sensors are called ChemFETs or chemisresistors, for the detection of glucose, 
cholesterol, dopamine and other biomolecules.  
2.5. DNA templated diagnostic platforms 
Foodborne diseases have emerged to be one of the major causes of illnesses and deaths 
throughout the world. Ingestion of contaminated and intoxicated foods and beverages are few of 
the most common sources of infections. Efficient strategies were developed for the prevention of 
foodborne diseases and most often depend on the use of molecular and cellular biological 
techniques. The Center for Disease Control and Prevention (CDC) identifies and categorizes the 
most significant causes that contribute to foodborne illnesses including; food from unsafe 
sources, inadequate cooking and improper holding temperatures of raw food, contaminated 
equipment and poor personal hygiene. Foodborne outbreaks were identified and reported to the 
CDC, including infections, hospitalizations and deaths associated with each outbreak. In 2007, a 
total of 698 outbreaks (497 confirmed and 201 suspected) were reported, resulting in 15,477 
illnesses with their etiological agents either confirmed or unidentified. Identifying the food 
18 
 
vehicle is one of the major steps in identifying any etiology. Unidentified etiological agents arise 
due to 1) irregular reporting of diseases to the health department, 2) different types of food 
consumed which makes it difficult to identify it as a single contaminated food vehicle and 3) 
human and food test results were unavailable due to improper documentation. Sudden population 
based-outbreaks were reported electronically which helps guide the surveillance system design a 
toolkit for investigation. Pathogenic bacteria associated with food products are a major concern 
for both the food industry and the governing bodies. Bacterial foodborne outbreaks caused in 
2007 were almost similar to the number of outbreaks in the 2002-2006 average.  
 
Figure 2-2 Number of food-borne related outbreaks and cases reported from 2002 to 2007 
Reported data shows that Campylobacter is one of the most common causes of acute 
bacterial enteritis globally. Among 12,767 illnesses due to outbreaks, bacteria with a single 
etiological agent caused 259 (52%) outbreaks with 6,441 (50%) illnesses. Additionally in 2007, 
Campylobacter was responsible for 372 outbreak related illness out of 7115 due to a bacterial 
outbreaks. 
19 
 
 
Figure 2-3 Breakdown of foodborne outbreaks by source from 2002 to 2007 
 
 
Figure 2-4 Foodborne pathogens responsible for transmitted diseases in 2007 
2.6. Campylobacter jejuni (C. jejuni) 
Although campylobacters were first recognized about 100 years ago, their widespread 
association with diseases in humans was observed in recent times [19]. C. jejuni is by far the 
most important human enteropathogen among Campylobacters. It is a micro-aerophilic, gram-
negative, flagellated spiral bacterium that are mostly slender, curved and motile rods, 0.2 ? to 0.8 
20 
 
?m wide and 0.5 to 5 ?m long. Cells of some species are predominantly straight rods while old 
cultures may form coccoid bodies that are a degenerative form in the bacterial cycle, than a 
dormant stage of the organism. Owing to its aerophilic nature, it requires 3 ? 5% oxygen and 2 ? 
10 % carbon dioxide for optimal growth. Also known as a antecedent of Guillain-Barr? 
syndrome. Two sub species are recognized within C. jejuni: C. jejuni subsp. jejuni and C. jejuni 
subsp. doylei. Both the strains differ from each other by the absence of a nitrate reduction, 
cephalothin susceptibility and often catalase activity. These species are respiratory and 
chemoorganotropic types that grow under microaerophilic conditions, typically between 30 and 
37 ?C. Hence, they are able to flourish across a wide range of dairy and poultry products [20].  
2.7. Pathogenesis of C. jejuni diseases 
C. jejuni is a type of proteobacteria and belongs to the Campylobacteriales order, and is a 
member of the Spirillaceae family because of its physiological and anatomical similarities [21]. 
They have small genomes (1.6 ? 2.0 megabases) and can establish long-term associations and 
pathogenic consequences with their hosts. In today?s world waterborne infections are less likely 
to infect any host, and animals turn out to be the prime source of human infection and diseases. 
Acquired from the ingestion of contaminated poultry products, the human response to C. jejuni is 
more symptomatic than that of poultry animals. There have also been reports of C. jejuni 
infections leading to septicemia, skin and soft tissue infection and meningitis. Additionally, 
many other outcomes of C. jejuni infection in humans are not well understood, due to the lack of 
suitable models that emulate the symptoms and diseases.  
 Humans are always at a risk of acquiring acute gastroenteritis even from very low 
concentrations of C. jejuni, which is accompanied by inflammation, abdominal pain and diarrhea 
and in most cases leads to Guillain-Barr? syndrome. Symptoms caused due to Campylobacter 
infection can be manifested in two ways; self-limiting inflammatory bloody diarrhea containing 
21 
 
leukocytes and non-inflammatory, watery diarrhea, predominantly among children. This 
virulence has been characterized by four major properties if C. jejuni namely: motility, 
adherence, invasion and toxin production. The motility and corkscrew morphology of C. jejuni 
facilitates moving towards the attaching sites of the epithelium. Following this, C. jejuni attacks 
the intestinal cells, by adhering to the cell wall and colonization. This is in turn characterized by 
the induction of proinflammatory cell-signaling proteins; an indication of Campylobacteriosis. 
Although adhesins and fimbrial structures are significant in colonization of bacteria, intracellular 
killing and growth occurs in vitro due to the secretion of proteinaceous toxins from the bacteria. 
These toxins can be classified in to two types based on their activity: enterotoxins and 
cytotoxins. Enterotoxins are secreted proteins that bind to the cellular receptor. They contain an 
enzymatic part A, and a receptor binding pentamer B. Deregulation of the cellular regulatory 
system occurs after receptor A binds to the target cell causing an uneven increase in intracellular 
cyclic AMP levels, leading to a change in ion flux, which secretes excess fluid from the body 
resulting in watery diarrhea.  
As compared to the above effects, cytotoxins are proteins that kill target cells. They are 
classified in to two types based on their level of toxicity. The first type which is called verotoxins 
contains two subunits; an enzymatic subunit A and a receptor binding subunit B. On attaching to 
the cell, subunit A proteolytically cleaves and induces depurinating activity by inhibiting protein 
synthesis and resulting in cell death. Contrarily, the second type of toxin called hemolysins, have 
the capacity to form pores in nucleated cells. In the course of infection, profound effects like 
lysis of erythrocytes and cytoskeleton dysfunction causes damage to leukocytes and 
granulocytes, which eventually reduces the immune system of the host [22]. One of the widely 
studied mechanisms during C. jejuni infection is its Cytolethal Distending Toxin (CDT) activity. 
22 
 
Although produced in several pathogens such as Escherichia coli, Shigella dysenteriae and 
Helicobacter spp., not all of them are implicated to enteric diseases. CDT activity is expressed as 
a result of the formation of a tripartite holotoxin complex encoded from three tandem genes; 
cdtA, cdtB and cdtC. The active component of the toxin is bound to the host cell using cdtA and 
cdtC, while cdtB encodes the active component of the toxin. A high percentage of retailed 
poultry products are contaminated with C. jejuni which is a major factor in human exposure to 
this pathogen. Introducing antibiotics in poultry feed reduces the levels of C. jejuni 
contamination. The most commonly prescribed antibiotics for Campylobacter infections are 
macrolides and fluoroquinolones. Quinolones were introduced in the 1960?s for treating 
Campylobacteriosis and C. jejuni infections. The FDA approved the use of two fluoroquinolones 
- sarafloxacin and enrofloxacin. However, by the 1980?s it was confirmed that these pathogens 
developed resistance to fluoroquinolones within a span of two decades [23-25]. Additionally, 
resistance to erythromycin and tetracyclines in human and animal Campylobacter isolates have 
also been well documented [26]. Although resistance to macrolides has remained at a very low 
level, a recent outbreak of macrolide-resistant C. jejuni in Finland has raised serious concerns. It 
has also been indicated that very few alternatives exist for treatment of Campylobacteriosis 
caused by this multi-drug resistant Campylobacter strain. Also studies have shown that patients 
infected with quinolone and erythromycin resistant Campylobacter strain have adverse effects 
than compared to antimicrobial susceptible drugs bacterial strains [27, 28]. The epidemiology of 
C. jejuni is unclear and it is believed that poultry and farm animals are the carriers of this 
organism, in most cases without showing any symptoms of the disease. It is also believed that the 
number of Campylobacter infections may exceed those caused by Salmonella spp., Shigella spp. 
and Vibrio cholera [29]. 
23 
 
Anti-microbial agent MIC (?g/ml) breakpoint of resistant isolates % Resistant 
Erythromycin 16 10 
Azithromycin 4 11 
Clindamycin 8 11 
Nalidixic acid 32 91 
Ciprofloxacin 4 92 
Norfloxacin 16 90 
Ofloxacin 8 90 
Levofloxacin 8 76 
Ampicilin 32 40 
Co-amoxiclav 32 42 
Chloramphenicol 32 1 
Imipenem 16 0 
Meropenem 16 0 
Gentamicin 16 2 
Tetracycline 16 61 
Tigecycline 0.5 0 
 
Table 2-1 Susceptibilities of clinical isolates of C. jejuni for different anti-microbial agents 
 Since, the discovery of the hippurate hydrolysis in 1980 [30, 31] to distinguish between 
C. jejuni (hippurate positive) and other Campylobacters (hippurate negative), triggered the 
research in to the taxonomical aspects of this thermophilic bacteria. The hippurate hydrolysis 
involves cleaving of N-Benzoylglycine to glycine and benzoic acids which are then, detected 
using a ninhydrin-based reagent system and precipitation with ferric chloride reagent. On the 
contrary, an occurrence of hippuricase-negative variants and size dependence of the phenotypic 
expression of enzymes led to the failure of assays in detecting low-level producers of 
hippuricase. Hence, the hippuricase activity was characterized and sequenced using deletion 
subclones and insertional inactivation permitting the development of a genetic probe specific 
only to C. jejuni and a practical approach to investigate the role of hipO gene in the activity [32, 
33].  
 
24 
 
2.8. Biosensors 
 Chemical sensors which utilize a biochemical mechanism in recognizing the analyte are 
termed biosensors, with only a bioreceptor, at a direct spatial contact with the transducer. Being 
the key component of any sensor platform, a recognition element imparts selectivity acquiring a 
dose response from any given group of analytes. Th?venot et al. [34] classified biosensors in to 
catalytic and non-catalytic (affinity based) sensors, while some of the newly devised sensor 
platforms use microorganisms as receptors. Classic examples of catalytic platforms are glucose 
biosensors, biosensors involved in detection and decontamination of pesticides and chemical 
warfare agents. Since then, significant advance has been made in biosensor technologies 
including nucleotides, aptamers and peptides as biorecognition elements. Nucleotide or nucleic 
acid sensors belong to the class of chemical sensors which transforms a biochemical stimulus in 
to a readable signal (induced by change in chemical structure or analyte concentration). Venture 
in to developing small sensing devices for biomedical applications is rapidly flourishing, with an 
enormous potential in decentralizing point-of-care and critical-care. Breakthroughs in the field of 
nanotechnology and biosensors have paved way for rapid, sensitive and ?on-the-spot? clinical 
diagnostics including miniaturized devices, rapid monitoring of dynamic events and induction of 
advanced sensing materials. Potential applications include; patient health care, brewing industry, 
pharmaceutical synthesis, food manufacture, waste water treatment and energy production. 
2.9. Biorecognition elements 
2.9.1. Enzymatic Biosensors 
In general, enzymes are employed as biocatalysts in reactions where the mode of action 
involves oxidation and reduction. They contain a prosthetic group that can bind itself to the 
substrate, and in most of the cases the detection is done electrochemically. Although, a very wide 
range of enzymes are available commercially with well defined assay characteristics, they are 
25 
 
expensive and the cost of extracting, isolating and purifying them is excessive. Also, they tend to 
lose activity and degrade after a relatively short period of time. Alternatively, plant and animal 
tissues were also used with minimal preparation. Presence of multiple enzymes makes them less 
selective than purified enzymes, but they undergo minimum degradation, which enables them to 
have a longer shelf-life. However, they have a very slow response and induce several interfering 
processes which are not desirable for any biosensor development. 
2.9.2. Antibody-Based Biosensors 
Being one of the most versatile biological selective agents, antibodies have been 
developed against a wide range of analytes. Although, they donot possess the catalytic activity as 
those of enzymes, antibodies have been long used as immunoassays, exhibiting higher binding 
power than enzymes have to their substrates, making them ultrasensitive recognition elements. 
This is due to the specificity of antigen-antibody interaction. Since the physiochemical change 
induced by antigen-antibody interaction does not produce any electrochemically detectable 
signal, the antibody or antigen is labeled with fluorescent compounds and electrochemically 
active species. Antibodies used are selective to the epitope on the antigen. A detectable signal is 
produced even if the epitope is present in live or a dead host. This is undesirable because a 
positive result from even a nonviable foodborne pathogen may raise a false alarm. Also, when 
adsorbed or covalently immobilized on the surface, they attach with a random orientation. 
Another pitfall in antibody based biosensors is the non-specific binding of proteins, instability 
and leakage of antibodies from the surface [35]. 
2.9.3. Aptasensors 
 Originating from in vitro selection across random sequence libraries nucleic acid 
aptamers are single-stranded oligonucleotides optimized for high affinity sensing. It is coined 
from the Latin ?aptus?, which means ?to fit? and involves the three-dimensional folding of a 
26 
 
peptidic bioreceptor with its target. This 3D folding falls under the category of stacking, shape 
complimentarity, electrostatic and hydrogen bonding. ?Aptasensors? are a novel class of 
receptors that bind to the target in picomolar and nanomolar concentrations which exceed the 
properties of antibody based biosensors. Aptamers are commonly used in therapeutic 
applications and one of the most commonly used model is Pegaptanib which is an aptamer 
directed against vascular endothelial growth factor (VEGF). The VEGF is a condition that causes 
blood-retina barrier breakdown and results in a reduction in fluid and electrolytes within the 
extracellular space. An anti-VEGF aptamer (PEGylated, short 2?-F- and 2?-methoxy-modified 
RNA oligonucleotide) is used as the nuclease resistant target [36]. 
 Other common applications are the use of aptamers in common diagnostic assays 
including blot assays, immunoprecipitation and flow-cytometry. Various types of proteins were 
detected using different configurations of aptamers as target molecules [37-41]. The main 
advantages of using aptamers as bio-receptors are: does not require biological raw materials, cost 
effectiveness, high thermal stability, wide range of surface chemistries and higher affinity. 
Although, aptamers are used to detect a wide spectrum of proteins and disease biomarkers, there 
are factors which limit it to the laboratory setting, including longer incubation time due to slow 
diffusion of the analyte through an unstirred recognition layer.  
2.9.4. Tethered DNA structures on 2D substrates 
 DNA serves as a very intriguing structure and forms the central icon of modern biology. 
Sensors using tethered DNA involve the selective interaction between immobilized receptors and 
its partner molecule and they are called affinity biosensors. Several types of biosensors have 
been developed, including electrochemical DNA sensors that use well defined plasmid nucleic 
acids. Synthetically prepared oligodeoxyribonucleotides (ODNs) are used as DNA probes in 
hybridization based sensing platforms. The end groups are labeled with thiols, amines or biotin 
27 
 
to be immobilized on to transducer surfaces. The probe sequence is generally designed 
complimentary to the target to be detected. As in the case of antibiotics, specific base-pairing 
between two strands of nucleic acids gives rise to a genetic code, which determines the 
replicating characteristics of the cell and thereby inherited properties of every living cell, 
proteins and enzymes. DNA receptors called ?Probes? can be used either in the ?short? synthetic 
form or in the ?long? cloned form. They can be used to detect cancers, viral infections and 
genetic diseases. DNA can also be used to improve sensor development by increasing enzyme 
yield by effectively duplicating the gene that codes an enzyme. Virtually any biological agent 
can be identified using hybridization based systems (genotyping). Any hybridization based 
system is characterized by its ability to form stable hybrid complexes with complementary DNA 
strands. The mechanism with which a complementary strand anneals to its probe adheres to the 
general Watson-Crick rules of base pairing.  
 
Figure 2-5 Sample preparation procedures for different types of DNA biosensors 
Ba ct e r i a l  cell s
Cell  l y si s Cell  l y si s
L y so z y m e /
Al k a l i
Cell  I so l a ti on
Pr ot e a se
Pr o te ase 
i n h i b i to r
Im m u n o  m ag n e tic 
sep ar ati on
DNA r R NA
In tr ac e l l u l ar
Ag
S u r face
Ag
28 
 
Dissociation of the hybrid is directly related to the melting temperature (Tm) of the hybrid 
and directly corresponds to the stability of the DNA hybrid. As a general rule, the Tm is 
equivalent to the sum of 2?C for each A?T base pair and 4?C for each G?C base pair. Besides, 
melting temperature can also be affected by change in ionic concentration, pH, length of the 
complementary sequence and mismatches between the strands. 
2.10. DNA as an alternate probe for biosensors 
 DNA sequencing technologies have turned out to be a laborious procedure involving 
electrophoretic size separation of labeled DNA fragments. Sequencing can also be achieved by 
hybridization of a specific target DNA with strands which have been attached to a surface [42]. 
In a typical DNA-based sensor array, single-stranded DNA (ssDNA) of different sequences 
called ?probe molecules? are attached to the surface. The array is then exposed to fluorescently or 
radioactively labeled ssDNA called ?target molecules? that hybridize with the probe molecules. 
Hence, DNA-based technologies hold great promise for diagnosis of genetic diseases and 
accurate sequence determination.  
Almost all self-replicating eukaryotes and prokaryotes can be discriminated on their 
nucleic acid sequence unique to that biological organism. The most challenging aspect of this 
issue is to develop a multi-assay sensing platform that recognizes all the potential biological 
variants. Based on this consideration, DNA arrays can be developed using two strategies: target 
amplification and direct target probing with signal amplification. The number of target molecules 
in vitro can be increased by incorporating amplification processes that greatly improve 
hybridization based systems. Ideally, nuclei-acid based assays should generate a gazillion of 
target copies starting with one, and detect a single ?probe molecule? organism. The sensitivity of 
nucleic-acid based assays is limited to 105?106 targets, which is very low considering the number 
of target molecules. In general rRNA-based detection systems have a low limit of detection 
29 
 
considering the number of cells available for growth. Saiki et al [43] proposed a novel 
amplification method called polymerase chain reaction (PCR), which has wide spread 
applications in clinical analysis, environmental microbiology and forensics. 
Recently, there have been reports of DNA assays developed for the detection of virulence 
gene in disease causing bacteria [44]. It involves a direct approach of coding genes that specify 
the virulence factor in biological threat agents. For instance, virulent strains in Bacillus anthracis 
such as ?Ames? and ?Vollum? are detected by targeting the corresponding genes lef, cya and pag 
that are present on its pXO1 plasmid. For Campylobacter jejuni the hippuricase gene encodes the 
specific enzyme activity, which contains a single open reading frame of 1149 bp, which is absent 
in other species [45]. The sequence of both strands of hipO were determined from similar clones, 
by the di-deoxy chain termination method using nested deletions obtained using an exonuclease 
III and a S1 nuclease. Besides, PCR-RFLP studies also indicate that the virulent gene (hipO) 
conserved among C. jejuni strains alone and thus detection of hipO gene using PCR based 
amplification methods can be a reliable method to detect C. jejuni [46]. 
2.11. DNA Immobilization 
2.11.1. Physisorption 
Dense nucleic acid (NA) layers obtained by immersing the substrate in the NA solution 
are the key in developing electrochemical DNA sensors on carbon electrodes. The negative 
phosphate backbone of nucleic acid binds to the carbon paste electrode or on highly oriented 
pyrolytic graphite, thereby having a strong effect on the adsorption of oligonucleotides, and is 
strong enough to conduct experiments in solution. Besides, a positive potential applied on the 
electrode during immobilization at a pH of 5.3 forms more condensed and a compact DNA 
lattice [47]. Physisorption can lead to inaccessibility of the immobilized DNA by a target 
molecule as the backbone is attached to the electrode, which results in poor hybridization 
30 
 
efficiency. Entrapment within cationic and conducting polymers enables stable immobilization, 
better accessibility and conductivity. However, immobilization in polymeric materials can lead 
to conformational passivity and high loading of the probe molecules.  
2.11.2. Covalent Immobilization 
In order to maintain the primary recognition ability of a DNA molecule, the sensing 
platform can be prepared by immobilization via one end, which in turn increases reusability and 
longer shelf-life. Millan et al. [48] reported a novel DNA biosensor which involves the covalent 
immobilization of amine functionalized DNA probes using 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide to glassy carbon electrodes (GCE). Henke et al. [49] designed a nucleic acid 
sensor using a amine terminal moiety extended from silanization of an optical fiber and 
covalently immobilizing DNA probes on the surface. DNA probes were also attached covalently 
attached using a self-assembled monolayer of a succinimidyl ester [50]. Recently, it was shown 
that nucleotides can be covalently immobilized on to aldehyde activated silanized alumina 
membranes, and detected using electrochemical impedance spectroscopy [51]. However, the 
reaction of these nucleotides with the corresponding amino groups has not been entirely 
documented.  
2.11.3. Affinity binding 
Highly specific interactions between avidin-biotin are used frequently to immobilize 
DNA probes biotinylated at the 5? end to avidinated surfaces. DNA sensors on 3D surfaces were 
designed using biotin-avidin-biotin bridge that can be photo-activated. Highly specific affinity 
DNA biosensor with better regeneration efficiency was designed using an intercalation of avidin 
on a copolymer electro-synthesized from polypyrrole-biotin and then immobilizing biotinylated 
DNA on to this surface. Giakoumaki et al [52] reported a novel DNA biosensor using surface 
plasmon resonance, where biotinylated DNA was immobilized on to a avidinated dextran 
31 
 
modified surface. Besides, an avidinated surface can act as a barrier that inhibits redox mediators 
and hence increases sensitivity in electrochemical detection. However, incorporating avidin-
biotin chemistry accompanies the problem of non-specific binding that produces a drift in most 
optical biosensors and is capable of producing a false positive result in most cases.  
2.11.4. Chemisorption 
 Adsorption can be classified in to two types based on the type of bonding to the surface 
of interest: physisorption and chemisorption. It involves the attachment of biomolecules through 
hydrophobic or electrostatic interactions, weak charge transfer forces or formation of van der 
waals forces and hydrogen bonds. Proteins can be adsorbed on a number of surfaces like, 
alumina, charcoal, clay, cellulose, kaolin, silica gel, glass and collagen. A classic example is 
attaching wild type proteins to a biochemical analysis surface such as polystyrene, 
polyvinylidene fluoride (PVDF), nitrocellulose membrane or a poly-Lys-coated glass plate 
traditionally used for immunoblot and phage display [53-55]. Mathematically, this phenomenon 
can be represented by Langmuir adsorption isotherm, where the fraction of the surface covered 
by the adsorbent is related to various kinetic parameters as given: 
rate of adsorption        (   )                                                                                                   (   ) 
                                                                                                                                      
rate of desorption                                                                                                                            (   ) 
At equilibrium, both the rates are equal, and hence: 
         
 
                                                                                                                                             (   ) 
 Nuzzo et al [56] reported the first spontaneous self-assembly of alkanethiols on gold. 
Since then, SAMs have grown to be a widely used research tool for various surface chemistries 
with molecular organization and defined stoichiometry over large surface areas.  
32 
 
 
Figure 2-6 Schematic of an ideal Self-assembled monolayer formed on a gold (111) surface 
SAMs are self-assembled nanostructures typically 1-3 nm in thickness and the most 
elementary form of any nano-meter sized thin-film material. In general, self-assembly involves 
the spontaneous organization of molecules in to reproducible arrangements in a drive to 
minimize interaction energy between molecules. Interfacial properties of metals and 
semiconductors can be tailored using this convenient system called self-assembled monolayers 
(SAM). These organic molecules assemble by the adsorption of molecular constituents from a 
solution or gas phase on to a solid platform in a regular array; or a spontaneous or epitaxial 
organization in to crystalline or semi-crystalline structures [57]. Additionally, the organic 
molecule acts as an electrostatic barrier against aggregation to minimize the interaction between 
surface atoms and behaves as an electrically insulation film. More specifically, proteins and 
single-stranded DNA align themselves and form a platform to sense its complement and hence to 
form a double-stranded helix. Hence a relatively strong and simple point self-assembled 
monolayer can be developed using chemisorption for effective DNA biosensors [58].  
M e ta l - S u l f u r  I n ter f a c e :
? S ta b i l i z e s  s u r f a c e  a to m s
? M o d i f i e s  e l e c tr o n i c  s ta tes
Or g a n i c  I n ter f a c e  ( 1 - 3  n m ) :
? Def i n e d  th i c kn e s s
? Ac ts  a s  a  p h ys i c a l  b a r r i e r
? Al ter s  e l e c tr o n i c  a n d  
o p ti c a l  p r o p e r ti e s
Or g a n i c  I n ter f a c e  ( 1 - 3  n m ) :
? De te r m i n e s  s u r f a c e  p r o p e r ti e s
? Ar e s  f o r  c h e m i c a l  f u n c ti o n a l  g r o u p s
S p a c e r  g r o u p
H e a d  gr o u p
T e r m i n a l  Fu n c ti o n a l  g r o u p
Au  ( 1 1 1 )
~4 . 9 7  ?
~3 0 ?
33 
 
High purity should be maintained during the surface preparation, as SAM formation is an 
exchange process such that, the thiol molecule needs to displace any adventitious material 
adsorbed on the surface to form a spontaneous assembly. The rate of desorption of these foreign 
materials should aid the attachment of the thiols to the surface. Degassing the solvent before 
SAM development also reduces oxidation thiols to oxygenated species. Chemical activation is 
required for the S-H bond to form a thiolated on the surface.  
Nuzzo et al [59] pioneered the studies behind the thermodynamics of SAM formation. 
Adsorption of a thiol is a fully reversible reaction, and the physisorption enthalpy was calculated 
to be ~33 kJ/mol [60, 61]. Also, the structural organization of thiols on Au (111) has been well 
documented, and is generally based on a (?3??3) R30? hexagonal closed packed structure, 
where R indicates rotated. Likewise the spacing between each alkane chain was found to be 
~4.97 ?, which is three times the van der Waals diameter of a sulfur atom (~1.85 ?). 
2.12. Structure-Property of biological ligands as SAMs 
 Although n-alkanethiols form ordered arrays of self-assembled molecules in a 
spontaneous or epitaxial fashion, the molecules used in biology and biochemistry differ 
substantially from these SAMs. More specifically, the factors that are not taken in to 
consideration are: size and shape of the ligands that form a SAM on gold, functional groups that 
counteract protein adsorption on to Au surface and interaction of the SAM with the aqueous 
media required for the biochemical reaction. Because of their large surface area (? 0.25 nm2) 
ligands and biomolecules are unable to form SAMs with the same organization as n-
alkanethiolates. Stearic hindrances from the bulk of the ligands induce disorders across the SAM. 
Paradoxically, if the ligands are able to form an ordered domain, the structural formation differs 
completely from that of alkanethiolates and creates pinholes and defects in the assembly. These 
defects can be overcome by using a monolayer comprised of a mixture of molecules known as 
34 
 
?mixed-SAMs?. It involves the incorporation of a molecular species whose length and size would 
commence the formation if a well-ordered assembly. Herne et al [62] reported the first ?mixed-
SAM? based platform for DNA hybridization. 6-Mercapto-1-hexanol (MCH) was used as the 
second thiol in order to prelude the formation of a well-ordered array of thiolated ssDNA on Au 
surface [57].  
2.13. Commercial C. jejuni testing kits 
 The latex agglutination tests using a limited amount of bacteria and the corresponding 
neutralizing agent and a reactive latex has been the  ?gold standard? for Campylobacter detection 
apart from the conventional culturing and staining methods [63]. However, the presence of 
antibiotics for suppressing bacterial growth and varying susceptibility of agar growth reduces the 
reliability of these tests. As a result, several commercial latex agglutination tests were conceived, 
including those incorporating polyclonal antibodies. In 1993, the Food and Drug Administration 
had approved ?Meritec-Campy?, which are able to detect antigenic outer membrane proteins or 
antigenic epitopes from flagella, for samples isolated from clinical and food samples. Other latex 
agglutination tests include PanBio-Campy, the Dryspot Campylobacter and the Microgen M46 
Campylobacter. These test kits showed reactivity to thermotolerant Campylobacter species, 
while the Dryspot Campylobacter showed difference by large clumps formation to other bacteria 
[64]. In order to avoid cross-reactivity and to detect as many campylobacter species, the 
appropriate test kit must be selected. However, these kits were not able to identify all of the 
campylobacter isolates, and had pooled serogroups. The change in antibody is not reliable 
enough to verify and confirm the diagnosis of a disease [65].  
2.14. C. jejuni biosensors 
 Biosensing platforms for direct detection of C. jejuni are obsolete compared to traditional 
microbiological methods such as multiplex polymerase chain reaction (mPCR), which initially 
35 
 
focuses on amplification of the gene of interest and then identifying the host cell. However, there 
has been seldom a report on direct detection of C. jejuni using a well defined sensor platform. 
For instance, Kuijf et al reported a sialylation based sensing platform using TLR-4 in order to 
detect C. jejuni, having a limit of detection of 35 pg/ml [66]. Dong et al [67] reported a surface 
plasmon resonance based detection of C. jejuni cells by biotinylated anti-C. jejuni antibody 
immobilized on an avidinated gold surface and a sensitivity of 107 cfu/ml was obtained. A 
commercial C. jejuni detection platform was also designed based on PCR-ELISA, where a 
chemically tagged nucleotide was incorporated in to a PCR amplicon and the product of the this 
PCR was detected with an antibody-enzyme conjugate [68]. Recently, a new enzyme 
immunoassay (EIA) called ?ProSpecT Campylobacter Microplate Assay? was developed for the 
detection of C. jejuni, which involves the detection of the uncharacterized Campylobacter?
specific antigen [69], which had a limit of detection varying from 3 ? 104 cfu/ml to 3 ? 105 
cfu/ml. Although, these methods prove to be sensitive they invariably require complicated anti-
C. jejuni antibody-enzyme interaction for specific detection on the surface.  
2.15. Transducers ? Why optical transducers? 
 Photon driven devices have mainly been based on photometric transducers, as in the case 
of spectroscopic methods. Research in optical transducers kicked off from the development of 
fiber optics, thus enabling greater flexibility and miniaturization. The most common optical 
transducer based techniques are absorption spectroscopy, fluorescence spectroscopy, 
luminescence spectroscopy, internal reflection spectroscopy, surface plasmon resonance and 
scattering of light. Transducers can be subdivided in to four main subdivisions as given. The 
main advantage of using optical transducers is because they can be used for different types of 
spectroscopy, with different spectro-chemical properties recorded, including amplitude, energy, 
polarization and decay time. 
36 
 
Transducer Techniques 
Optical Absorption, fluorescence, phosphorescence, Raman, SERS, 
refraction, dispersion spectrometry, Surface Plasmon Resonance 
spectroscopy, Luminescence 
Electrochemical Amperometric, Potentiometric, Voltammetric, Impedimetric and FET 
based sensors 
Mass Surface acoustic wave, love wave sensor, flexural plate wave, shear-
horizontal acoustic wave (SH-APW) 
Thermal Catalytic gas sensors, thermistors, thermal conductivity devices 
 
Table 2-2 Different types of transducers employed in sensor development 
Amplitude is the most commonly measured parameter, as it is directly related to the 
concentration of the analyte. Intramolecular atomic vibrations and conformational changes in the 
local environment can be monitored with change in energy (i.e. Raman or FT-IR Spectroscopy). 
Besides this, the interaction of a free molecule with any surface can be studied using polarization 
measurements. This is because the polarization of emitted light due to a molecule remaining 
fixed to a surface is constant. Time decay emissions can be used to obtain information about 
molecular interactions as the decay time and length are very much dependent on the excited state 
of the molecules. Additionally, the phase of any emitted radiation changes the refractive index of 
the medium during a biomolecular event. This phase change on the impinging radiation is due to 
the change in medium when the molecule binds to the surface. There were several reports of 
novel optical transducers in the past. Vo-Dinh et al [70, 71] reported a novel phase-resolved fiber 
optic fluoroimmunosensor (PR-FIS), which can differentiate a metabolite and the corresponding 
carcinogen based on fluorescence lifetimes. Plowman et al [72] reported an etched thin-film 
waveguide fluoroimmunosensor that was able to detect analyte molecules in the femto-molar 
concentrations. Besides, Balasubramanian et al [73] reported a novel immunosensor based 
37 
 
surface plasmon resonance spectroscopy for the detection of bacterial cells using lytic phage as a 
recognition element. 
Surface plasmon resonance (SPR) biosensors are based on optical transduction and offer 
?label-free? and ?near real-time? detection of the analyte of interest. The principle of this method 
has been discussed extensively in literature [74-76]. Total internal reflection (TIR) occurs at the 
interface between two completely different media. An evanescent wave infiltrates through the 
interface which is modified with a 50 nm gold film, and couples with the surface plasmons. As a 
result, the reflectivity and resonance angle changes, which is directly correlated to the change in 
refractive index (RI) of the medium. The change in RI which is continuously monitored as a 
function of time is due to the change in surface concentration of the interacting ligands. 
Typically, a response of 1000 RIU or 0.1? change in angle corresponds to a change in surface 
protein concentration of 1 ng/mm2. SPR, coupled with the right surface chemistry, has proven to 
be a highly sensitive technique for the analysis of biological systems as labeling of ligands or 
receptor molecules are not required and can be used to determine kinetic parameters of the 
interacting molecules.  
Diffraction Optics Technology (DOT) biosensing is similar to SPR based sensors, except 
that it is a grating-based light diffraction method. Probe molecules are immobilized on an 
optimized grating that produces a strong diffraction pattern when a coherent light strikes an array 
of molecules immobilized on the surface. Diffraction efficiency is improved when biomolecules 
bind to a patterned surface, which in turn increases the signal intensity and hence a measurable 
change in signal is produced. Being a self-referencing method, it is considered a robust sensor 
platform for analysis. In simplistic terms, the diffraction efficiency depends on the relative height 
difference between the patterned and non-patterned areas, thus non-specific binding to both the 
38 
 
patterned and non-patterned regions will not affect the signal significantly. This attribute 
provides a considerable advantage over other optical biosensor systems in which any surface 
binding event will cause a spurious increase in signal [77].  
2.16. Thesis organization 
 Pathogenic bacteria should be eliminated from poultry and dairy products in order to 
avoid any potential health hazards and combat disease transmission. Although, there are several 
methods to detect bacteria, most of them are labor intensive when large samples have to be 
prepared. All microorganisms can be identified and differentiated based on a virulent gene that 
causes diseases. Biosensor platforms using immobilized ssDNA have the potential for quickly 
sequencing and detecting DNA samples by selective hybridization. Additionally DNA 
diagnostics find applications from sequencing the entire human genome (The Human Genome 
Project), detecting disease-causing food-contaminating microbes and forensic and environmental 
applications. However, applications of ssDNA in SPR and DOT based platforms for direct 
detection of virulent ssDNA is minimal. Hence a proof of concept was developed to show that 
ssDNA directly tethered to a substrate without complex surface chemistries can be used to 
capture the hipO gene of C. jejuni without losing the sensitivity after several trials and with the 
same hybridization efficiency after several trials of regenerating the sensor surface, by 
denaturation of the double-helix structure formed during detection. Finally, a novel non-
destructive technique was developed to analyze the electron transfer and charge injection through 
SWNT based nanomaterials, where dsDNA has been used as a dispersant for SWNTs; using 
scanning electrochemical microscopy. These nanomaterials were developed using a ?bottom-up? 
approach with critical control for anti-bacterial applications. 
Thus, the main objectives of the present study were: 
39 
 
I. Investigation of the prospects of DNA immobilized on a SPR and DOT sensor platform 
for the label-free detection of the hippuricase (hipO) gene in Campylobacter jejuni. Here 
we use ssDNA as a nanoscale scaffolds for ?real-time? detection of pathogenic bacteria. 
The detection was additionally verified with the immobilization chemistry using 
electrochemical impedance spectroscopy (EIS). DNA scaffolds were also developed 
using a long and a short probe to verify the prospects of using long chained ligands 
directly immobilized on the sensing surfaces.  
II. The preparation of supramolecular adducts using DNA as a dispersing agent in order to 
prepare an aqueous phase of SWNTs with uniform length. Accordingly, DNA can also be 
used as a precursor and a building block for macromolecular electronics. An advanced 
analytical technique - scanning electrochemical microscopy (SECM), is used as a novel 
approach to measure the lateral (in-plane) and cross-film electron transport in nano-
composite films developed using DNA and SWNT adducts. Energetics of the entire 
charge injection process can be quantified using SECM, with sensitivity across every 
single nanotube layer. 
 
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48 
 
3. Scanning Electrochemical Microscopy ? Principle & Experimental Setup 
3.1. Scanning Electrochemical Microscopy (SECM) 
 Measurement of an electric current through an ultra-microelectrode (UME) when moved 
in a solution in the vicinity of a substrate that instigates an electrochemical response from the tip. 
This perturbation then provides information about the nature and properties of the substrate. It 
has played a vital role in electrochemical sciences for the past two decades. As an 
electrochemical tool, it can be used to study the heterogeneous and homogeneous reactions and 
imaging the chemical reactivity and topography of a wide range of surfaces. Because of it sub-
micron and nanometer-sized spatial resolution, SECM aids in monitoring local kinetics and 
interfacial phenomena. The most commonly used operational mode in the SECM is the tip 
generation-substrate collection (TG/SC) mode, where a reactant generated by the tip is detected 
at the substrate electrode. An alternate method is the substrate generation-tip collection (SG/TC) 
mode where the concentration profiles near the substrate can be studied without rastering or 
imaging the surface.  
 Although the SECM resembles a scanning tunneling microscope (STM), in the method 
used to raster the tip over the substrate, they differ from each other in the principle of operation 
and range of applications. Unlike the STM where the current between the tip and the substrate is 
non-faradaic, the current is defined by a redox process between the tip and substrate. This current 
is electron transfer controlled at the interface and mass transfer controlled across the bulk 
solution [1]. 
 
49 
 
3.2. Ultramicroelectrodes (UME) 
 Ultramicroelectrodes give a quantitative analysis of the electrochemical measurements 
that can be performed using SECM. Fig. 3-1 shows a simplified setup of a SECM. The solution 
contains a species, O, whose concentration is c, and also contains a supporting electrolyte which 
reduces the solution resistance and ensures a diffusion controlled reaction between the electrolyte 
and the substrate.  
 
Figure 3-7 Schematic of a cell for ultramicroelectrode voltammetry 
A platinum (Pt) auxiliary electrode completes the circuit. When the circuit is closed, a reduction 
reaction takes place at the tip,  
 
resulting in a current flow and an oxidation reaction at the auxiliary electrode. Meanwhile the 
potential across the tip is monitored with respect to a standard silver/silver chloride (Ag/AgCl) 
reference electrode, which is represented by a standard voltammogram for an ultramicroelectrode 
(Fig. 3-2).  
I V
U M E
P t Co u n t e r  
e l e ct r ode
R e f e r e nc e  
e l e ct r od e
O R
O
O
O
O + ne ? R 
50 
 
 
Figure 3-8 Typical voltammogram for a 10 ?m ultramicroelectrode 
The limiting current obtained as a result of diffusion of species, O, from the bulk to the electrode 
surface is completely mass-transfer controlled. For a conductive disk electrode of radius a, this 
steady state current when the tip is at an infinite distance from the substrate is give by: 
                                                                                                                                                    (   ) 
Although electrodes with other shapes, hemispheres and cones can be used, most of the SECM 
experiments are carried out with disk electrodes because of their high sensitivity. Besides, the 
current is independent of the radius of the insulating glass sheath, rg, which is also represented 
as: 
                                                                                                                                                             (   ) 
Likewise, convection has little or no influence on the tip current because the diffusive flux of 
species O is relatively large compared to the size of the UME. Apparently, if the potential at the 
tip is sufficient enough to reduce species O to R, at a diffusion limited rate, then electrode 
51 
 
reaches a steady state at a relatively short time. Numerous applications list out when SECM is 
coupled with biological systems. These include imaging of biological structures, kinetics of 
enzymatic reactions to name a few. Measurements are usually carried out in buffered solutions, 
with the UME that does not interact with the immobilized substrate unlike most of the scanning 
probe techniques. 
3.3. Feedback Mode 
  In the feedback mode, a steady-state diffusion controlled current iT,?, is measured at the 
UME, where the ? implies the distance between the substrate and the tip. A hemispherical flux is 
used to actuate the current from the bulk to the electrode. If the tip electrode is far from the 
substrate, the infinite steady-state current is due to hemispherical diffusion (Fig. 3-3A). 
However, the diffusion layer will be hindered and the tip current decreases if the substrate is an 
insulator, and hence iT < iT, ?. As a result, the value of iT becomes smaller, as it gets closer to the 
substrate (Fig. 3-3B). At the limit when the distance between the tip and the substrate, d, 
approaches zero, iT reduces to zero. This is termed as ?negative feedback?. On the contrary, if the 
substrate is a conductor, and maintained at a potential where R can be recycled back to species 
O, the tip current will increase. Although there is blockage of the diffusion of species O, it is 
regenerated by the oxidation of R at the substrate and diffuses to the tip, which in turn increases 
the current (Fig. 3-3C). Thus for a conductive substrate, where iT > iT, ?, as d approaches zero, 
electron tunneling occurs at the tip and hence the current increases drastically, which is termed as 
?positive feedback?. In order to completely understand and conceive the mechanism occurring at 
the tip and substrate, a quantitative explanation for entire feedback phenomena should be 
formulated.  
52 
 
 
Figure 3-9 Basic principles of SECM: A) Far from the substrate [Hemispherical diffusion] B) 
Near the substrate [Feedback Diffusion-conductive] and C) Near the substrate [Hindered 
diffusion-insulating] 
3.4. Theoretical Predictions 
3.4.1. First-Order Heterogeneous Reaction for reaction between tip and substrate  
 In the feedback mode, the species O is reduced at the tip and can be reoxidized at the 
substrate. The process occurring between the tip and the substrate can be written as: 
                     (UME)                                                    
OR
HEMISP HERICA L  DI FFUS I O N
FEEDB A CK DIFFUS IO N
HIN DERED DI FFUS I O N
C O NDUCT I VE SU BS T R A T E
I NSU L A T I NG  SU BS T R A T E
(A )
(B )
(C)
53 
 
                     (Substrate)                                               
Theoretical predictions for the SECM include a combination of a cylindrical diffusion to the 
ultramicroelectrode (UME) and a thin-layer type diffusion space. As a result, this quasireversible 
prediction under non-steady state conditions can be given as: 
   
   
    
    
    
    
 
 
   
                                                                                                                     (   ) 
with boundary conditions: 0 < T, 0 ? R, 0 < Z < L 
Other dimensionless variables are given as:  
                                                                                                                                                               (   ) 
                                                                                                                                                               (   ) 
      
  
                                                                                                                                                        (   ) 
                                                                                                                                                             (   ) 
If the diffusion coefficients of both the species O and R are considered equal, then the problem 
can be described in terms of a single species. Then, the boundary conditions are given by: 
0 < T, 0 ? R < 1, Z = 0                         (UME surface) 
   
              (    )                                                                                                         (   ) 
0 < T, 1 ? R ? RG, Z=0                        (insulating sheath) 
    
                                                                                                                                                          (   ) 
0 < T, 0 ? R ? h, Z = L                         (substrate) 
   
              (    )                                                                                                        (    ) 
54 
 
R > RG, 0 ? Z ? L: 
                                                                                                                                                           (    ) 
where,    and    are the normalized fluxes of the species at the tip and substrate. Hence, it can be 
represented as: 
  
   
   
   
 
                                                                                                                                            (    ) 
  
   
   
   
 
                                                                                                                                           (    ) 
                                                                                                                                                         (    ) 
                                                                                                                                                            (    ) 
where    is the radius of the tip-insulating material and    is the substrate radius. Considering 
the definition of the problem, the initial condition is, 
T = 0, 0 ? R, 0 ? Z ? L;                                                                                                            (    )                                                                        
On the contrary, if the substrate is an insulator, then    ? 0. Also, the reactions occurring at the 
tip and the substrate are electrochemical in nature. Hence, the rate constants for reduction and 
oxidation are given by Butler-Volmer equations: 
        [   (     )]                                                                                                              (    ) 
        [(   ) (     )]                                                                                                     (    ) 
Hence, the solution of the problem can be solved in terms of dimensionless currents   ( ) and 
  ( ) and can be given by, 
  ( )     ?   
 
 
(   )                                                                                                                 (    ) 
55 
 
  ( )     ?   
 
 
(   )                                                                                                                  (    ) 
where    and    are the physical currents due to normalization of the tip current,     
  
 
 This general formulation involves a mixed diffusion control for one of the electrodes and 
hence,  
0 < T, 0 ? R ? 1, Z = 0;                                                                                                            (    ) 
can be substituted with the boundary condition in Eq. 8, and Eq. 10 can be replaced with a much 
simpler boundary condition: 
0 < T, 0 ? R ? h, Z = L;                                                                                                           (    ) 
Hence the time dependent SECM problem was solved using a semi-analytical two dimensional 
equations as explained in [2] and [3]. For a conductive substrate,       , and it is always 
larger than the value calculated using the predicted ?thin-layer cell? theory (TLC). On the other 
hand, for an insulating substrate,       , and hence smaller the tip-substrate separation, the 
earlier the transients between SECM and the tip electrode occur.  
3.4.2. Steady-State Conditions considered for reaction at SWNT nanocomposites 
 A steady state problem in SECM can be given by the following Laplace equation, with its 
respective boundary conditions, for oxidized or reduced form of the mediator [4].  
   
    
   
    
 
 
  
                                                                                                                            (    ) 
where,         ,     ,     , and   is the radius of the SECM tip (10 ?m) (Fig. 3-4). The 
following boundary conditions generally apply: 
 (   )                                
  
                                            
56 
 
 (   )                                         
 (   )                                     
  
                                                 
  
                                           
 
Figure 3-10 Geometry of the diffusion domain and the parameters defining the diffusion problem 
in SECM 
where,   is the normalized tip-substrate distance and     . The boundary conditions for the 
electron transfer reaction between redox mediator and DNA-SWNT/Lysozyme-SWNT assembly 
is given by 
                   (     ) ( )       (     )(      ( ))                                (    ) 
d
a
r
g
r
1l
1
z
U ME
SUBS TRA TE 
57 
 
where,       is the diffusion coefficient of the redox mediator and   is the surface concentration 
of the SWNTs, and      is the maximum surface concentration. But, if this equation is made 
dimensionless, then 
  
         (   )     (   )                                                                                               (    ) 
where,                ? ,                ?  and        ? . Lateral charge transport 
across the SWNT nanocomposites can be given by the following equation, assuming a thermally 
active mechanism 
     ( 
  ( )
    
 
 
  ( )
  )      (     ) ( )       (     )(      ( ))    
From the above equation, it is clear that       is the parameter that characterizes lateral charge 
transport though the SWNT based material. The dimensionless form can be given as, 
   
    
 
 
  
   
 
   (  (   )     (   ))                                                                     (    ) 
where        (   )? , the maximum surface concentration and              ? . During a 
steady-state SECM reaction, the response from the tip is sensitive to the ratio of the surface and 
solution diffusion coefficients and the maximum surface concentration of SWNTs. Therefore, 
the normalized tip current during feedback mode can be predicted as 
 
            
 
 ? 
  
    
 
 
                                                                                                             (    ) 
As the amount of positive feedback is given by the dimensionless parameter      , the diffusion 
coefficient characterizing the electron transport across the film can be correlated to the 
conductivity of the film as 
58 
 
       
      
     
             
                                                                                                   (    ) 
Hence this theoretical explanation gives the effect of lateral charge transfer across nanometer 
sized thin films including back diffusion across the plane of the microelectrode, and is related to 
the difference between the standard potentials of the SWNT nanomaterials and redox mediator. 
3.4.3. Ohmic behavior at the substrate during charge injection                                                                   
 As discussed above, the SECM response observed at steady state is due to the 
combination of diffusion flux in the solution and the flux due to lateral charge transport in the 
nanotube film. As the diffusion flux is directly proportional to the concentration of the redox 
mediator, in order to observe a measurable effect due to lateral charge transfer, a sufficiently low 
concentration of the redox couple is to be used. Since, SWNTs have large coulomb gap, the 
nanotubes deposited on the surface can be considered as a multivalent redox species. 
Additionally, the transport mechanism of electrons through the layer is considered to be 
thermally activated tunneling. Besides, if the thickness of the nanocomposites film is extremely 
thin compared to the diameter of the tip, then it will not be polarized in the z-direction.  
 The layers of nanotubes are developed on an insulating substrate and hence hindered 
diffusion is observed at high redox mediator concentrations. At reduced concentrations, the 
relative contribution of mediator regeneration due to lateral electron transport in the film 
increases. Hence, the behavior changes from an insulator (negative feedback) to a conductor at a 
given concentration. As a result of this behavior change due to concentration dependence, the 
lateral charge transport model can be adopted. The model predicted, assumes that the monolayer 
exhibits ohmic behavior, i.e. the current at the tip electrode is directly proportional to the 
electrochemical potential gradient, which is valid in the absence of a coulomb blockade and low 
59 
 
potential gradients. Thus, the electrochemical potential of electrons in the nanotube monolayer   
is given as 
      ( ( )                                                                                                                             (    ) 
where   the standard is electrochemical potential,   is the charge,   is the radial coordinate and 
 ( )  is the local electrostatic potential in the film. Taking this in to consideration, the 
equilibrium electrochemical potential is given by 
           (         ? )                                                                                                               (    ) 
where       and      are the bulk concentrations of the reduced and oxidized species,   
Boltzmann constant and   the absolute temperature. The conductivity due to electron transfer is 
adopted from Ohm?s law, 
                                                                                                                                                       (    ) 
which can be expressed as the flux of the electrons,   as a function of the electrochemical 
potential gradient.  
     
   
 (    )
                                                                                                                            (    ) 
Introducing the following dimensionless quantities, 
 ?     
 
                                                                                                                                                (    ) 
       
   
   
  
  
    
                                                                                                                           (    ) 
     ?                                                                                                                                                   (    ) 
    
   
                                                                                                                                                 (    ) 
60 
 
          
      
                                                                                                                                      (    ) 
Combining these dimensionless quantities, the electrochemical mass-transport on the substrate 
coupled with the diffusion equation can be given by 
   ?
    
 
 
  ?
   
  
 ((   ) 
 ?  ?     ?  ? )                                                                                (    ) 
Where,    is the dimensionless standard rate constant of the electron-transfer reaction between 
the redox mediator and the nanotube layer,   is the dimensionless concentration of the redox 
mediator and   is the dimensionless conductivity of the film [5, 6].  
3.5. Theoretical fitting of conducting and insulating approach curves 
 As discussed in the previous section, the equations representing a diffusion-controlled 
SECM process explains two limiting cases of the steady-state problem, i.e., 1) pure positive 
feedback produced by rapid generation of the reactant species, 2) pure negative feedback when 
no mediator regeneration occurs at the substrate. Hence, from the above theoretical 
considerations, for RG values from 1.1 to 10, we have 
 ( )
  
(                       (        ))
                                                                   (    ) 
for an insulating substrate with        and thus a negative feedback. On the other hand, if 
       for a positive feedback, we have 
 ( )                         (        )                                                                       (    ) 
Where,      and   being the tip-substrate distance and   the tip radius. 
61 
 
 
3.6. SECM setup 
 Introduced by Bard and coworkers in 1989 [7], the Scanning electrochemical microscope 
is an instrument that can analyze the chemistry at high resolution near interfaces. It is based on 
the reactions that occur between the small electrode and the close proximity of the substrate. 
SECM can be used to obtain reactivity images and quantitative measurements of reaction rates. 
Pointedly, a current flows through an ultramicroelectrode, near a conductive or an insulating 
substrate immersed in an electrolyte solution. In order to probe the diffusion layer, the UME can 
be perpendicular to the surface along the z-axis, and can be scanned at constant z-value across 
the surface (x and y-axes). The tip and sample are a part of the electrochemical cell, including 
the reference electrode (Ag/AgCl) and counter electrode (platinum electrode). The tip moves 
with a given resolution towards the substrate by means of piezoelectric elements or stepping 
motors driving differential springs. The instrument used in this research is a CHI900B Scanning 
Electrochemical Microscope consists of a digital function generator, a bipotentiastat, a high 
resolution data acquisition circuitry, a three dimensional nano-positioner with a combination of 
stepper motors and a XYZ piezo block, a stepper motor, piezo controller and a sample-cell 
holder. A 3D nano-positioner allows a spatial resolution of 1.6 nm and a maximum travelling 
distance of 2.5 cm. The instrument has a potential control range of ?10 V and a substrate 
potential of ? 10 V versus ? 3.276 V and a current range of ?10 mA, with a current measurability 
up to 1 pA (Fig. 3-5). The input impedance of the reference electrode is 1012 ? and a compliance 
voltage of ? 12 V with the current measurement range from 1?10-12 ? 0.001 A/V in 10 ranges. 
Speed of the ADC controller is 16-bits at 25 KHz and the dual high resolution ADCs have 20-bit 
62 
 
(1 kHz) and 24-bit (10 Hz) controllers. Other features include, an automatic iR compensation 
controller, with a RDE control giving a 0-10 voltage output. 
 
Figure 3-11 Schematic of an SECM electrochemical cell setup 
3.6.1. Tip preparation and operation 
 Disk-in-Glass microelectrodes are the most conventionally used microelectrodes in 
SECM. A fine platinum wire 10 ?m in diameter is placed in a 1 mm id Pyrex tube sealed at one 
end. The other end of the tube is heated in a nichrome wire helix in vacuum for an hour, 
followed by sealing the glass followed by inspecting the glass for any impurities and bubbles in 
the glass. The sealed end is then polished with sandpaper, until the cross section of the Pt wire is 
exposed. Electrical connections to the sealed end are made using silver paste. The glass wall 
A g /Ag Cl  
R e fer e n c e
Pt Wi r e  
Co u n te r  
S E CM  c e l l
(Te fl o n )
S u b str ate
Ti p  Holde r
P t U M E
Wor ki n g
63 
 
(insulating part) is conically sharpened with emery paper until the diameter of the flat glass 
section if 10 more than that of the Pt UME (RG = 2.2) (Fig. 3-6). 
 
Figure 3-12 SEM image of the 10 ?m Pt UME showing the insulating sheath and the tip (RG ? 
2.2) 
 Before using the UME for SECM experiments, the electrode is polished with 3 ?m, 0.03 
?m and 0.05 ?m alumina polishing liquid (Buehler, USA). The electrode is then washed with 
water and then washed in 0.5 M sulfuric acid (H2SO4) and 0.5 M nitric acid (HNO3) for 5 
minutes each. The UME is again washed with water and verified for impurities and 
imperfections at the surface. The next step is to fix the UME in the holder and connect the UME, 
reference and auxiliary electrode to their respective leads. A cyclic voltammogram is obtained 
between -0.2 to 0.5 V with a sensitivity of 1?10-9 and a scan rate of 0.01 V/s and a quiet time of 
5 s. 
 At this point, the experiment parameter setup has been completed. The UME has to be 
positioned over the substrate electrode. A probe approach enables one to approach the tip quickly 
100 ? m
64 
 
to the substrate and based on the increase or decrease in tip current. The current variation implies 
as to how much the substrate is biased relative to the UME reaction. Before starting the probe 
approach, the potential values from the voltammogram are entered, with the sensitivity of the 
probe electrode set at 1?10-9 A. Probe stopping current is the value at which the tip electrode 
stops inching towards the substrate and should be set at 125 %. Maximum increment is the 
distance that the tip moves for each step and is set at 5 ?m. Using the probe control, the tip is 
placed over the substrate. With a total travel distance of ~300 ?m and a scan rate of 3 ?m/s, the 
tip is scanned towards the surface in order to obtain a feedback curve.  
3.6.2. Protocol for SECM setup 
 The following protocol is routinely used to analyze nanomaterials using SECM. 
1. The UME is cleaned with 3 ?m, 0.3 ?m and 0.05 ?m alumina particles for 30 s each 
2. Wash with copious amount of DI Water 
3. Rinse with 0.5 M sulfuric acid (H2SO4) and 0.5 M nitric acid (HNO3) for 5 minutes each 
4. Blot the surface with Kim wipe 
5. Fix the UME to the holder and switch on the SECM controllers 
6. Add 1 mM Ferrocenemethanol in the SECM electrochemical cell and position the tip in 
the solution such that it is partly immersed 
7. Obtain a cyclic voltammogram in the range -0.4 to 0.4 V at 10 mV/s scan rate and in the 
presence of Ag/AgCl reference electrode 
8. Switch to SECM technique and enter the peak potential values obtained from the 
voltammogram 
9. Using the probe  controller, position the tip close to the substrate and conduct a probe 
approach curve at a sensitivity of 1?10-9 A. Set the probe stopping current to 125 % 
65 
 
10. Once the tip reaches the substrate and shows a feedback response, retract the curve by 
~300 ?m and obtain a probe scan curve at a scan rate of 3 ?m/s 
11. After obtaining the feedback curves, record the data and report the results, using the 
equations discussed above, based on the type of feedback obtained 
 
3.7. References 
1. Bard, A.J., et al., Scanning Electrochemical Microscopy. Introduction and Principles. 
Anal. Chem, 1989. 61(2): p. 132-138. 
2. Mirkin, M.V. and A.J. Bard, Multidimensional integral equations: a new approach to 
solving microelectrode diffusion problems: Part 2. Applications to microband electrodes 
and the scanning electrochemical microscope J. Electroanal Chem, 1992. 323(1-2): p. 
29-51. 
3. Bard, A.J., et al., Scanning electrochemical microscopy. 12. Theory and experiment of the 
feedback mode with finite heterogeneous electron-transfer kinetics and arbitrary 
substrate size. J. Phys. Chem., 1992. 96(4): p. 1861-1868. 
4. Ruiz, V., et al., Probing Conductivity of Polyelectrolyte/Nanoparticle Composite Films 
by Scanning Electrochemical Microscopy. Nano Lett., 2003. 3(10): p. 1459-1462. 
5. Liljeroth, P., et al., Electron Transport in Two-Dimensional Arrays of Gold Nanocrystals 
Investigated by Scanning Electrochemical Microscopy. J. Am. Chem. Soc., 2004. 
126(22): p. 7126?7132. 
6. Ruiz, V., et al., Molecular Ordering and 2D Conductivity in Ultrathin Poly(3-
hexylthiophene)/Gold Nanoparticle Composite Films. J. Phys. Chem. B, 2005. 109(41): 
p. 19335?19344. 
66 
 
7. Bard, A.J., et al., Scanning electrochemical microscopy. Introduction and principles. 
Anal. Chem, 1989. 61 (2): p. 132?138. 
67 
 
4. Diffraction Optics Technology (DOTLAB) 
4.1. Optical Transducers 
 Several years ago, optical sensing devices called ?optrodes? were introduced besides 
electrochemical platforms, originally to monitor pH and CO2 concentrations. This kick started 
the novel field in optical sensors and a number of different optical transducers have been 
developed. Interactions between radiation and matter, dispersion, reflection, scattering, changing 
transmission or fluorescence are used to monitor probe and target interaction. Advantages 
include, high sensitivity, miniaturization of the sensor platform and cost efficient. Optical sensor 
platforms are capable of measuring surface concentration to about 25 ng/ml. Likewise advanced 
concepts such as total internal reflection and evanescent-field based light propagation have made 
a far-reaching improvement in optical sensor development. Two basic signal transduction are 
involved in optical sensor development, a) probing the surface for minute changes in refractive 
index by evanescent wave techniques such as surface plasmon resonance and b) fluorescence 
assays by labeling the sample [1]. 
4.2. Detection using diffraction based sensing  
 Diffraction based sensing can be used to observe biomolecular interactions between 
probe and target molecules. A diffraction pattern is obtained when a laser light impinges on a 
pattern of repeating parallel lines which could yield an image with equally spaced spots in a row. 
The addition of target molecules helps interact with the diffraction pattern, which in turn causes 
an increase in height or refractive index of the patterned substrate and apparently causes an 
increase in intensity of the diffraction image. Diffraction based sensing involves, grating-based 
68 
 
light diffraction and immobilized affinity surfaces for a sensitive platform without the use of 
chemi-luminescent and fluorescence labels [2].  
4.2.1. Diffraction measurement and ?self-referencing? 
 Referencing plays an important role in developing label-free biosensors, which includes 
building a reference channel (RC) similar to the working channel (WC), and requires an extra set 
of electronic and fluidic system. Bailey et al., [3] reported a micro-patterned technique to 
generate a surface grating that consist of functional and non-functional surfaces.  
 
Figure 4-13 Schematic representation of the binding phenomena to patterned surface of 
polystyrene sensor. The illustration shows that diffraction based sensing is 'self-referencing' 
A v i d i n ate d  sp o t
B i o tinyl ate d  p r o b e  m o l e c u l e
Pr o b e  m o l e c u l e  +  Tar g e t ssD N A
h
? h +  h
Pr o b e  m o l e c u l e  +  Tar g e t ssD N A  +  N o n - sp e c i fi c  b i n d i n g
? h +  h
do t Lab O t her  op t ic al  pla t f or ms
69 
 
The receptor molecules bind only to the functional surface and not to the passivated non-
functional surface. Analyte association or dissociation modulates the amplitude of the recorded 
diffraction intensity. Meanwhile, the bulk effects that alter the refractive index across the 
functional and non-functional surfaces do not induce any change in diffraction intensity. Hence, 
the surface is said to be ?self-referencing? (Figure 4-1). Additionally, surface plasmon field 
enhancement can be used to boost the sensitivity of the diffraction based sensor platform [4].  
4.2.2. Sensitivity 
 Nakajima et al., [5] proposed a model to describe the intensity of diffracted beam through 
a transmission grating. As the sensor consists of two parts, a transparent portion and a semi-
transparent region that partially absorbs light. During analyte adsorption, the transmitted beam is 
diffracted, as it passes through a grating and a change in intensity is observed. The intensity of 
the diffracted beam was given by 
                  (    
 
)                                                                                                    (   ) 
Where,    is the difference in intensities of the beams and    and   are the beam intensities 
before and after transmission. But the intensity of transmitted light is given by Beer-Lambert?s 
law  
     (   )                                                                                                                                               (   ) 
Substituting Eqn. 4.2 in Eqn. 4.1, we obtain the following relation 
  (  ? )  (      )                                                                                                                         (   ) 
Although, diffraction based sensing can be related to fluorometry, both have different sensitivity 
levels and quantum yields. The efficiency of light collection in diffraction methods is of the 
order of 0.5; while most fluorescence based methods have an efficiency of 0.1-0.001. Hence, 
70 
 
these techniques are more universal, highly sensitive and show minimum fluorescence, which 
results in an infinitesimal signal loss.  
4.3. dotLab system 
 dotLab system (DOT) was developed by Axela Biosensors Inc. (Toronto, Canada) and 
will be used for diffraction based experiments. In the DOT system, a coherent light strikes the 
regular pattern of capture molecules on the sensor surface creating interference patterns that 
produce a well defined image. When target molecules interact with the receptor surface, the 
height of the pattern increases which in turn increases the diffraction intensity. This change in 
intensity is monitored by a photo diode. As the process of diffraction across patterned surfaces is 
self-referencing, the transduction of binding events will occur exclusively for target binding to 
the probe surface, which minimizes non-specific binding. This is a characteristic advantage of 
DOT over other biosensing platforms. The DOT system is based on a near real-time and total 
internal reflection scheme which allows for 95% of the signal to be detected [6].  
A 670 nm laser diode, class 3R with a maximum output of 4.0 mW and a beam 
divergence of 5.0 mRad was used as the light source, to produce a diffractive pattern. One end of 
the sensor was connected to a fluidic panel, which is responsible for circulating the buffer 
solutions and the other end is connected to a sensor manifold which aspirates the analyte from 
the well. The sensor chip was made of optically clear polystyrene with an interspersed prism 
situated below the flow channel. The linear flow channel which can hold a volume of 10.0 ?l, 
consisted of eight, circular assay spots, each 2.0 mm in diameter, with 15.0 femto moles of 
avidin deposited on each spot (Figure 4-2) [7]. The sensor chip has an integrated prism which is 
responsible for the diffraction of the laser light (Fig. 4-3). 
 
71 
 
 
Figure 4-14 Cross section of a polystyrene dotLab sensor showing the microfluidic setup 
 
 
Figure 4-15 Schematic representation of a dotLab system 
R e fl e c te d  m ai n  b e am
D i ff r ac te d  b e am
A ssay  
S p o t
In l e t
Fl o w c h an n e l
Sen s o r  1    (S1)
Sens o r  3    (S3 )
Bul k  B
W a s te
Sens o r  4    (S4 )
Sen s o r  2    (S2)
Bul k  A
Bul k  C
Bul k  B
A i r
Sy ri ng e 2
Sy ri ng e 1
6 - po rt v a l v e
Sens o r  bl o ck
A uto - Sa m pl er  r o bo t
96 - w el l  pl a te
A uto - Sa m pl er  a rm
W a s te co l l ecto r
W a s h to w ers
72 
 
Every sensor is purchased from the manufacturer at a low cost (US$ 15 per chip) and has 
up to eight assay features along each linear flow channel. Each spot has a diameter of 2 mm and 
a depth of 0.25 mm. Neutravidin is immobilized on the diffraction pattern, in order for biotin 
labeled bio-probes to be attached as receptors for sensor development. The DOT system 
introduces the analyte sample through the channel using a high precision fluidic controller. 
Different reagents can be passed over the surface by introducing an air gap between each 
reagent, which in turn leads to a transient spike in the signal. dotLab software version 1.1.1 
provided by Axela monitors the change in diffraction intensity near the sensing surface 
calculates the statistical noise in the signal and displays a self-referenced signal relative to 
biomolecular interactions in all the eight spots. The signal is generally displayed in diffraction 
intensity unit (DI) [8]. 
4.4. Protocol for sensor setup 
 The following protocol is routinely used to prepare the dotLab sensor for biosensor 
experiments. 
1. Place the polystyrene sensors at room temperature and allow thawing for 2 hours.  
2. Remove the sensor from its cladding and is dock it firmly in one of the four probe 
stations on the dotLab,  
3. When fixed, a ?click? sound is heard from the sensor which indicates that the sensor in 
secured in place with optical platform 
4. Open the dotLab software and start a new program. A schedule is designed according to 
the user?s requirements, which includes three priming steps and one tip wash step before 
the start of each experiment. 
5. The analyte to be immobilized on the avidinated polystyrene surface and the one that has 
to be detected are added to the 96 well holder over the auto sampler. 
73 
 
6. Start monitoring the signal with the immobilization process (~45 minutes) and follow the 
change in DI vs. time with each analyte introduced. 
7. Record the data in the data sheet and report the results 
 
4.5. References 
1. Brecht, A. and G. Gauglitz, Recent developments in optical transducers for chemical or 
biochemical applications. Sens. Actuators B: Chem., 1997. 38(1-3): p. 1-7. 
2. Wolfbeis, O.S., Analytical chemistry with optical sensors Fresenius' J. Anal. Chem., 
1986. 325(4): p. 387-392. 
3. Bailey, R.C. and J.T. Hupp, Large-scale resonance amplification of optical sensing of 
volatile compounds with chemoresponsive visible-region diffraction gratings. J. Am. 
Chem. Soc., 2002. 124: p. 6767?6774. 
4. Yu, F. and W. Knoll, Immunosensor with Self-Referencing Based on Surface Plasmon 
Diffraction. Anal. Chem., 2004. 76(7): p. 1971?1975. 
5. Nakajima, F., et al., Diffractive Optical Chemical Sensor Based on Light Absorption. 
Anal. Chem., 1999. 71(13): p. 2262-2265. 
6. Goh, J.B., et al., A quantitative diffraction-based sandwich immunoassay. Anal 
Biochem., 2003. 313(262-266). 
7. Cleverley, S., I. Chen, and J. Houle, Label-free and amplified quantitation of proteins in 
complex mixtures using diffractive optics technology. J Chromatogr B Analyt Technol 
Biomed Life Sci., 2010. 878(2): p. 264-270. 
8. Borisenko, V., et al., Diffractive Optics Technology: A Novel Detection Technology for 
Immunoassays. Clin. Chem., 2006. 52(11): p. 2168-2170. 
 
74 
 
5.  Nanoelectronic Properties of Single-Walled Carbon Nanotubes based Antimicrobial 
Films 
5.1. Introduction 
Silicon semiconductor technology has exponentially increased in terms of production, 
with an increase in number of transistors per chip and thereby performance of the devices. 
Unfortunately, the limit of assembling transistors in the tera-scale may end due to the 
fundamental physical or economic limitations. Additionally silicon poses certain key material 
limitations with which it has to be replaced in the near future for tera-scale integration, including 
transit time, switching energy, thermal conductivity and dielectric constant of the interconnect 
material. Indeed, it has been predicted that even after four decades of advances in silicon 
semiconductor technology, a systematic analysis of the echelons of the limits of device 
manufacture; enormous potential lies in the tera-scale integration of multitrillion transistor chips.  
 Nanorods and nanotubes have been predicted to be a significant alternative in integrating 
nanostructured devices for efficient electron and exciton transport. Hierarchical assembly of 
these one dimensional nanomaterials paves way for the next generation of self-aligned devices. 
Theoretical predictions and experimental measurements show that carbon nanotubes form 
different forms and shapes; and that?s their electrical or optoelectronic properties will exceed the 
performances of wide-band gap semiconductor materials. Although carbon nanotubes, more 
specifically single-walled carbon nanotubes (SWNTs) are considered to be highly anisotropic 
rigid rods, the major challenge lies in the assembly and processing of individually dispersed 
SWNTs in to usable devices, which has thwarted the development of suitable materials and 
interconnects for nanoelectronic devices [1-3]. Individual dispersion of carbon nanotubes can be 
75 
 
done using surfactants, polymers and biomolecules. More specifically, Smalley et al proposed a 
method to disperse SWNTs using various anionic and cationic surfactants. Kotov et al reported a 
novel method to disperse SWNTs using amphiphilic compounds. Dispersion using biomolecules 
has attracted mainly for its ease of preparation and versatility. The unique structure of DNA 
allows the sonication-assisted dispersion of SWNTs. As a result, it can be used to prepare 
fascinating nanoscale architectures based on layer-by-layer assembly. 
 Charge transport studies through DNA based nanomaterials paves way for a number of 
molecular electronic devices and insight in to the electrical properties of DNA. Various 
techniques were used to analyze the electron transport through DNA based surfaces and 
nanomaterials, including scanning tunneling microscopy, transient absorption spectroscopy and 
low-energy electron point source microscopy (LEEPS). We have used DNA as a biopolymer to 
individually disperse and wrap SWNTs, and have shown that the electrical and charge transport 
through nanocomposites can be verified using scanning electrochemical microscopy, where the 
conductivity through DNA wrapped SWNTs contributes to the change in steady state current and 
prevents insulation of consecutive layers, thereby enabling a response through a redox reaction 
[4].  
5.2. Coupled self-assembly of SWNT based biopolymers 
Controlled scalable bottom-up assembly of nanorods across the nano- to macro- length 
scales is one of the key obstacles to the fulfillment of their promising nanoscale properties. Both 
controlled placement and orientation are required for nanorod?s anisotropic properties to be fully 
manifested in macro-scale materials. Carbon nanotube-based devices have been prepared using 
random deposition, electric field assisted assembly, flow alignment and chemical patterning [5-
7]. However, none of these techniques can readily be extended to large scale manufacturing with 
controlled orientation and assembly. This prediction has motivated extensive efforts aimed at 
76 
 
developing new device concepts and fabrication approaches that may enable integration to go far 
beyond the limits of conventional microelectronics technology [8]. Similarly, dry deposition 
methods have been reported for micro-scale assembly of nanotubes using photolithographic 
techniques [9], but this process is complicated and requires a multitude of steps. Significant 
challenges remain for achieving large area assemblies; approaches to overcoming this challenge 
include patterned growth [10] and solid phase processing directly from the reactor [11] or from 
grown forests of materials [12, 13]. While these approaches are promising, particularly for 
smaller scale electronics, devices and sensors, fluid phase processes such as those developed for 
polymers are considered promising for their scalability and potential economic viability [14, 15]. 
Fluid phase processing requires unlocking the nanoscale promise of SWNT by dispersing them 
as individual nanotubes prior to reassembling them in a controlled manner. Because of SWNTs? 
ambiphobic nature and the large 500 eV/?m van der Waals attractions between them SWNTs 
tend to form large aggregates of entangled ropes. Superacids, dicholorobenzene, N-methyl 
pyrrolidone and surfactants are the most commonly used dispersants for SWNT.  Stabilized 
dispersions of individual SWNT have also been developed using polymers and biomaterials [16-
19]. Biopolymers such as DNA and Lysozyme provide attractive alternatives because of their 
greener chemistry and the ability to enable additional multi-functionality such as antimicrobial 
activity or sensing capability. Controlled self-assembly and proper integration of these high-
performance building blocks is necessary to harnessing their diverse functional properties and 
allowing a wide range of applications from 3D integrated electronics to antimicrobial coatings 
[20, 21]. 
Here we report the results of extensive characterization of the electrochemical interfaces 
of DNA-SWNT-LSZ coatings made by the LBL assembly process on an insulating substrate. 
77 
 
Lateral charge transport measurements made by scanning electrochemical microscopy (SECM). 
SECM analysis showed that increasing the number of layers resulted in positive feedback and 
that biocoating assembled on an insulating substrate acted, as a conducting film. Lateral charge 
transport was due exclusively to the composite film alone. Whether the terminating layer of the 
coating was LSZ or DNA resulted in significant differences in lateral charge transport. CV and 
EIS confirmed the electrical conductivity of the films and the stability of their response. As 
expected the feedback current from SECM increases with number of layers and our findings 
indicate that there is no insulation between the interlaced layers of SWNT-DNA and SWNT-
LSZ. These results demonstrate that the process used to form these mechanically robust 
antimicrobial coatings had the additional benefit of providing a conductive coating. This process 
can be extended to other SWNT-biological systems readily enabling the assembly of large area 
devices and sensors. 
5.3. Experimental Section 
5.3.1. Reagents 
All aqueous solutions were prepared using Milli-Q reagent water (Millipore Corp., 
resistivity > 18 M? cm). Purified HiPCo SWNTs were purchased from Rice University 
(Houston, TX). Lysozyme from chicken egg whites and DNA from salmon testes were obtained 
from Sigma-Aldrich (St. Louis, MO). Clean glass microscopy slides (VWR International, West 
Chester, PA) were used as substrates. SWNT-LSZ and SWNT-DNA dispersions were prepared 
using the method of Nepal et al [22]. All chemicals were used as received. First, glass slides 
were cleaned using a piranha solution composed of 3:1 96% H2SO4/30% H2O2. Slides were then 
subsequently alternately immersed for 15 min each in an aqueous dispersion of SWNT-LSZ and 
SWNT-DNA. 
78 
 
5.3.2. Apparatus 
SECM (CH-900, CH Instruments Inc., Austin, TX) was used to evaluate charge injection 
kinetics and lateral charge transfer through multilayer (SWNT-LSZ/SWNT-DNA)n films. A 
three electrode system was employed with a platinum wire serving as the counter electrode, 
Ag/AgCl as a reference electrode and a 10 ?m platinum ultra-micro electrode (UME) as the 
working electrode. Optical micrographs indicated a 2.2 ratio of SECM tip radius to platinum disk 
electrode (RG) radius. The solutions were de-aerated with nitrogen prior to use. The UME was 
held at a potential of +0.25 V vs. Ag/AgCl and approach curves were obtained at a scan rate of 
30 ?ms-1. The current at the UME was recorded as a function of distance as the SECM tip 
approached the glass substrate. 
Impedance spectroscopy measurements were taken using a Solartron 1260 frequency 
response analyzer (Farnborough, UK) with a frequency range of 0.001?10 Hz and signal 
amplitude of 5 mV. Measurements were carried out under open-circuit voltage with a two-probe 
configuration in which a separate reference electrode was not used. All impedance spectra were 
fitted to equivalent electrical circuits using ZView software (Huntington Beach, CA). Cyclic 
voltammetric and EIS were performed using a BAS CV-100W potentiostat/galvanostat. A one-
compartment 2 mL glass cell with a working volume, Ag/AgCl reference electrode, and a 
platinum wire auxiliary electrode was used for the measurements. 
5.3.3. Layer-by-Layer assembly 
 DNA-SWNT and LSZ-SWNT were prepared according to protocol [22], and zeta 
potential measurements of both the solutions indicated that the cationic and anionic nature of 
LSZ-SWNT (+22 mV) and DNA-SWNT dispersions (-30 mV) provided an excellent platform 
for the formation of the nanocomposites [(LSZ-SWNT)-(DNA-SWNT)]n.  
79 
 
 
Figure 5-16 Schematic showing the Layer-by-Layer assembly of SWNT nano-coating 
The orientation of the nanotubes was controlled in each layer by applying a directed air 
stream between each deposition step (Figure 5-1). Additionally, the air stream enabled shear 
alignment of SWNTs within each individual layer, and hence each individual layer had a distinct 
orientation. 
5.4. Results and Discussion 
5.4.1. Charge injection through the nano-film 
We investigated the lateral conductivity of different layers using SECM steady-state 
feedback mode approach curves. Most previous work on SWNT SECM characterization has 
focused on SWNTs? potential use as a probe but here we have used it as a coating. The 
mechanism of charge injection is as shown in Figure 5-2. The UME is set at approached towards 
the substrate at a constant scan rate. Since, we expect the film to be conductive; there is a lateral 
(in-plane) and cross-film electron transport in the multilayer polymer/nanoparticle film.  
Elect r oc hemic al Analy sis using  SE CM
I n su l a ti n g  g l a ss sl i d e
S W NT - LSZ
(+ 22  m V)
S W NT - DNA
( - 30  m V)
S W NT - LSZ
S W NT - DNA
(+ 22  m V)
( - 30  m V)
Di r e ct e d  A i r  Str e a m
Pl ai n  Gl ass
6 10 14 20
(N u mb e r o f l a y e rs )
LS Z - S W N T DN A - S W N T
80 
 
 
Figure 5-17 Schematic of the proposed mechanism of charge transfer through the nano-coating 
One of the principle advantages of using SECM for investigating conductivity of 
nanotube assemblies is that, the method is non-invasive, highly localized and does not require the 
film to be externally biased. Besides, the film substrate is an insulating glass slide, and hence the 
response obtained is solely due to the charge injection across the film. Figure 5-3 shows typical 
UME experimental approach curves (microelectrode tip current versus distance from the film 
surface) as a function of SWNT-LSZ and SWNT-DNA layer numbers as coating using the same 
concentration of redox mediator (1 mM FcMeOH + 0.1 M KCl) was used. A clear increase of the 
steady-state current is observed as the tip approaches the film [23, 24]. The regeneration of the 
electro-active species is driven by the lateral conductivity within the film and the kinetics of the 
redox process. An increase in tip current is therefore attributed to the lateral conductivity across 
the SWNT-LSZ and SWNT-DNA. Positive feedback increases with the number of layers and 
therefore layers are not insulated from another. 
Di r e ct i on  of  p r ob e
Ox R ed
S WNT - D NA
S WNT - L SZ
L a t e r a l  Ch a r g e  T r a n sp or t
d
In su l ating gl ass 
81 
 
 
Figure 5-18 Approach curves at identical concentrations of redox mediator to nano-composite 
films composed of different number of SWNT-LSZ/SWNT-DNA layers 
Increased current is a result of fine carbon nanotube assembly control, indicating that 
transport along the layer is dominated by single-electron transport along a localized area in the 
nanotube or a whole lot of bundles [19, 25]. Clearly, the tip-generated oxidized mediator 
FcMeOH is capable of oxidizing the multilayer and thereby regenerating FcMeOH+, which 
diffuses back to the tip. Figure 5-4 shows approach curves for a substrate with ten alternating 
layers of SWNT-LSZ/SWNT-DNA at different bulk concentrations of FcMeOH. Positive 
feedback becomes more pronounced as a result of lateral charge transfer within the film when 
the concentration of the redox mediator is decreased. The observed tip current increase is due 
solely to lateral charge transfer and not background charge accumulation. Positive feedback with 
FcMeOH is a clear effect of the coatings present on top of the glass slide, confirming that 
regeneration of the mediator comes from the multilayer. 
82 
 
 
Figure 5-19 Approach curves for 10 layers of SWNT-LSZ/SWNT-DNA at different 
concentrations of redox mediator. The concentration of the solution redox mediator from top to 
bottom is 100 ?M, 330 ?M and 1 mM 
A similar result has been obtained using a two-dimensional network of SWNT [26, 27]. 
Approach curves were also recorded for plain glass slides acting as controls. Control slides gave 
negative feedback at all times. Background measurements were performed by swapping the 
mediator with the supporting electrolyte alone to allow observations of current intensity 
variation. The approach curve exhibits negative feedback in the absence of multilayers, verifying 
a insulate surface. The biocoating proved to be very robust, giving consistent electrochemical 
results over periods of up to six continuous hours. 
5.4.2. Nanoelectronic properties of alternate layers 
Investigation of the effects of the number of layers and whether the top layer was SWNT-
LSZ or SWNT-DNA showed that the magnitude of positive feedback depends on the uppermost 
deposited layer (Figure 5-5). A slightly higher current was observed for 10 layers terminating in 
83 
 
SWNT-DNA than 11 layers terminating in SWNT-LSZ. Lysozyme has a high dielectric response 
when dissolved in water and may not transfer electrons at neutral pH. At neutral pH, LSZ has a 
net charge of +8 if charge distribution in the side chains is considered [28]. The positive nature 
of LSZ results in minimal diffusion to the SWNT backbone due to electrostatic repulsion. Thus, 
electron transfer from FcMeOH is blocked with respect to the redox couple. Alternatively, DNA 
can facilitate electron transfer to dissolve the mediator [4]. We conclude that the regeneration of 
mediator is accentuated as SWNT-DNA is deposited. SECM analysis on non-SWNT coupled 
DNA and LSZ layers assembled on glass slides was also conducted to verify lateral charge 
transfer. This is in agreement with Lie et al [29] who observed negative current in the approach 
curves for DNA monolayers immobilized on silicon substrates.                                                              
To further investigate the effect of the outermost layer composition, impedance 
spectroscopy was used to determine the impedance associated with the electrode reactions rather 
than the inherent electrical resistance of the material. Fig. 3 shows that the resistance increased 
from 6 to 18 k? cm2 with decreasing numbers of layers demonstrating higher resistance, which 
is consistent with the evidence obtained by SECM. However, it was observed that when the last 
layer deposited was SWNT-LSZ there was slightly increased resistance, which was contrary to 
observations for SWNT-DNA. The SWNT-DNA appeared to provide additional sites for the 
electrochemical reaction and effectively enhance electronic transfer it has been proposed by 
different authors that DNA can facilitate electron tunneling [4]. In contrast, our observations 
suggest that LSZ block the active sites and increase electrode resistance. EIS measurements were 
performed in the presence of FcMeOH as redox couple ions. Figure 4 shows the electrochemical 
impedance spectra (Nyquist plots, Z? vs. Z?) for the layers. The diameter of the semicircle 
represents the charge-transfer resistance (Rct) at the electrode surface. 
84 
 
 
Figure 5-20 Approach curves for alternating layers of SWNT-LSZ/SWNT-DNA assembly 
As shown in Figure 4, the diameter of the semicircle, indicating the corresponding Rct, 
decreases as the number of layer is increased. This change can be attributed to increased 
repulsion between the redox marker ions and the electrode surface; the repulsion impedes the 
charge-transfer through the interface. The calculated values of charge transfer resistance 
increased in the range of 600 and 1700 ? cm2 with the number of layers, resulting in an 
increased electrostatic repulsion of the charged probe and hence the inhibition of the electron 
transfer. 
5.4.3. Nano-bioelectrode 
Electrochemical characterization of the coating was carried out by cyclic voltammetry of 
the 20 layers at different scan rates in 0.1 M of KCl in the presence of 1 mM FcMeOH is shown 
in Figure 5-6. The electrode exhibits a couple of redox peaks (V vs. Ag/AgCl) and the peak 
current increases linearly with scan rate. The small peak-to-peak potential separation and the 
peak potential dependence on the scan rate indicate a reversible electrochemical process. The 
85 
 
inset in Figure 5-6 shows the effect of the scan rate on the anodic peak currents. Scan rate was 
varied between 10 mV/s (1) and 250 mV/s (2). The response is linear with slopes of 0.7 
indicating an anodic surface electrochemical reaction with a diffusion component for the anodic 
process; a diffusion controlled reaction. The cyclic voltammogram further indicated that films 
are conducive and have a stable response. The biocoatings are capable of working in a wide 
potential range, from ?1 V to +1 V, in aqueous solution using both reductive and oxidative 
mediators. Further detailed studies on these effects, and other details of the electrochemical 
response, are under investigation for future reporting. Electrochemical Impedance Spectroscopy 
was carried out in the same redox mediator, in the frequency interval 50 KHz to 30 mHz with 
respect to Ag/AgCl electrode (0.57 V). As shown in Figure 5-7, the Nyquist plots indicated that 
as the number of layers increases, the charge transfer resistance (Rct) decreases, from 1700 ?cm2 
to 600 ?cm2. 
 
Figure 5-21 Cyclic coltammogram at different scan rates shows that the coating behaves as a bio-
electrode 
86 
 
5.4.4. Impedance Analysis 
To further investigate the effect of the outermost layer composition, impedance 
spectroscopy was used to determine the impedance associated with the electrode reactions rather 
than the inherent electrical resistance of the material. Figure 5-8 shows that the resistance 
increased from 6 to 18 k? cm2 with decreasing numbers of layers demonstrating higher 
resistance, which is consistent with the evidence obtained by SECM. However, it was observed 
that when the last layer deposited was SWNT-LSZ there was slightly increased resistance, which 
was contrary to observations for SWNT-DNA. The platform which houses the substrate was 
custom built using conductive soldering metal and copper connects. Both ends of the holder were 
sputter coated with gold for increased conductivity (Figure 5-9). The SWNT-DNA appeared to 
provide additional sites for the electrochemical reaction and effectively enhance electronic 
transfer it has been proposed by different authors that DNA can facilitate electron tunneling. In 
contrast, our observations suggest that LSZ block the active sites and increase electrode 
resistance. 
5.5. Conclusion 
In conclusion, we have applied a simple and reliable technique for the biocoating fabrication 
using layer-by-layer technique, and we have used a complementary suite of methods to provide 
the first electrochemical characterization of this system. The positive feedback response obtained 
with SECM shows that the presence of the SWNT-biological coating causes conducting surfaces 
to behave with finite electrode kinetics. In addition to enhanced electron flow, impedance 
spectroscopy demonstrated the decrease of charge-transfer resistance measurements at number of 
layer. These results show that the combination of Nature?s toolbox and single-walled carbon 
nanotubes enable multifunctional conductive coatings where the charge transport can be tailored 
based on the number of layers and the composition and polarity of the outermost layer. 
87 
 
 
Figure 5-22 EIS spectra for different layers of assembly 
  
 
Figure 5-23 Representative impedance responses of the different number of layers at open circuit 
88 
 
 
Figure 5-24 Schematic of the custom built platform to hold the glass substrates for impedance 
analysis 
Expected advancements in the separation of metallic and semiconducting SWNTs will 
further increase flexibility and performance. This research indicates that combination of SWNTs 
with biopolymers offer great promise for creating new, sustainable bio-interfaces with 
controllable electrical and mechanical parameters. We believe that these approaches can be 
extended to the creation of new SWNT-biological coatings for a variety of electrochemical 
processes, and applications in electronics and sensing. 
5.6. References 
1. Huang, Y., et al., Directed Assembly of One-Dimensional Nanostructures into Functional 
Networks. Science, 2001. 291(5504): p. 630-633   
2. Vigolo, B., et al., Macroscopic Fibers and Ribbons of Oriented Carbon Nanotubes. 
Science, 2000. 290(5495): p. 1331-1334   
3. Rao, S.G., et al., Large-scale assembly of carbon nanotubes. Nature, 2003. 425: p. 36-37. 
A
A
A  ? to  Im p e d an c e  an al y ze r
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4. Lie, L.H., et al., Electrochemical detection of lateral charge transport in metal complex-
DNA monolayers synthesized on Si(111) electrodes. J. Electroanal. Chem., 2007. 603: p. 
67-80. 
5. Shim, B.S. and N.A. Kotov, Single-Walled Carbon Nanotube Combing during Layer-by-
Layer Assembly: From Random Adsorption to Aligned Composites. Langmuir, 2005. 21: 
p. 9381-9385. 
6. Wang, H., et al., Shear-SANS study of single-walled carbon nanotube suspensions. Chem. 
Phys. Lett., 2005. 416: p. 182-186. 
7. Reches, M. and E. Gazit, Controlled patterning of aligned self-assembled peptide 
nanotubes. Nat. Nanotechnol., 2006. 1: p. 195-200. 
8. Duan, X., Y. Huang, and C.M. Lieber, Nonvolatile Memory and Programmable Logic 
from Molecule-Gated Nanowires. Nano Lett., 2002. 2: p. 487-490. 
9. Huang, S., L. Dai, and A.W.H. Mau, Controlled Fabrication of Large-Scale Aligned 
Carbon Nanofiber/Nanotube Patterns by Photolithography. Adv. Mater., 2002. 14(16): p. 
1140-1143. 
10. Shao, M., et al., Karman vortex street assisted patterning in the growth of silicon 
nanowires. Chem. Commun., 2007. 8: p. 793-794. 
11. Li, Y., I.A. Kinloch, and A.H. Windle, Direct Spinning of Carbon Nanotube Fibers from 
Chemical Vapor Deposition Synthesis. Science, 2004. 304: p. 276 - 278. 
12. Zhang, M., et al., Strong, Transparent, Multifunctional, Carbon Nanotube Sheets 
Science, 2005. 309: p. 1215 - 1219. 
13. Zhang, M., K.R. Atkinson, and R.H. Baughman, Multifunctional Carbon Nanotube Yarns 
by Downsizing an Ancient Technology Science, 2004. 306(5700): p. 1358-1361. 
90 
 
14. Tang, Z., et al., Biomedical Applications of Layer-by-Layer Assembly: From Biomimetics 
to Tissue Engineering. Adv. Mater., 2006. 18(24): p. 3203?3224. 
15. Glotzer, S.C., et al., Self-assembly of anisotropic tethered nanoparticle shape 
amphiphiles. Curr. Opin. Colloid Interface Sci., 2005. 10: p. 287-295. 
16. Niyogi, S., et al., Selective Aggregation of Single-Walled Carbon Nanotubes via Salt 
Addition. J. Am. Chem. Soc., 2007. 129(7): p. 1898?1899. 
17. Ginic-Markovic, M., et al., Synthesis of New Polyaniline/Nanotube Composites Using 
Ultrasonically Initiated Emulsion Polymerization. Chem. Mater., 2006. 18(26): p. 6258?
6265. 
18. Park, C., et al., Actuating Single Wall Carbon Nanotube?Polymer Composites: Intrinsic 
Unimorphs. Adv. Mater., 2008. 20(11): p. 2074-2079. 
19. Li, Z., et al., Comparative Study on Different Carbon Nanotube Materials in Terms of 
Transparent Conductive Coatings. Langmuir, 2008. 24(6): p. 2655?2662. 
20. Nepal, D., et al., Strong Antimicrobial Coatings: Single-Walled Carbon Nanotubes 
Armored with Biopolymers. Nano Lett., 2008. 8(7): p. 1896?1901. 
21. Kaul, A.B.M., K. G., P. von Allmen, and R.L. Baron Single, aligned carbon nanotubes in 
3D nanoscale architectures enabled by top-down and bottom-up manufacturable 
processes Nanotechnology, 2009. 20(7): p. 75303. 
22. Nepal, D., et al., Supramolecular Conjugates of Carbon Nanotubes and DNA by a Solid-
State Reaction. Biomacromolecules, 2005. 6(6): p. 2919?2922. 
23. Zhang, J., et al., Combined scanning electrochemical microscopy?Langmuir trough 
technique for investigating phase transfer kinetics across liquid/liquid interfaces 
modified by a molecular monolayer. Electrochem. Commun., 2003. 5: p. 407. 
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24. Mandler, D. and P.R. Unwin, Measurement of Lateral Charge Propagation in 
Polyaniline Layers with the Scanning Electrochemical Microscope. J. Phys. Chem. B, 
2003. 107(2): p. 407-410. 
25. Strano, M.S., et al., Electronic Structure Control of Single-Walled Carbon Nanotube 
Functionalization Science, 2003. 301(5639): p. 1519 - 1522. 
26. Wilson, N.L., et al., Assessment of the Electrochemical Behavior of Two-Dimensional 
Networks of Single-Walled Carbon Nanotubes. Anal. Chem., 2006. 78(19): p. 7006?
7015. 
27. Gardner, C.E., P.R. Unwin, and J.V. Macpherson, Correlation of membrane structure 
and transport activity using combined scanning electrochemical?atomic force 
microscopy. Electrochem. Commun., 2005. 7: p. 612-618. 
28. Cheng, A.K.H., B. Ge, and H. Yu, Aptamer-Based Biosensors for Label-Free 
Voltammetric Detection of Lysozyme. Anal. Chem., 2007. 79(14): p. 5158?5164. 
29. Lie, L.H., et al., Immobilisation and synthesis of DNA on Si(111), nanocrystalline porous 
silicon and silicon nanoparticles. Faraday Discuss., 2004. 125: p. 235. 
 
 
 
 
 
 
92 
 
6. Affinity Based DNA Optical Sensors for the Detection of hipO Gene from 
Campylobacter jejuni 
6.1. Introduction 
Campylobacter jejuni is a significant cause of human bacterial gastroenteritis [1-3] and 
can be acquired from contact with farm animals or ingestion of contaminated poultry products, 
raw milk or impure water. This pathogen has been implicated in 80 outbreaks in the US alone 
[4], and the Centers for Disease Control and Prevention estimate that campylobacteriosis affects 
over 2.4 million people annually in the US. In 2008, 5,825 cases of campylobacteriosis were 
reported through FoodNet, with 12.68 cases per 100,000 people [5]. Campylobacteriosis has also 
been associated with the development of illnesses such as Guillian-Barre syndrome, reactive 
arthritis, Reiter syndrome, diarrheal infections, intestinal malignancy and dehydration [6, 7]. 
Traditional laboratory procedures are used for Campylobacter detection, which include 
plate culturing or extracting the bacteria from samples followed by serotype or genotype based 
diagnostic assays [8, 9]. Although these methods are sensitive, they can take up to 96 hours for 
the analysis to be done. Therefore, development of sensors capable of offering a rapid and 
inexpensive alternative, coupled with real-time monitoring and specific detection of low 
concentrations of C. jejuni in food products, is needed to ensure public safety. 
Numerous biosensor platforms have been developed for the detection of food-borne 
pathogens [10]. Nucleic acid-based sensing platforms have been widely described, including 
piezoelectric sensors that measure frequency changes resulting from the hybridization of 
immobilized probes with a corresponding target [11, 12], chemi-luminescence based systems 
where fluorescently labeled probes are immobilized onto transducer surfaces [13], aptamer-based 
93 
 
sensing using DNAzymes to design functional chimera structure-like probes [14]. However, 
these methods are rigorous, time-consuming and possess low sensitivity in comparison to other 
detection platforms [15, 16]. 
Recently, there have been reports of detection of pathogenic food-borne bacteria using 
disease causing genes as probe molecules [17, 18]. Detection using Polymerase Chain Reaction 
(PCR) is very selective but may lack the required sensitivity for the rapid screening of samples 
[19]. Electrochemical DNA microarrays are advantageous over PCR-based assays for their high 
sensitivity, portability and also better performance without labeling the probes. They can also be 
used for multiple target preparation. This method is quite sensitive but produces high background 
signals that are difficult to separate, and the sensing platform is destroyed after every 
measurement. This is problematic for the reusability, reliability and robustness of the surface 
structures that are developed over the electrodes [20]. An alternative to these methods is DNA-
based biosensors, which can be selective, specific and robust. For example, self-assembled 
monolayers of ssDNA probes specific to targeted ssDNA can be immobilized on sensor surfaces, 
allowing indirect detection of a bacterium of interest [21].  
Hybridization-based sensing platforms were developed using DNA probes labeled with 
fluorophores. Although these methods are specific and sensitive, they may suffer from photo-
bleaching and have limited differentiation between hybridized and non-hybridized sequences in 
the mixture. Moreover, signals that arise from non-specific adsorption and diffusion across the 
colloid are difficult to analyze. While, DNA sequences can be labeled with nano-particles for 
effective signal enhancement, a disadvantage is the attachment of nano-particles by thiol linkage 
that which can form dense layers of DNA on each particle [22]. Covalent bond formation on 
94 
 
gold is employed to develop anti-fouling biosensor surface, often via thiol-gold linkages or 
avidin-biotin chemistries [11, 23].  
The basis for the difference between C. jejuni and any other bacteria is the expression of 
the N-benzoylglycine amidohydrolase (hippuricase) gene. The hallmark of the hippuricase 
activity is the conversion of hippuric acid into benzoic acid and glycine. This gene is the key 
feature in discriminating C. jejuni from other similar Campylobacter species, such as C. coli. 
Diffraction Optics (DOT) sensors are widely used for label-free detection and real-time 
monitoring of bio-molecular interactions. This technique has been widely employed for the 
detection of proteins and small molecules [24], and live cells [25, 26]. A variety of biotin labeled 
ligands including proteins, antibodies and DNA can be immobilized on the DOT sensor chip. 
Our present work accounts the development of hybridization-based Diffraction Optics 
Technology (DOT) and Surface Plasmon Resonance (SPREETA) sensing platforms. False 
positive responses due to analyte concentration and bulk refractive index are eliminated in the 
DOT and only the diffraction phenomena across the grating is recorded, which are precisely 
advantageous over other label-free optical biosensor technologies. Accordingly, a low limit of 
detection of 5 nM was obtained. A range of surface chemistries and the extensive possibilities in 
label-free and amplified modes, coupled with low cost disposable sensor chips makes it an 
attractive optical sensing device. The application of this coupling strategy to two devices was 
conducted with the aim of exploiting its applicability in different instrumentations using the 
same sensor formats. 
6.2. Experimental 
6.2.1. Reagents 
Potassium phosphate monobasic, Phosphate buffered saline (pH 7.4) containing 0.138M 
NaCl and 0.0027M KCl (PBS) and 6-Mercapto-1-hexanol were obtained from Sigma-Aldrich 
95 
 
(St. Louis, MO). Tween-20, EDTA, sodium phosphate dibasic and sodium chloride were used as 
received from Fisher-Scientific (Pittsburgh, PA). Reductactryl? (Cleland?s reagent) was 
obtained from Calbiochem (San Diego, CA). 
6.2.2. Sensor Platforms 
DOT sensors chips (Axela Inc., Toronto, Canada) were used for diffraction experiments 
(Fig. 1). A 670 nm laser diode, class 3R with a maximum output of 4.0 mW and a beam 
divergence of 5.0 mRad was used as the light source, to produce a diffractive pattern. One end of 
the sensor was connected to a fluidic panel, which is responsible for circulating the buffer 
solutions and the other end is connected to a sensor manifold which aspirates the analyte from 
the well. The sensor chip was made of optically clear polystyrene with an interspersed prism 
situated below the flow channel. The linear flow channel which can hold a volume of 10.0 ?l, 
consisted of eight, circular assay spots, each 2.0 mm in diameter, with 15.0 femto moles of 
avidin deposited on each spot [27]. These spots constituted the grating where light is diffracted in 
a predictable manner.  
The SPREETA (Texas Instruments, Dallas, TX) is a robust, two-channeled SPR system  
comprised of a multi-channel SPR sensor, flow cell, and 12-bit three-channel DSP electronic 
control box (ECB). The sensing region is coated with a semi-transparent gold film (~50 nm) with 
a Cr-adhesion layer (1-2 nm). A two-channel polypropylene flow cell coupled with a silicone 
gasket was used to establish a flow system along with a peristaltic pump (Ismatec, Cole-Parmer 
Instrument Co., Chicago, IL) at a flow rate of 25.0 ?l/min at 25oC. Multi-channel SPREETA 
software version 10.83 (Texas Instruments) monitored the changes in RI near the sensing 
surface, calculated the statistical noise in the signal and displays the results. The signal is 
generally displayed in response unit (RU) (1 response unit = 10?6 refractive index unit) [28]. 
96 
 
6.2.3. DNA Probes 
Primers and probes were designed from the hippuricase gene (hipO) of C. jejuni 
(accession number Z36940.1). The forward (5?- GAC TTC GTG CAG ATA TGG ATG CTT-3?) 
and reverse (5?-CGA TAT TGA TAG GCT TCT TCG GTA GTA-3?) primers amplified a 344 
bp segment. The short (SP, 5?- GGT GGT GCT AAG GCA ATG ATA G-3?) and long (LP, 5?-
GGT GGT GCT AAG GCA ATG ATA GAA GAT GGA TTG TTT GAA AAA TTT GAT AG-
3?) probes attached to the 344 bp segment. Primers and probes were synthesized by Integrated 
DNA Technologies (Coralville, IA). Reverse and complementary ssDNA (IDT) corresponding to 
the amplified segment were diluted in TE Buffer (10 mM Tris, 0.1 mM EDTA, pH 8.0). The 
disulfide bonds of thiolated DNA probes were cleaved by resuspending in TE buffer along with 
Reductactryl? 1:50, allowing formation of self-assembled monolayers (SAM) with higher 
surface density and better reproducibility [29]. This mixture was then agitated at room 
temperature (RT) for 15 min followed by syringe filtration (0.2 ?m). 
Experiments were also conducted using a scaffold of LP (5?-GGT GGT GCT AAG GCA 
ATG ATA GAA GAT GGA TTG TTT GAA AAA TTT GAT AG-3?) and short target (5?-CTA 
TCA TTG CCT TAG CAC CAC C -3?). Equimolar mixture of the probe and target were mixed 
together and annealed in up to 95oC. A DNA probe with a 22-mer double strand and 28-mer 
single strands was formed. This was followed by slow cooling of the mixture [DNA scaffold 
colloids paper]. Reduction of the DNA was done using Reductactryl? in order to cleave the 
disulfide bonds present in the long probe. This mixture was then agitated at room temperature 
(RT) for 15 min followed by syringe filtration (0.2 ?m). Experiments were also conducted by 
attaching a long probe (LP) (5?-GGT GGT GCT AAG GCA ATG ATA GAA GAT GGA TTG 
TTT GAA AAA TTT GAT AG-3?) and a second 22-mer (5?-CTA TCA TTG CCT TAG CAC 
CAC C -3?) ssDNA. 
97 
 
 
Figure 6-1 Illustration of the formation of DNA scaffolds using a 50-mer long probe and 22-mer 
short probe by annealing the complimentary regions in both the strands 
Equimolar mixture of the probe and target were mixed together and annealed up to 95oC. A 
DNA probe with a 22-mer double strand and 28-mer single strand was formed (Figure 6-1). This 
was followed by slow cooling of the mixture [30]. Disulfide bonds in the long probe were 
cleaved with Reductactryl?. 
6.3. Immobilization of DNA probes 
6.3.1. dotLab  
Analyte is passed at a standard flow rate of 500.0 ?l / min is maintained all throughout 
the experiment in the mixer mode. Flow of reagents was controlled by the method that was 
programmed before the start of the analysis (Table 6-1). 500 ?L of a mixture of 1X PBS and 
0.0025% Tween (PBST) was primed over the neutravidin modified polystyrene surface. The 
non-specific area across the flow channel was blocked with BSA (5.0 mg/ml) for 3 minutes. 
Subsequently, biotinylated DNA probes (1.0 ?M) were injected for 45 minutes followed by 
+
A nnea l i ng a t  95
o
C
4 min
DNA  Sc a f f ol d
T a r g e t
r e cept or  
r e g i on
D N A   SA M  
te th e r i n g
T a r g e t  ssDN A
22 - m er 50 - m er  th i ol p r ob e
In c i d e n t an g l e
RU
98 
 
washing with buffer. Changes in diffraction intensity were monitored using dotLab? software. 
After a sequence of washing steps, the target was injected sequentially in graded concentrations 
through a precise fluidic system and pre-defined incubation time.  
6.3.2. SPREETA 
Gold surface of SPREETA? sensors were carefully cleaned with piranha solution 
followed by argon plasma cleaning. The uniqueness of SPREETA allows real-time monitoring of 
the specific interactions between the probe and target using sensing and reference channels. 
Thiolated probes (1.0 ?M) suspended in the immobilization buffer were passed over the gold 
surface for 3 hours.  
 
Figure 6-2 Response curve for DNA immobilization and hybridization using SPREETA sensor 
Short immobilization time ensured no degradation of DNA occured during assembly on 
the surface [31]. Apparently, a low pH was required to form a self-assembled monolayer [32]. 
After DNA immobilization, the surface was thoroughly washed and 1 mM 6-Mercapto-1-
hexanol (MCH) was injected. Long DNA probes (1.0 ?M) were resuspended in immobilization 
99 
 
buffer and injected over freshly cleaned surface for 3 hours. MCH was not immobilized on the 
surface because the DNA scaffolds presumably formed a tightly packed monolayer which 
prevents non-specific binding [33]. The surface was washed after immobilization in order to 
remove loosely bound probes. This short time was selected because DNA is present in a low pH 
buffer which rapidly degrades ssDNA in solution [34]. Apparently, a low pH is required to 
develop the self-assembled monolayer. Hence the assembly of thiolated DNA probes should be 
done within a short period of time. Binding of the probes (1 ?M) to the gold surface is facilitated 
by passing the ssDNA at a flow rate of 25 ?l / min in recycling mode. The thiolated probes are 
injected for 3 hrs, and the change in RU after washing the surface was found to be 1380 ? 15 
(Figure 6-2). The SPR response is as follows, (a) priming the surface with water, (b) establishing 
a baseline with immobilization buffer, (c) modification of surface by passing 1 ?M thiol-labeled 
ssDNA probes (d) washing the surface with buffer to remove unbound ssDNA probes. Surface is 
then washed with water at (e), because of the 1 mM aqueous MCH that is passed in order to 
minimize non-specific binding (f). (g) washing the surface with water to remove unbound MCH, 
(h) establishing baseline with hybridization buffer, (i) injection of target ssDNA from 2.5 nM to 
1280 nM, (j) regenerating the surface using 1 mM HCl, (k) establishing a baseline again and also 
removing any HCl present, (l) target injection after regeneration of surface, (m) denaturation of 
dsDNA, (n) washing the surface with hybridization buffer for 3rd injection. Loosely bound DNA 
is removed by a 30 second wash after passing the DNA. It is also speculated that the 22 bp 
ssDNA forms a densely packed crystalline or semi-crystalline monolayer, due to Van der Waals 
forces between the ssDNA. 
100 
 
 
Table 6-1 Summary of method for the detection of hipO gene programmed using dotLab 
software 
6.4. Detection of target DNA 
6.4.1. dotLab  
 All solutions were prepared in PBST buffer. In order to determine the limit of detection, 
target DNA was serially diluted from 0.005 ?M to 1.28 ?M and introduced sequentially, starting 
101 
 
with the lowest concentration. Complementary target DNA had the sequence, 5? ? CTA TCA 
TTG CCT TAG CAC CAC C - 3? and the non-complimentary DNA is 5? ? CCG TCG TAG 
ACA TAC TAC ATC G - 3?. Each dilution was passed for 5 minutes until saturation of target 
binding. Between injections of each aliquot, the surface was washed thoroughly with PBST. The 
difference in signal between the PBST baseline and respective intensity was determined for each 
concentration. Biotinylated DNA probes diluted in PBST is introduced at a flow rate of 500 
?l/min. Auto sampler aspirates the analyte from the tray, which is controlled by the two syringe 
pumps. Volume and flow rate is defined in the method and analyte location is assigned in the 
schedule. Loosely bound DNA was removed by priming the surface with PBST, which showed a 
decrease in signal. Before the molecules bind to the surface, a weak intensity image is produced 
upon illumination. However, when the probe molecules are immobilized on to the surface, an 
increase in height of the diffraction pattern is produced, which in turn corresponds to the increase 
in intensity. Probes are immobilized without fluorescent and chemi-luminescent labels, over a 
highly sensitive and accessible platform for the detection of biomolecular interactions. Each 10 
?l flow channel is connected to the fluidic system and the analyte is passed at a standard flow 
rate of 500 ?l / min is maintained all throughout the experiment in the mixer mode for 5 minutes, 
with a data acquisition rate of 1 Hz. Detection of the binding phenomena can be monitored by 
inquiring with the laser light in a total internal reflection mode, without the laser passing through 
the flow channel, so that 95 % of the signal can be measured. The signal is generally displayed in 
diffraction intensity (DI). The protocol was established as recommended by the manufacturer. 
Non-specific binding spots were covered using 5 mg/ml BSA in PBST buffer. Buffers and the 
analyte are injected through the automated fluidic system.   
102 
 
6.4.2. SPREETA  
Hybridization experiments were conducted in hybridization buffer (150 mM NaCl, 20 
mM Na2HPO4, 0.1 mM EDTA, 0.005 % Tween 20, pH ? 7.4). Target DNA was serially injected 
from lowest (2.5 nM) to highest (1280 nM) concentrations for 5 minutes. Sensors were washed 
after every injection to remove any loosely bound DNA. Origin Pro 8 (OriginLab Corporation, 
MA) software was used to calculate the net response by subtracting the working channel signal 
(baseline) from the control channel. The loosely bound DNA is washed with buffer for 30 
seconds. Net response was calculated by deducting the control signal from sensing signal. This 
allows the elimination of the signal generated due to non-specific adsorption of cells to the 
sensor surface, as well as change in RI due to bulk refractive index of the solution. Specificity of 
the system was tested using a non-complimentary target ssDNA which was injected from 5 nM 
to 1280 nM. Similarly, a non-complimentary nucleotide was also passed through to check for 
specificity and there was no detectable signal from the control channel. As in the case of the 
dotLab system, a dose-response curve was plotted with different concentrations of the target 
ssDNA from 2.5 nM to 1280 nM. 
In the case of longer probes, the target DNA is serially passed over the surface starting 
from the lowest concentration (10 nM to 640 nM) for 5 minutes. A non-complimentary target is 
also passed over the surface to verify specificity. 
6.4.3. Electrochemical Impedance Spectroscopy (EIS) 
  AC electrochemical impedance spectroscopy was carried out using an FRA2 - 
?AUTOLAB ? TYPE III electrochemical impedance analyzer (Metrohm USA Inc., Westbury, 
NY). A one-compartment 2.0 mL glass cell with a working volume, Ag/AgCl reference electrode 
and a platinum wire auxiliary electrode was used for the measurements. The sensing surface was 
characterized over a wide range of frequency (10 Hz to 100 MHz; E0?= 180 mV versus Ag/AgCl) 
103 
 
in the presence of 5 mM K3Fe(CN) 6 + K4Fe(CN)6 in 0.1 M KCl. A linear region for the bare gold 
electrode implies at low frequency shows that the reaction across the electrode is diffusion 
controlled [35]. 
6.5. Results and Discussion 
6.5.1. Immobilization of Biotinylated DNA probes (dotLab) 
 The response curve for probe binding at different concentrations is shown in Figure 6-2. 
A net response (Figure 6-3) was determined by deducting the control signal from the sensing 
signal. Probe optimization was done by passing a wide range of concentrations over the surface. 
The working range lies between 0.5 ?M and 1 ?M. Hence this was chosen as the probe 
concentration as there is very minimum difference in intensity across these concentrations. This 
confirmed the high specificity of the system using biotinylated ssDNA probes. Using the same 
concentration (1 ?M)) for 20 cycles of hybridization/regeneration on the same chip in 7 days, the 
average value was 4.7 x 10-2 ? 1.16 x 10-2 DI units (96% relative standard deviation, R2 = 0.995) 
(data not shown). This is comparable with diffraction-based assays or surface plasmon resonance 
platforms [36, 37]. The ability of cross-linking biotinylated ssDNA was examined using the 
dotLab system. The dotLab system is a relatively new optical biosensor based on diffractive 
optics technology. This system uses a sensor chip with an integrated prism situated below the 
flow channel, which monitors biomolecular interactions in real time within a complex media. 
Because diffraction is inherently self-referencing, nonspecific binding to both the patterned and 
unpatterned regions will not affect the signal. Such characteristic offers an important advantage 
over other optical biosensor systems in which any surface binding event will cause non-specific 
binding. Besides, the dotLab biosensor is a cost-effective platform that has the format flexibility 
to provide a quick and simple solution to both assay development and routine analysis. 
104 
 
6.5.2. Immobilization of Thiolated DNA probes (SPREETA) 
  Using previously established methods [28, 38]; the surface coverage of DNA probes was 
determined to be 3.64 x 1012 ? 0.15 molecules/cm2, with a surface coverage density of 0.44 ? 
0.12 ng/mm2 [32]. Change in RU after washing the surface was found to be 1380 ? 15. The 
charge transfer resistance (Rct), after immobilization of DNA on gold electrode was 8.0 k?. It is 
speculated that 22 bp ssDNA probes form a densely packed semi-crystalline monolayer, due to 
Van der Waals forces between the ssDNA [39, 40].  
 
Figure 6-3 Sensor response to probe immobilization. There was a continuous increase in net 
change, which was calculated by subtracting the subsequent PBST baseline 
EIS was performed for each step of DNA immobilization and hybridization to better 
understand the immobilization of thiolated probes on the surface and to characterize the 
electrical properties of the interfacial interaction between DNA probes before and after 
incubation with target DNA (Figure 6-4). 
105 
 
 
Figure 6-4 Dose-Response curve for probe binding. The net response curves were plotted by 
subtracting the response signal from the base line. 
 
Figure 6-5 Faradaic response for immobilization of thiolated ssDNA on SPREETA. Frequency 
intervals: 10 mHz to 100 KHz and measurements carried out at 0.32 V vs. Ag/AgCl. 
106 
 
 
Figure 6-6 Nyquist plots for the electrode in a 5.0?10?3 mol L?1 [Fe(CN)6]3?/4?+ 0.1 mol L?1 KCl 
aqueous solution. Faradaic response for detection of target ssDNA 
 
 
Figure 6-7 Dose-Response curve for impedance spectrum obtained during target binding 
107 
 
Incubation of MCH resulted in a decrease in Rct (4 k?), indicating that the inclusion of 
MCH spacer thiol allows redox molecules to freely reach the electrode, thereby reducing 
tunneling distance where no redox process occurs. Figure 6-5 shows the EIS of the DNA 
hybridization using complimentary target. Clearly, Rct increases with target concentration. This is 
attributed to the increase in repulsive electrostatic interactions along the backbone of the SH-
ssDNA which later doubles with double-stranded DNA [41, 42]. Additionally, the limit of 
detection using EIS was verified to be 5 nM (Figure 6-6) from a curve which is a sigmoidal fit 
with a regression coefficient of 0.94. 
6.5.3. DNA detection - dotLab 
Here, we show the application of dotLab as a biosensor by using ssDNA specific to the 
hippuricase gene of C. jejuni. An interaction between immobilized probe and target causes a 
shift in intensity and a gradual increase in signal (Figure 6-7). DNA hybridization occurs through 
concentrations 5 nM and 1280 nM. When injecting the hippuricase target gene at different 
concentrations, a shift resulting from DNA hybridization was detected. The net response for each 
concentration was calculated by subtracting the non-detectable response (?DI ~ 2.94 x 10-3) 
from specific signal. Non-detectable hybridization signals were obtained from this target. It can 
be seen that DNA has hybridized (concentrations 0.005 ?M to 1.28 ?M), indicated by the rise in 
signal. On analyzing the binding profile, the base line from where binding starts is approximately 
4.26 x 10-3 DI units and the amount of binding in each step is taken as a reference from this point 
The limit of detection, determined as the lowest concentration of target DNA that produced a 
distinguishable net response (signal/noise>3), was 5.0 nM (Figure 6-8) and demonstrates the 
high specificity of the dotLab. Spurious signals due to non-specific binding are eliminated, as the 
diffraction efficiency depends on the difference in height between the patterned and non-
patterned areas [43]. Since diffraction phenomena on the dotLab are inherently self-referencing, 
108 
 
nonspecific binding to both the patterned and unpatterned regions will not affect the signal. Such 
characteristic offers an important advantage over other optical biosensor systems in which any 
surface binding event will cause non-specific binding. Besides, the dotLab biosensor is a cost-
effective platform that has the format flexibility to provide a quick and simple solution to both 
assay development and routine analysis [44, 45]. 
 
Figure 6-8 Representative response curves for the detection of the hippuricase gene. The target 
was serially diluted and introduced over the sensing surface, starting with the lowest 
concentration.  
6.5.4. DNA Detection - SPREETA 
In order to evaluate the performance of the device, a calibration curve has been obtained 
with different concentrations of target ssDNA injected serially from 2.5 nM to 1280 nM (Figure 
6-9). The limit of detection was estimated to be 2.5 nM (Figure 6-10). The introduction of target 
109 
 
resulted in an initial drop in RU, which is attributed to the bulk refractive index change as the 
amount of drop is similar in both channels. 
 
Figure 6-9 Dose-Response curve for hippuricase gene target binding.  
This is attributed to the bulk refractive index change as the amount of drop is similar in 
both channels. Following the initial drop, the RU signal in the sensing channel increased with 
DNA binding, while the reference channel remained constant. For instance, when the target 
solution was injected, the change in RU at a concentration of 320.0 nM was found to be 101.45 ? 
10 (n = 10), with a non-detectable signal of ?DI < 2 from the non-complimentary target ssDNA. 
The same surface can be regenerated using 2.5 mM HCl and reused more than 20 times over one 
week with only a small change in CV (11%). The sensor surface was regenerated by a 2 minute 
treatment with 2.5 mM HCl followed by a buffer wash.  Regeneration efficiency was found to be 
between 89-95 % across the concentration range from 2.5 nM to 1280 nM. In the case of longer 
probes, the target DNA was serially passed over the surface from the lowest to highest 
110 
 
concentrations (10.0 nM to 80.0 nM) with a complimentary nucleotide on the reference channel 
(Figure 6-11).  
Finally, as observed from the range of concentration and analytical curves, the sensitivity 
of the SPREETA and dotlab in the detection of the hipO gene of C. jejuni is higher than that of 
the BIAcore or amperometric detection platforms. The calculated lower limit of detection was 
2.5 nM for SPR and 5.0 nM for dotlab.  
 
Figure 6-10 Representative response curves for the detection of the hippuricase gene. Graded 
concentrations of ssDNA were introduced to both, sensing channel and control channel (both 
blocked with spacer thiol). 
111 
 
 
Figure 6-11 Dose-Response curve for hippuricase gene target binding 
 
Figure 6-12 A representative response curve for long DNA probes immobilized on gold surface. 
The concentration of probes used was 1 ?M. Inset shows calibration curve for the same. 
112 
 
6.6. Conclusion 
We have reported a comparative study of immobilization techniques based on direct 
coupling of thiolated probes and covalent binding of biotinylated DNA to a patterned avidinated 
surface. Detection of DNA segments from the hippuricase gene of C. jejuni using diffraction 
optics technology (dotLab) and surface plasmon resonance was achieved. Development of self-
assembled monolayers was used as the primary sensing mechanism, using by covalent 
immobilization of thiolated and biotinylated probes. Probes were not labeled and were 
immobilized using simple physical adsorption on to the surface of polystyrene or a gold sensor 
thereby avoiding complex and expensive surface chemistries for fast, responsive DNA sensors. 
Further experiments are underway to improve sensitivity and reusability of these optical sensing 
platforms using ssDNA from asymmetric PCR and real samples. 
 
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7. Overall Conclusion 
Surface science coupled with nanotechnology has the potential to bring about the next 
revolution in nanoelectronics and medical research. Nevertheless, development of 3D 
nanoarchitectures and macromolecular devices still remains a challenge. ?Bottom-up? 
nanofabrication techniques appear to be an inexpensive and efficient alternative to ?top-down? 
approaches that escalate costs for precision manufacturing. Biological templates such as 
aptamers, DNA and proteins provide a more sophisticated means to develop organized self-
assembled nanomaterials templates for ?bottom-up? nanofabricated macromolecular devices. 
Extensive ruggedness and the apt chemical functionalities make DNA a suitable tool to develop 
DNA-templated nanomaterials. Single-walled carbon nanotubes have turned out be a promising 
candidate for the development of efficient electronic and sensing devices due to their high 
conductivity and efficient charge transfer. Moreover, being the central icon in modern biology, 
and having extensive applications in genomics and gene sensing; it can also be used as a 
nanomaterial template to building macromolecular electronic devices and in medical diagnostics.  
This research mainly focuses on the ?bottom-up? surface engineering of DNA in 
developing molecular scaffolds and templates for nanoelectronics and biosensing. We have 
investigated the application of DNA as a dispersant and nanomaterial template for single-walled 
carbon nanotubes in developing 3D superstructures for macromolecular electronics and 
analyzing their electrochemical and charge transfer kinetics. Scanning electrochemical 
microscopy (SECM) was used to analyze the electrical and charge transfer kinetics across these 
nanomaterials. The steady state positive feedback obtained from the ultramicroelectrode clearly 
119 
 
indicates that the presence of DNA aids in electron hopping across two adducts, and these layers 
are not insulated from one another. Besides, the response curves at varying concentrations of the 
redox mediator indicate that the current response is solely due the charge injection through 
multiple layers and that these layers are not insulated from one another.  
Finally, a portable and reliable optical DNA biosensor platform has been developed for 
the detection of the hippuricase gene from pathogenic bacteria with high sensitivity and 
specificity. Our main aim in this part of the research was to develop a prototype biosensor for the 
detection of the disease causing gene from this bacteria at low levels, which is of great 
importance due to the economic and health concerns that rises from C.jejuni outbreaks. 
Although, antibody and enzymatic sensors serve as a versatile biosensing platform, they are not 
desired because most of the systems remain expensive, require a high level of purity and 
cumbersome procedures to prepare and may in some cases give a false positive response. Here, 
we have shown the versatility of tethered DNA molecular structures and scaffolds for responsive 
and ?real-time? detection of the highly specific hipO gene from C. jejuni. Single-stranded DNA 
was immobilized on to gold surface on the SPREETA platform and an avidinated polystyrene 
surface in the DOT via chemisorption with minimal time in developing the biosensor surface. 
The detection limit was found to be 2.5 nM and 5 nM respectively. A competitive inhibition 
assay using non-complimentary gene demonstrates that the detection was highly specific and 
selective. In conclusion, a unique blend of DNA with nanomaterials combined with its superior 
attributes can be used in developing futuristic macroscale electronic devices and clinical 
diagnostic systems.  
 
 
120 
 
8. Future Recommendation 
8.1. Localized Surface Plasmon Resonance (LSPR) based biosensors 
  As discussed in this research, three dimensional nanostructures have a significant 
contribution in the growth of novel technologies for biosensing and medical diagnostics. The 
elementary steps for charge transfer, molecular rearrangement and chemical reactions take place 
at the nano-scale. Manifestations of these mechanisms require a controlled fabrication of 
nanomaterials to harness their properties to the fullest. Such designs are popular in the 
development of optical communication devices and light routers. Essentially, it also has 
widespread applications in the fabrication of biosensor platforms based on the type of 
biomolecular interactions that take place at any interface [1].  
When an electromagnetic radiation impinges on the conduction electrons of any 
nanoparticle, then coherent surface oscillations occur, which is termed as surface plasmon 
resonance (Figure 8-1).  
 
Figure 8-13 Schematic representation of localized surface plasmon resonance across a metallic 
nanoparticle 
121 
 
By definition, any nanoparticle capable of a negative real and positive imaginary 
dielectric constant is capable of exhibiting the phenomena of surface plasmon resonance. 
Consequently, when an electromagnetic wave interacts with nanoparticles which are much 
smaller than its own wavelength, then the particles resonate with a frequency known as localized 
surface plasmon resonance. Further analysis of the properties of LSPR led to the development of 
surface-enhanced Raman scattering (SERS). SERS is a powerful tool in identifying specific bio-
molecules using their unique vibrational spectrum. Gold nanoparticles (AuNPs) have a distinct 
spectral signature and this signature depends on the size, shape and orientation of these particles. 
The main aim of developing 3D nanostructures for LSPR sensing is to provide the 
maximum surface area for biomolecular immobilization. Carbon nanotubes prove to be better 
templates for attaching AuNP and also act as a binding scaffold for better assembly. There have 
been a few major strategies for 3D assembly of LSPR platforms. AuNPs were assembled using 
alternate deposition of bi-functional cross-linkers.  
 
Figure 8-14 Schematic of the setup for LSPR sensing using a transmission mode UV-Vis 
Spectrophotometer 
A u NP /M W NT - NH
2
sli d e
Ligh t p r o b e
Ligh t sour c e
Spectr o p h o t o me t er
122 
 
Although, they claim that this technique provides better sensitivity, it may hinder target 
molecules to penetrate through the tiny crevices and reach the probe receptors immobilized on 
the AuNP surface [2]. We report the layer-by-layer assembly of a novel carbon nanotube/gold 
nanoparticle (CNT/AuNP) based sensing platform for label free DNA sensing and enzymatic 
biosensors [3-5]. Ionic nature of gold nano-particles and poly-cationic nanomaterials facilitates 
in the layer-by-layer assembly of 3D nanoarchitectures [6]. This 3D structure provides a highly 
accessible AuNP structure, with high composite density and sufficient path for the diffusion of 
target molecules to their respective receptors. Besides, LSPR phenomena can be observed on a 
conventional UV-Vis spectrophotometer in transmission mode [7]. Work is still in progress in 
order to optimize the size and shape of nanoparticles and also in selecting the right template that 
would increase the aspect ratio for biomolecular immobilization and better communication 
between the probe and target.  
 
8.2. References 
1. Willets, K.A. and R.P. Van Duyne, Localized surface plasmon resonance spectroscopy 
and sensing. Annu. Rev. Phys. Chem., 2007: p. 267-297. 
2. Ye, J., et al., Enhanced localized surface plasmon resonance sensing on three-
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123 
 
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