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Optimizing surface modifications for quantum dot self assembled monolayers (-via surface mediated DNA… Sundar, Rochita 2018

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OPTIMIZING SURFACE MODIFICATIONS FOR QUANTUM DOT SELF ASSEMBLED MONOLAYERS (-VIA SURFACE MEDIATED DNA HYBRIDIZATION) ON MONOCRYSTALLINE GOLD BEAD ELECTRODES USING ELECTROCHEMISTRY COUPLED FLUORESCENCE IMAGINGby Rochita Sundar B.Tech., Indian Institute of Technology Guwahati, 2016A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Chemistry) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) October 2018 © Rochita Sundar, 2018 The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, a thesis/dissertation entitled: Optimizing Surface Modifications for Quantum Dot Self Assembled Monolayers (-via Surface Mediated DNA Hybridization) on Monocrystalline Gold Bead Electrodes Using Electrochemistry Coupled Fluorescence Imaging submitted by Rochita Sundar in partial fulfillment of the requirements for the degree of Master of Science in Chemistry Examining Committee: Dr. Dan Bizzotto Supervisor  Dr. David Chen Supervisory Committee Member Dr. Keng Chou Supervisory Committee Member iiAbstractSurface hybridization of DNA strands on electrode surfaces (probes) with complementary DNA strandsin solution (targets) forms the basis of several electrochemical biosensors used to detect nucleic acidsof interest. This work aims to construct a site-selective assembly of probe strands on only some partsof the electrode surfaces to study specific and non-specific surface interactions of target strands dur-ing the hybridization step. The target strands used in this work have been precisely tailored onto aQuantum-dot (QD) surface. This ties to the larger goal of the project of assembling QDs on electrodesurfaces. Such a surface has potential widespread bio-sensing application owing to QDs unique opti-cal and surface properties. Monocrystalline gold bead electrodes displaying different surface featureswere coated in 11-Mercapto-1-undecanol solutions (or MUDOL). MUDOL was reductively removed byapplying negative potentials only from some surface features of the electrodes. Subsequently, AF488fluorophore tagged DNA strands were assembled onto these surface features either directly or followingan assembly of 6-Meracpto-1-hexanol (or MCH) spacer molecules. The value of the applied negativepotential, the DNA concentration used and time of electrode immersion in DNA solution were optimizedto form low probe surface coverages and low probe surface densities. Preliminary hybridization experi-ments in the absence or presence of divalent Magnesium ions in the target solutions (containing either0.2 DNA strands per QD, 2 DNA strands per QD or 10 DNA strands per QD bioconjugates) at roomtemperature or at an elevated temperature of 45°C were attempted. These surfaces were studied usinga fluorescence microscope coupled with an electrochemical control. The results indicate that some ofthe targets interact with the surface probes non-specifically through weak van der Waals forces or mayexist in a partially hybridized state. A small fraction of targets is able to hybridize (specifically interact)with the surface probes, and a still smaller fraction is stable at applications of negative potentials. Itmight be possible that the negative potentials induce electrochemical melting or dehybridization readilyas the bulky nature of the QDs prevent a stable hybridized state.iiiLay SummaryMore versatile and robust electrochemical and luminescent biosensors to detect/ measure different ana-lytes/ biomolecules of interest are needed with diverse applications in agricultural, environmental, foren-sic and medical fields. Quantum dots (QDs)- small nanometer sized luminescent semi-conductor par-ticles offer a large surface area to volume ratio that can be tailored with suitable bi-functional ligandsand polymers to detect virtually any biomolecules of interest. Hence, a controlled assembly of QDs onelectrodes is ideal in the development of an ubiquitous biosensor. This can be achieved by buildingon current biosensors that utilize DNA modified Au electrodes to detect specific sequences of comple-mentary DNA strands. Complementary DNA strands were attached to the QD surface in a controlledmanner and allowed to hybridize with DNA modified Au electrodes. In this work, the specificity, selectiv-ity of the QD assembly has been optimized on Au electrodes with different surface chemistry at differentexperimental hybridization conditions.ivPrefaceThe experimental designs and the data analysis steps were developed in collaboration with the researchsupervisor, Dr. Dan Bizzotto and the results presented in this thesis are the original and unpublishedworks of the author. The procedure used to prepare the surfaces in Chapter 5 are a modification of thetechnique employed by Elizabeth Fisher, a previous graduate student of the same research group. TheQD-DNA bioconjugates used in Chapter 6 for the hybridization experiments were supplied by Hyungki(David) Kim from Dr. Russ Algar’s research group, UBC Chemistry Department. The glassware for theelectrochemical/ spectro-electrochemical set-ups were prepared by Brian Ditchburn, UBC ChemistryDepartment glassblower.vTable of ContentsAbstract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Lay Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ivPreface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vTable of Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . viList of Figures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xNomenclature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xix1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.1 Rationale . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2 Scope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32.1 Electrochemistry of a metallic interface in an electrolyte . . . . . . . . . . . . . . . . . . 42.2 Electrochemical Measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52.2.1 Differential capacitance of the double layer . . . . . . . . . . . . . . . . . . . . . . 52.2.2 Cyclic voltammetry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52.3 Fluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72.3.1 Influences on fluorescence intensities . . . . . . . . . . . . . . . . . . . . . . . . . 82.3.1.1 Metal mediated quenching . . . . . . . . . . . . . . . . . . . . . . . . . . 82.3.1.2 FRET . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82.3.1.3 Influence of O2 and light . . . . . . . . . . . . . . . . . . . . . . . . . . . 92.3.2 Fluorescence microscopy imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . 9viiiixviTable of Contents2.4 Gold electrode surface crystallography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112.5 Self-assembled monolayer (SAM) on electrodes . . . . . . . . . . . . . . . . . . . . . . . 122.5.1 Alkylthiol SAM on Au electrodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132.5.2 Electrochemical characterization of alkylthiol SAM . . . . . . . . . . . . . . . . . 142.5.3 DNA SAM on Au electrodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152.5.4 DNA hybridization on Au electrodes . . . . . . . . . . . . . . . . . . . . . . . . . . 162.6 Quantum dots (QDs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192.6.1 Properties and surface chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192.6.2 QD SAM on Au electrodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202.7 Supplementing the cartoon . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 213 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243.1 Glassware and Teflon cleaning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243.2 Monocrystalline gold bead electrodes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243.2.1 Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243.2.2 Cleaning . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253.3 Differential capacitance measurement of Alkylthiol SAMs . . . . . . . . . . . . . . . . . . 273.4 DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283.4.1 DNA reduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293.4.2 DNA purification post reduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293.4.3 DNA quantification post reduction and purification . . . . . . . . . . . . . . . . . . 293.5 DNA SAM on Au(111) facets . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 293.5.1 MUDOL SAM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313.5.2 Etret potential to selectively desorb MUDOL SAM off Au(111)s . . . . . . . . . 313.5.3 MCH SAM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313.5.4 DNA SAM . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323.6 QD-DNA bioconjugates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323.7 QD-DNA bioconjugates hybridization with ssDNA SAM on Au(111)s . . . . . . . . . . . 333.7.1 Control samples . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333.7.2 Preliminary hybridization samples . . . . . . . . . . . . . . . . . . . . . . . . . . . 343.7.3 Hybridization at room temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . 353.7.4 Hybridization at 45oC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35viiTable of Contents3.8 Fluorescence microscopy spectro-electrochemical set-up . . . . . . . . . . . . . . . . . 353.9 Potential profiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373.9.1 Potential modulation profiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373.9.2 Reductive potential step profiles . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393.10 Image analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 403.10.1 Background correction procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . 403.10.2 Normalized ΔFS calculation procedure . . . . . . . . . . . . . . . . . . . . . . . . 413.10.3 Correcting for spectral bleed-through . . . . . . . . . . . . . . . . . . . . . . . . . 444 Electrochemical characterization of alkylthiol SAMs . . . . . . . . . . . . . . . . . . . . . . 464.1 MUDOL and MUDA SAMs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 464.1.1 Alkylthiol SAM reproducibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 464.1.2 Electrochemical alkythiol SAM stability . . . . . . . . . . . . . . . . . . . . . . . . 484.2 Selective reductive desorption of MUDOL SAM from the (111) facets . . . . . . . . . . . 495 Spectro-electrochemical characterization of selectively modified alkylthiol-DNA SAMs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 535.1 Approach1: Without intermediate MCH back-fill . . . . . . . . . . . . . . . . . . . . . . . . 535.1.1 Background corrected fluorescence intensities (at -0.4V) . . . . . . . . . . . . . 545.1.2 Normalized ΔFS calculations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 565.2 Approach2: With intermediate MCH back-fill . . . . . . . . . . . . . . . . . . . . . . . . . . 585.2.1 Background corrected fluorescence intensities (at -0.4V) . . . . . . . . . . . . . . 585.2.2 Normalized ΔFS calculations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 615.2.3 Characterization using capacitance measurements . . . . . . . . . . . . . . . . . 645.3 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 656 Spectro-electrochemical characterization of QD SAMs on Au electrodes . . . . . . . . 666.1 Surface hybridization at room temperature (with targets in TBS) . . . . . . . . . . . . . . 666.1.1 Control experiments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 666.1.2 Preliminary hybridization experiment . . . . . . . . . . . . . . . . . . . . . . . . . . 716.2 Surface hybridization at 45oC (with targets in IB) . . . . . . . . . . . . . . . . . . . . . . . 736.2.1 Control experiment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 746.2.2 Preliminary hybridization experiments . . . . . . . . . . . . . . . . . . . . . . . . . 75viiiTable of Contents6.3 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 817 Summary & future work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 837.1 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 837.2 Future work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87AppendicesA Normalized ΔFS calculation procedure (Image optimization) . . . . . . . . . . . . . . . . . 95B Spectral bleed-through correction (for AF488) . . . . . . . . . . . . . . . . . . . . . . . . . . 98ixList of Figures2.1 A cartoon depiction of the system: QD- complementary ssDNA biconjugates (targets)hybridized with ssDNA (probes) on selective parts of the gold bead electrode (not drawnto scale). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32.2 Cartoon depiction of the electrical double layer, and the distance dependent potentialdecay profile for a charged metallic electrode in an electrolyte. By Elcap [CC0], fromWikimedia Commons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42.3 Cyclic voltammetry scan of a clean Au bead electrode in a 50mMpH~8.2 phosphate buffer(scan rate of 20mV/s). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62.4 Jablonski diagram which depicts a radiative fluorescence and possible non-radiative (in-ternal conversion and vibrational relaxation) decay pathways of an excited electron. . . 72.5 Cartoon depiction of the system: FRET pair between AF488 (donor) modifiedDNA alkylth-iol hybridized with QD (acceptor)- DNA bioconjugate. . . . . . . . . . . . . . . . . . . . . 92.6 Schematic depiction of the filters used in the fluorescence microscope instrumentation. 102.7 Crystallographic planes andmiller index notation for a cubic system. By Indices_miller_plan_exemple_cube.png: Cdangderivative work: McSush (Indices_miller_plan_exemple_cube.png) [CC-BY-SA-3.0 (http://creativecommons.org/licenses/by-sa/3.0/) or GFDL (http://www.gnu.org/copyleft/fdl.html)], via Wikimedia Commons . . . . . . . . . . . . . . . . . . . . . 112.8 Brighfield image of a monocrystalline gold bead electrode with the (111) and (100) facetsclearly indicated. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 122.9 Approximate cartoon depiction of a mixed alkylthiol SAM formation by using a multistepelectrochemical desorption procedure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142.10 Cartoon depiction of a non-specifically and specifically bound DNA on Au electrode. . 152.11 Cartoon depiction of ’flipping’ of a bound DNA between a ’standing’ state and a ’lying’state in response to negative and positive potential applied to the electrode. . . . . . . 162.12 Cartoon depiction of surface mediated DNA hybridization mechanism. . . . . . . . . . . 17xList of Figures2.13 Broad absorption band and narrow excitation dependent symmetrical emission spectraof QDs employed in this work. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192.14 Cartoon depiction of the system (QD-DNA biconjugates hybridized with ssDNA on Auelectrode) with the dimension estimates (Not drawn to scale). . . . . . . . . . . . . . . . 233.1 Cyclic voltammetry scan of a Au electrode in 0.1M KOH (scan rate of 20mV/s) with distinctAu oxidation and reduction peaks. The inset shows an enlarged CV scan of the doublelayer with hydroxide adsorption and desorption peaks at approximately -0.2V. . . . . . 253.2 Cyclic voltammetry scan of a Au electrode in 0.1M H2SO4 (scan rate of 20mV/s) withdistinct Au oxidation and reduction peaks. The inset shows additional set of peaks at 0.4Vand 0.3V which could probably be due to the adsorption and desorption of the impurity inAu respectively. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263.3 Cyclic voltammetry scan of a clean Au electrode in 0.1M H2SO4 (scan rate of 20mV/s)with distinct Au oxidation and reduction peaks. The inset shows an enlarged CV scan ofthe double layer with sulphate adsorption and desorption peaks at approximately 0V. . 273.4 A decreasing potential step profile with a potential step of -50mV and each potential beingapplied for 15s. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 283.5 (a) AlexaFlur 488 fluorophore functionalization and (b) C6alkyl disulfide functionalizationat the 3’ and 5’ end respectively of the custom synthesized DNA oligonucleotide. . . . . 283.6 Flowchart depicting the two preparation approaches to prepare selectively modified ss-DNA probe surfaces. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303.7 Gluthathione structure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323.8 Gel electrophoresis data (1% TBE Agarose,100 V, 30 minutes in 1X pH 8.3 TBE buffer)for: Well 1: QD Only (10 uL), Well 2: 0.2 DNA strands per QD, Well 3: 2 DNA strands perQD, Well 4: 10 DNA strands per QD, Well 5: 50 DNA strands per QD, Well 6: QD Only(~3 uL). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333.9 Schematic of the first control experiment to study QD-DNA bioconjugates interactions withMUDOL SAM. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 333.10 Schematic of the second control experiment to study QD-DNA bioconjugates interactionswith MCH SAM on the (111) facets. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343.11 Schematic of the preliminary hybridization experiments to study specific adsorption ofQD-DNA bioconjugates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34xiList of Figures3.12 Absorption and emission spectra of AF 488 and QD and the Chroma filters used as part ofthe spectroscopic instrumentation for their detection. QD spectra was measured using anAgilent Cary Eclipse fluorimeter. AF488 spectra was generated from the manufacturer’swebsite. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363.13 Potential modulation profile. ’+’ corresponds to a fluorescence image captured by thecamera. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 373.14 Modified potential modulation profile-1. ’+’ corresponds to a fluorescence image capturedby the camera. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383.15 Modified potential modulation profile-2. ’+’ corresponds to a fluorescence image capturedby the camera. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 393.16 Reductive potential step profiles correlating the fluorescence image numbers and the po-tentials applied to the working electrode. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 403.17 Background correction steps for a fluorescence image taken at a step potential of -0.4V. 413.18 ΔFS calculation steps for step potentials (S)=+0.35V, 0V and -0.40V with base potentials(B)=+0.35V. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 423.19 Normalized ΔFS calculation steps for step potentials (S) =+0.35V, 0V and -0.40V with themost negative step potential=-0.40V. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 433.20 Optimization of normalized ΔFS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 443.21 Fluorescence images of a surface modified with ssDNA AF488 without QD modificationcaptured using GF and DIM filter cube sets. . . . . . . . . . . . . . . . . . . . . . . . . . . 454.1 Capacitance values (measured at 0V) for MUDOL and MUDA coated electrodes for threedifferent immersion times (30 minutes, 90minutes, 240minutes). . . . . . . . . . . . . . 474.2 Capacitance values (uF/cm2) plotted as a function of a decreasing potential step profilefor MUDA and MUDOL coated electrodes for three different immersion times (30 minutes,90 minutes & 240 minutes). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 484.3 Capacitance change as a function of the time of application of different Etret potentialson MUDOL coated electrodes (prepared by immersion for 90 minutes). . . . . . . . . . 504.4 (a) Capacitance change as a function of the time of application of different Etret po-tentials (multiple experiment replicates to test reproducibility of results in Fig: 4.3) (b)Average capacitance values measured after Etret potential application for 400s acrossreplicates. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51xiiList of Figures4.5 Fraction of MUDOL SAM desorbed as a function of Etret potential (applied for 400s). 525.1 Average (111) fluorescence intensities (counts) as a function of Etret potentials for sur-faces prepared using Approach1: Without intermediate MCH back-fill. . . . . . . . . . . 555.2 (FS- F+0.35V )/F−0.40V images for three different step potentials (S=-0.40V, 0V and +0.35V)for a surface prepared using Approach1: Without intermediate MCH back-fill with anEtret potential of -0.925V and 10 minutes DNA immersion time. . . . . . . . . . . . . . 575.3 Average (111)ΔFS/F values as a function of the step potentials (S) for surfaces preparedusing Approach1: Without intermediate MCH back-fill. . . . . . . . . . . . . . . . . . . . . 575.4 Average (111) fluorescence intensities (counts) as a function of Etret potentials for sur-faces prepared using Approach2: With intermediate MCH back-fill. . . . . . . . . . . . . 605.5 (FS- F+0.35V )/F−0.40V images for three different step potentials (S=-0.40V, 0V and +0.35V)for surfaces prepared using Approach2: With intermediate MCH back-fill. . . . . . . . . 625.6 Average (111)ΔFS/F values as a function of the step potentials (S) for surfaces preparedusing Approach2: With intermediate MCH back-fill. . . . . . . . . . . . . . . . . . . . . . . 635.7 Capacitance (uF/cm2) as a function of the reductive potential step profile (Fig: 3.16)for surfaces prepared using the two approaches: Without- (red points) and With- (blackpoints) intermediate MCH back-fill. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 646.1 DIM fluorescence image of the first control after hybridization with 0.2 DNA strands perQD bioconjugates (in TBS) at room temperature. . . . . . . . . . . . . . . . . . . . . . . . 676.2 DIM fluorescence images of the second control after hybridization with 0.2 DNA strandsper QD bioconjugates (in TBS) at room temperature. . . . . . . . . . . . . . . . . . . . . 686.3 Intensities from the (111) facets as a function of step potentials (S) (& at base potentials(B) of +0.35V before stepping to the corresponding step potentials) for several potentialmodulation profiles (similar to Fig: 3.13). DIM fluorescence images (uncorrected for back-ground scattering) corresponding to the first base potential and the last step potential foreach potential modulation profile is also shown. These results are of the second controlhybridization experiment with 0.2 DNA strands per QD bioconjugates (in TBS) at roomtemperature. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 696.4 Intensities from the (111) facets as a function of several potential modulation profiles (fromFig: 6.3) on a time axis. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70xiiiList of Figures6.5 DIM fluorescence images of the third control after hybridization with 0.2 DNA strands perQD bioconjugates (in TBS) at room temperature. . . . . . . . . . . . . . . . . . . . . . . . 716.6 GF and DIM fluorescence images of the preliminary hybridization experiment with 10 DNAstrands per QD bioconjugates (in TBS) at room temperature. . . . . . . . . . . . . . . . 726.7 Intensities from the Left(111) facet of DIM & GF fluorescence images as a function of steppotentials (S) (& at base potentials (B) of +0.35V before stepping to the corresponding steppotentials) of a potential modulation profile. DIM & GF fluorescence images (uncorrectedfor background scattering) corresponding to the first base potential and the last step po-tential of the potential modulation profile are also shown. The results are of preliminaryhybridization experiment with 10 DNA strands per QD bioconjugates (in TBS) at roomtemperature. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 736.8 (a) Assemblage of DIM fluorescence images (uncorrected for background scattering) withQD-DNA bioconjugates interactions highlighted as yellow ROI’s (regions of interest) as aresponse to modified potential modulation profiles. (b)Subsequent DIM fluorescence im-age after overnight immersion in IB. The results are of a control surface after hybridizationwith 2 DNA strands per QD bioconjugates (in IB) at 45oC. . . . . . . . . . . . . . . . . . 756.9 (a) Assemblage of DIM fluorescence images (uncorrected for background scattering) af-ter preliminary hybridization with 2 DNA strands per QD bioconjugates (in IB) at 45oCas a response to open-circuit potentials and modified potential modulation profiles. (b)Subsequent DIM fluorescence image after overnight immersion in IB. . . . . . . . . . . 766.10 DIM fluorescence images collected after applications of modified potential modulationprofiles and subsequent overnight IB immersion for control and preliminary hybridizationexperiment with QD-DNA bioconjugates (in IB) at 45oC. . . . . . . . . . . . . . . . . . . . 776.11 (a) A sequence of modified potential modulation profiles -1 & -2 (b) DIM intensities fromthe (111) facets as a function of the applied sequence of modified potential modulationprofiles for cases in Fig: 6.10. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 786.12 DIM intensities from the (111) facets as a function of modified reductive potential stepprofile for cases previously discussed in Fig: 6.10 and Fig: 6.11. . . . . . . . . . . . . . 80xivList of Figures6.13 (a) Fluorescence spectra of 2 DNA strands per QD and 10 DNA strands per QD biocon-jugates (in IB), of the IB solution and equivalent unmodified QDs (b) Gel electrophoresisdata (1% TBE Agarose,100 V, 30 minutes in 1X pH 8.3 TBE buffer) for: Well 1: 2 DNAstrands per QD, Well 2: 10 DNA strands per QD, Well 3: unmodified QDs. The resultsare of QD-DNA bioconjugate solutions following hybridization experiments at room tem-perature (for 20+hours) and at 45oC (for 2 hours). . . . . . . . . . . . . . . . . . . . . . . 817.1 Cartoon depiction of the system: QD-DNA biconjugates (targets) indirectly hybridized withssDNA (probes) on the electrode surface via linker DNA strands (not drawn to scale) - adifferent approach to assemble QDs on Au electrode. . . . . . . . . . . . . . . . . . . . . 86A.1 Summary of normalized ΔFS fluorescence images at step potentials (S) of +0.35V, 0Vand -0.40V after modification with Accurate Gaussian Blur filters of different radii andDespeckle filters. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 96A.2 Average (111) normalized fluorescence response as a function of step potentials (S) forchosen cases (corresponding to the edge corners of matrices in Fig: A.1). . . . . . . . 97B.1 Fluorescence images captured using a GF filter cube set (Left) and a DIM filter cube set(Right) of AF488 tagged ssDNA probe layer - with 6 ’ROIs’ marked on the images forcalculation of spectral bleed-through factor. . . . . . . . . . . . . . . . . . . . . . . . . . . 98B.2 Intensity response of the (111) facets from background corrected GF fluorescence imagesin response to a reductive potential step profile for the surface (MUDOL, Etret=-0.925V,MCH, 0.4μM and 6hours DNA AF488). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99xvNomenclatureα Half of the aperture angle for a given objective, page 10εo Vacuum permittivity, page 14εr Relative permittivity of adsorbate, page 14η Refractive index, page 10λ Wavelength of light, page 10〈〉 Average, page 21ω Frequency of sinusoidal perturbation, page 5θ Angle between tangents at two points, page 21θ Fractional surface coverage, page 15AFM Atomic force microscopy, page 22B Base potentials, page 41BRET Bioluminescence resonance energy transfer, page 19C Capacitance value of MUDOL coated electrode post Etretppcton,pge 52Cθ=0 Capacitance value of a completely clean, eletrolyte coated, bare electrode (18μF/cm2),pge 52CΘ=1 Capacitance value of a completely MUDOL coated electrode (1.910 μF/cm2),pge 52Ckytho Capacitance of an alkylthiol coated electrode, page 15Cbre−god Capacitance of an electrolyte (or water) coated bare gold electrode, page 15CD Capacitance of the diffuse layer, page 4Cd Capacitance of the double layer, page 4xviNomenclatureCH Capacitance at the outer-Helmholtz plane, page 4CRET Chemiluminescence resonance energy transfer, page 19CV Cyclic Voltammetry, page 5d Length of adsorbate, page 14do Resolution limit of the microscope, page 10DIM Filter cube set to detect QD, page 36DNA Deoxyribonucleic acid, page 1ds Double stranded DNA, page 3Etret Electrochemical treatment potential, page 30ECL Electrochemiluminescence, page 20EM gain Electron-Multiplying Gain, page 36fcc Face centered cubic, page 11FRET Fluorescence resonance energy transfer, page 8FTIR Fourier-transform infrared spectroscopy, page 13FTMS Fourier Transform Mass Spectrometry, page 13GF Filter cube set to detect AF488, page 36GSH Gluthathione, page 32im Imaginary current, page 5ire Real Current, page 5IB Immobolization buffer, page 29L Distance between two points, page 21MCH 6-Mercapto-1-hexanol, page 15MUDA 11-Mercaptoundecanoic acid, page 46xviiNomenclatureMUDOL 11-Mercapto-1-undecanol, page 22NA Numerical aperture of the microscope, page 10NEXAFS Near edge X-ray absorption fine structure, page 13NSET Nanoparticle surface energy transfer, page 19P Persistence length, page 21QD Quantum Dot, page 1Ro Förster radius, page 8S Step potentials, page 41SAM Self assembled monolayer, page 12ssDNA Single stranded DNA, page 2TBS Tris-buffered saline, page 34TCEP Tris(2-carboxyethyl) phosphine, page 28Vc Amplitude of the sinusoidal potential perturbation, page 5XPS X-ray photoelectron spectroscopy, page 13xviiiAcknowledgmentsI am grateful to my supervisor, Dr. Dan Bizzotto for introducing me to the field of surface spectro-electrochemistry. Thank you for your guidance and support in the development of this thesis. I wouldlike to extend my thanks to my fellow labmates: Elizabeth Fisher, Issac Martens, Kaylyn Leung andTianxao Ma and to my colleagues in Dr. Russ Algar’s group: Hyungki (David) Kim and Hsin-Yun Tsaifor their valuable inputs.I am also grateful to Mitacs for bestowing me with the Globalink Graduate Fellowship award and forpartially funding my study at UBC. I would like to thank all UBC Chemistry department professors andstaff that have taught me and for whom I have worked for during the course of my program for enrichingmy educational experience.I am thankful to UBC’s St. John’s College (residence) for accepting me as a Juinor fellow for the pasttwo years and providing me with invaluable cultural and international perspectives. I would like to extendmy thanks to UBC’s IGSA (Indian graduate student association) for helping me connect with like-mindedpeers and alumni from India.Finally, I would like to thank my parents- my pillars of strength for their patience and advice while Idealt with the uncertainties of research life.xixChapter 1Introduction1.1 RationaleThis thesis describes the development of a new platform for a coupled electrochemical and optical sen-sor by attaching QDs to gold electrodes via DNA surface mediated hybridization. QDs have not yet beenexplored as probes for electrochemical biosensors. This is despite having superior photostability, multi-plexing capabilities (on account of broad absorption spectra and narrow symmetrical emission spectra)and unlimited surface bioconjugation capabilities to detect virtually any biomolecule of interests.1 Thereis little to no scientific literature that has studied immobilizing QD SAMs on Au electrodes at fixed (eas-ily tailorable) separations which might be important not only from a biological sensing perspective butalso to study the biophysics and energy transfer mechanism of QDs at close proximity to a large metalsurface.DNA surface mediated hybridization between immobilized DNA strands on electrodes (probes) withcomplementary strands in solution (targets) has been previously studied in depth for development ofdiagnostic devices and DNA based biosensors.2–4 This process can occur either through a direct ap-proach where the targets from the solution hybridize with the surface probes (3-dimensional) or via anindirect approach mediated through non-specific interactions of the target strands onto the surface whichdiffuses across the 2-dimensional electrode space searching for a probe to bind with. It is possible thattargets may adsorb non-specifically, diffuse across the electrode surface and desorb from the surfacewithout undergoing any hybridization with the surface probes.5,6The kinetics and thermodynamics of the surface hybridization process is also complex and dependson the approachibility of the target strands to the the probe strands. Efficiency of this process can beinfluenced by the probe surface coverage7, probe surface density, the surface chemistry8, and dimen-sions/ confirmations of the targets.In this thesis, the probe strands have been tagged with a fluorophore AF488 while the complementarytarget strands have been tailored onto a QD surface. A rational experimental approach to designing a11.2. Scopeselective ssDNA probe assembly on only some parts of the electrode surface has been utilized to studyhow the underlying surface chemistry affects non-specific adsorptions of the targets. A fluorescencemicroscope coupled to an electrochemical set-up was used to detect non-specific and specific adsorp-tion of targets onto electrode surfaces. Since, there is a possibility that an unstable hybridized duplexmay also undergo melting or de-hybridization of the targets onto the electrode surface6, stability of thehybridized duplex state was also studied using the coupled spectro-electrochemical set-up.1.2 ScopeIn Chapter 2, a literature review and background information relevant to understand this thesis is pre-sented. Chapter 3 is the methods and materials section and gives an overview of the experimentaldesigns and the characterization techniques employed in this work. This thesis aims to develop ssDNAassembly on only some select features of the gold monocrystalline bead electrodes (results discussedin Chapter 4). The probe DNA surface coverage and density have been studied as a function of differentpreparation conditions using a fluorescence microscope coupled with an electrochemical control (resultsdiscussed in Chapter 5). For these surfaces, surface mediated hybridization with different QD-DNA bio-conjugate solutions at room temperature and at an elevated temperature of 45oC has been attempted(results discussed in Chapter 6). Finally, a brief conclusion of important results has been presented inChapter 7.2Chapter 2BackgroundThis chapter introduces the background information required to understand the system studied in thisthesis and the spectroscopic and the electrochemical techniques used for its characterization. As shownin Fig: 2.1, the system consists of preparing ssDNA (probes) on selective parts of the gold bead electrodewhich is hybridized with QD-complementary ssDNA bioconjugates (targets) to form dsDNA.Figure 2.1: A cartoon depiction of the system: QD- complementary ssDNA biconjugates (targets) hy-bridized with ssDNA (probes) on selective parts of the gold bead electrode (not drawn to scale).Applying a potential to an electrode system that is introduced in an electrolyte results in a detectableelectrical current characteristic of the electrode system/ electrolyte interface. Some necessary contextregarding the electrode/ electrolyte interface has been discussed next.32.1. Electrochemistry of a metallic interface in an electrolyte2.1 Electrochemistry of a metallic interface in an electrolyteWhen a potential is applied to a metallic electrode placed in an electrolyte, charge is accumulated onlyon the outer surface of the electrode because a conductor can support no electrical fields within it. Onthe solution side, this charge is balanced by a layer of adsorbed ions and another layer of diffused ionswith concentrations of the opposite charge to that on the electrode in excess as compared to the chargeon the electrode. This is depicted in Fig: 2.2. The center of the adsorbed ions either specifically boundthrough covalent bonds or non-specifically through van der Waal interactions forms the inner-Helmholtzplane while the the center of hydrated ions, farther from the electrode interface forms the outer-Helmholtzplane (depicted by a distance of d2 in Fig: 2.2). The layer of ions from the outer-Helmholtz plane until thethe point at which the total charge on the solution side balances the charge on the metal (indicated asthe line labeled ’separator’ in Fig: 2.2) forms the diffuse layer which has a certain thickness.9 Together,the Helmholtz and the diffuse layer are referred to as the double layer.Figure 2.2: Cartoon depiction of the electrical double layer, and the distance dependent potential decayprofile for a charged metallic electrode in an electrolyte. By Elcap [CC0], from Wikimedia CommonsThis double layer can be modeled accurately as two capacitors in series, one for each of the layers.Mathematically, the capacitance of the double layer (Cd ) is given as a sum of two capacitors in seriesusing the formula: 1Cd =1CH+ 1CD where CH and CD denotes the capacitance at the outer-Helmholtz planeand the diffuse layer respectively.10 Under the assumption that no net free charge exists in the Helmholtz42.2. Electrochemical Measurementsplane, CH would be analogous to a capacitor with a fixed dielectric constant (constant for a chosenelectrolyte) and consecutively is thought to contain a constant electric field. Therefore, the potentialfrom the electrode surface decreases in a linear fashion till the outer-Helmholtz plane is reached. Usinga Maxwell-Boltzmann statistics for the ions in the diffuse layer, the potential decreases approximately inan exponential fashion in the diffuse layer as the distance from the electrode is increased.10,112.2 Electrochemical MeasurementsIn this thesis, two different electrochemical techniques namely: differential capacitance (of the doublelayer) and cyclic voltammetry have been employed for characterizing the modified electrode surface. Abrief description of these techniques is presented next.2.2.1 Differential capacitance of the double layerFor an electrode-electrolyte interface, the change in it’s stored charge in response to a small potentialperturbation applied to it, is reported as its differential capacitance. A small AC sinusoidal potential per-turbation at a single frequency ’ω’ (i.e. Vcsin(ωt)) is applied to the electrode and the resultant in-phaseand out-of-phase components of the sinusoidal current response (ireand im respectively) are recorded.The differential capacitance value is calculated as Cd= mVcω”1+ ( rem )2—. The use of a single frequencymeasurement of impedance is appropriate for these simple electrochemical systems that do not havea faradaic component. In this case, the electrochemical impedance spectra would result in the samemeasured capacitance if a comparatively low frequency (ω) of AC sinusoidal potential perturbation isused. The value of the double-layer capacitance is a characteristic measure of the electrode-electrolyteinteraction and can be used to study changes at the interface.10,122.2.2 Cyclic voltammetryAnother electrochemical technique typically performed is cyclic voltammetry. The potential is linearlycycled multiple times between two different potential limits (referred to as the switching potentials) at aspecific scan rate/sweep rate (typically chosen between 10mV/s to 100mV/s).This linear potential profileas a function of time is applied to the electrode and the resultant current is measured as a function ofthe potential applied (and consequently as a function of time). CV is a useful electrochemical techniqueto study faradaic reactions (like oxidation and reduction reactions), the adsorption/ desorption reactions52.2. Electrochemical Measurementson the electrode surface, charging of the double layer capacitance etc.10 A cyclic voltammetry scanfor a clean Au electrode between the hydrogen evolution and the oxidation regimes of Au with distinctphosphate adsorption and desorption peaks in a pH~8.2 phosphate buffer is shown in Fig: 2.3.13,14Figure 2.3: Cyclic voltammetry scan of a clean Au bead electrode in a 50mM pH~8.2 phosphate buffer(scan rate of 20mV/s).The electrochemical techniques discussed above provides an average measurement value for theentire electrode surface. However, as mentioned previously ssDNA has been assembled only on someparts of the electrode surface for the system studied in this thesis. ssDNA with a fluorophore AF488modification at the free end has been utilized in this work to enable selective characterization of parts ofthe electrode surface by employing a fluorescence microscope. A coupled electrochemical and spectro-scopic (fluorescence microscopy) technique can together provide more comprehensive characterizationof the modified electrode surface. The next section focuses on fluorescence and the fluorescence mi-croscope instrumentation.62.3. Fluorescence2.3 FluorescenceLight absorbed by a molecule results in an electronic transition from the lowest vibrational state of theground electronic state (S0) to a vibrational state of the excited electronic state. This electron quicklydecays to the ground vibrational state of the excited electronic state (order of picoseconds) due to non-radiative vibrational relaxations mediated by inter-molecular collisions. A release of energy in the form ofphoton as this electron relaxes to one of the vibrational states of the ground electronic state is measuredas the fluorescence energy (Fig: 2.4). Understandably, the fluorescence emission occurs at a largerwavelength compared to the excitation wavelength (difference is referred to as the Stokes shift).15,16Figure 2.4: Jablonski diagramwhich depicts a radiative fluorescence and possible non-radiative (internalconversion and vibrational relaxation) decay pathways of an excited electron.An alternative and less likely form of radiative decay is phosphorescence. The electron in the excitedsinglet electronic state (S1) can transfer to a vibrational state of an intermediate triplet electronic state(T1) with similar energies. This electron undergoes vibrational relaxations to the ground vibrationalstate of the triplet electronic state and eventually back to the ground singlet electronic state (S0) withan emission of photon but over a comparatively long duration since the transition is spin forbidden.Fluorescence typically occurs on a scale of 10s of nanoseconds while phosphorescence occurs on ascale of a few microseconds to several seconds.15,1672.3. Fluorescence2.3.1 Influences on fluorescence intensitiesDifferent experimental factors can influence (i.e increase or decrease) the fluorescence measurements.Some of the factors relevant to this work have been discussed below:2.3.1.1 Metal mediated quenchingThe decrease of fluorescence intensity of a fluorophore near a metal surface can follow different path-ways depending on the distance of the fluorophore from the electrode surface. For intermediate dis-tances between the fluorophore and the metal surface (<100nm), the excited state fluorophore can bethought of as an oscillating dipole emitter that induces surface plasmons (or oscillating electrons) in themetal. The loss of the the excited state emission by coupling of the fluorophore with the surface plas-mons in the metal occurs without the emission of a photon and hence the fluorescence is thought to bequenched. These surface plasmons created for a short fluorophore separations are often trapped dueto optical properties and eventually decays as heat. For these distances, the fluorescence decay ratewas found to be inversely related to the third power of the metal-fluorophore separation.17–192.3.1.2 FRETFRET is a non-radiative energy transfer mechanism between an excited state of a donor molecule to theexcited state of an acceptor molecule mediated through long range dipole-dipole coupling interactionswithout the emission of fluorescence energy as a photon. The acceptor molecule usually absorbs in thewavelength band similar to donor emission and may or may not undergo subsequent emission itself.20The efficiency of this process depends on several factors such as the distance between the acceptorand the donor molecule, the spectral overlap between the donor emission and the acceptor absorbanceand also on the orientation of the donor dipole with respect to the orientation of the acceptor dipole.21 Itis inversely proportional to the sixth power of the distance between the acceptor and the donor molecule.Sufficient FRET usually occurs for a separation distance between 0.5Roand 1.5Rowhere Ro is the dis-tance for which efficiency is 50% and is typically between 3-6nm.22The modified electrode surface studied in this thesis (Fig: 2.5) employs an AF488 modified ssDNAprobe on a gold electrode surface and a QD labeled target DNA. After target-probe DNA hybridization,a FRET pair can possibly form between the the AF488 fluorophore (as the donor) and the quantum dot(as the acceptor). This is because there is small overlap between the AF488 emission spectrum tailand the absorption spectra of the quantum dot (Spectra shown in Fig: 3.12 of Chapter 3). Excitation of82.3. FluorescenceAF488 when near a QD will result in a decreased emission from AF488 due to energy transfer to theQD. Emission from the QD will increase because of this energy transfer.Figure 2.5: Cartoon depiction of the system: FRET pair between AF488 (donor) modified DNA alkylthiolhybridized with QD (acceptor)- DNA bioconjugate.2.3.1.3 Influence of O2 and lightPresence of a quencher molecule like O2 is found to de-activate an organic molecule’s fluorescent stateeither by oxidizing the fluorophore or by promoting an inter-system crossing of the excited state electronto the triplet state.23 Excess amounts of light (photo bleaching) has also been reported to cause photo-induced chemical destruction of the fluorophore resulting in a non-radiative quenching of excited stateelectron emission.242.3.2 Fluorescence microscopy imagingAn epifluorescence microscope where both the excitation and emission light pass through the sameobjective has been used to analyze the samples in this work. A filter cube set with distinct excitation& emission filter and dichroic mirror is used for selecting the wavelength for fluorophore excitation anddetection. A schematic depiction of the filters used in the microscope has been shown in the Fig: 2.6.A mercury lamp acts as the fluorescence light source. An excitation filter was employed to block all thewavelengths of light except the specific wavelength band required for excitation. A dichroic mirror allowsthe excitation wavelength band to pass through the objective, reflecting the remaining wavelengths. Thisthen excites the sample which emits light isotropically. Some of the emitted light makes its way into the92.3. Fluorescenceobjective. A portion of the excitation light is also scattered and reflected back into the objective. Thedichroic mirror directs only the emitted light towards an emission filter. The emission filter specificallyallows only the light corresponding to the emission wavelength band to pass through to the detector.25Figure 2.6: Schematic depiction of the filters used in the fluorescence microscope instrumentation.The numerical aperture (NA ) of the microscope objective is defined as ηsnα where η is the refrac-tive index of the medium between the sample and the objective and α is half of the total aperture anglefor a given objective. The aperture opening for a given objective is defined by the maximum angle ofthe light diffracted isotropically from the focus that can enter the objective. The resolution limit of themicroscope (do) is defined as λ2NAwhere λ is the wavelength of light. This is the minimum separationbetween two points that can be imaged distinctly.26,27 The objectives of different magnifications (i.e.5X, 20X etc) have different numerical apertures with the value being higher for higher magnification ob-jectives. Hence, the higher magnification objectives have a greater resolving power (smaller do value).Since the resolution is diffraction limited, two distinct points are actually imaged as a blurred spot sur-rounded by diffraction rings of alternating constructive and destructive interference (’Airy disk’) and dois the minimum distance between the maxima of any two nearest airy disks.28102.4. Gold electrode surface crystallographyA coupled electrochemical and spectroscopic methodology has been employed in this work to simul-taneously capture fluorescence images and record differential capacitance measurements.292.4 Gold electrode surface crystallographyAtoms of gold in the solid state exist as a face-centered-cubic (fcc ) arrangement with 8 gold atomsoccupying the edge points of a unit cell and 6 gold atoms occupying the center of each of the faces of aunit cell. Several unit cells are joined together to form a 3-dimensional repeating crystal lattice structure.Any given plane that intersects the three main orthogonal axes (X, Y, Z) at points (m,0,0) , (0,n,0) &(0,0,p) can be represented using miller indices (h,k,l) where h~1/m , k~1/n & l~1/p and are the smallestpossible integral values. Truncation of the 3-D lattice creating different (h,k,l) planes results in differentarrangements of exposed surface atoms.30,31 The schematic of the three low index crystallographicplanes, (100), (110) and (111) can be seen in the Fig: 2.7. All planes that are geometrically identical (like (001), (100), (010) ) have not been distinguished in this work.Figure 2.7: Crystallographic planes and miller index notation for a cubic system. By Indices_miller_plan_exemple_cube.png: Cdangderivative work: McSush (Indices_miller_plan_exemple_cube.png) [CC-BY-SA-3.0 (http://creativecommons.org/licenses/by-sa/3.0/) or GFDL (http://www.gnu.org/copyleft/fdl.html)], via Wikimedia Commons112.5. Self-assembled monolayer (SAM) on electrodesThemonocrystalline gold bead electrodes prepared for use in this work exhibits a few crystallographicfacets of the possible fcc exposed surface structures.32 The brighfield image of one such monocrys-talline gold bead electrode with the two low index crytsallographic facets marked is shown in Fig: 2.8.AFM results previously reported in literature by former group member confirms the surface structure ofthe (111) facets on such monocrystalline bead electrodes.32Figure 2.8: Brighfield image of a monocrystalline gold bead electrode with the (111) and (100) facetsclearly indicated.The density of broken bonds can be calculated individually for each of the (h,k,l) facets as the numberof broken bonds with the nearest neighbours as a result of truncation per unit area of the the unit cell.This is a characteristic indication of the surface energy values. For the three low index facets (111), (110)& (100), truncation exposes a set of surface atoms with 3, 5 and 4 broken nearest neighbor interactionsper exposed surface atom respectively. The (111) facet has a smaller density of broken bonds andconsequently a smaller surface energy when compared with other low index facets (like the (100) & the(110)).332.5 Self-assembled monolayer (SAM) on electrodesA typical SAM sub-unit consists of a head group that can form a stable chemical bond with the solidsupport, a backbone that packs together well through van der Waals/ hydrophobic interactions andfinally a terminal group that confers specific properties to the SAM. Multiple sub-units spontaneouslyassemble either from a gaseous or a liquid phase onto a solid support to form a SAM.34For this work, primarily alcohol terminated alkylthiols with different backbone chain lengths & DNAmodified alkylthiols have been used as the SAM sub-units while a monocrystalline gold bead electrode122.5. Self-assembled monolayer (SAM) on electrodeshas been used as the solid support. This particular section provides the background to understandalkylthiol and DNA SAM formation and surface mediated DNA hybridization reaction.2.5.1 Alkylthiol SAM on Au electrodesAu is an inert material with specific interactions with sulfur which allows monolayer formation of severalalkyl functionalized thiols. The formation of self-assembled monolayer of alkylthiols on clean Au elec-trodes follows a two step process. The first step involves an oxidative addition of the -SH to the goldsurface forming thiolates. The second step is thought to involve a reductive elimination of H2.35R− S−H+A⇒ R− S−A+ 12H2The fact that the adsorbing species is a thiolate has been confirmed experimentally using a variety oftechniques like XPS 36, Raman37, FTIR & FTMS 38, electrochemistry39 etc. RS-Au is a strong bond witha bond energy of 40Kcal/mol and the net reaction is exothermic with a calculated value of -5Kcal/mol.40Kinetic studies of alkylthiol adsorption on gold (111) surfaces describes the process as consisting oftwo distinct kinetic steps.35 The first step is relatively fast where a SAM coverage of 80-90% is achievedon the order of a few minutes. This step is strongly correlated to the initial concentration of the alkylthiolsolution (lasting a few minutes for a 1mM conc. vs. 100s of minutes for a 1μM conc.).40 The secondstep, which is relatively slow can last several hours and involves re-organization and annealing of thedisordered layer to form a 2-D crystalline lattice. The two step kinetic process of alkylthiol adsorptionhas been confirmed using techniques like XPS, NEXAFS etc.40,41Longer chain alkylthiols (n>9) also forms more densely packed monolayers with a faster adsorp-tion kinetics when compared to monolayers formed with shorter chain alkylthiols (n<9).35 The alkythiolmonolayers were also found to be stable indefinitely at room temperatures but begin to lose stability whenheated to high temperatures (like 80oC) in solvents like n-hexane. Also, using ethanol as a solvent forthe alkylthiols was found to result in consistent monolayers.35Applying a sufficiently negative potential in an electrolyte of high pH (to push water reduction to hy-drogen reaction to even more negative potentials to separate the two reactions) can reductively removealkylthiol from Au electrodes. The mechanism of reductive desorption of alkylthiol was found to start pri-marily on defects & grain boundaries and further as small patches (nucleation) and subsequent outwardgrowth of these patches.42132.5. Self-assembled monolayer (SAM) on electrodesRS−A+ + H2O(q)+ e− ⇒ RS−(q)+A(H2O)(q)Different crystallographic facets have different characteristic reductive desorption potentials with Au(111)having the least negative reductive desorption potential value when compared to other low index planeslike Au(100) & Au(110).43,44 Another alkylthiol SAM can be deposited subsequently selectively on theparts of the Au electrode that was previously desorbed to form a mixed alkylthiol SAM.45,46 A cartoondepiction of this process can be seen in Fig: 2.9.Figure 2.9: Approximate cartoon depiction of a mixed alkylthiol SAM formation by using a multistepelectrochemical desorption procedure.2.5.2 Electrochemical characterization of alkylthiol SAMThe differential capacitance value (Cd) measured for an alkythiol SAM coated gold electrode is signifi-cantly different than that of a bare gold electrode. As discussed before, Cd is a series combination oftwo different capacitors CH & CD.Mathematically, CH = εrεod , where εo is the vacuum permittivity, εr is the relative permittivity & d isthe thickness of the adsorbed species. The value of εr for alkylthiols is approximately 2 and for wateris approximately 80. Hence, the calculated value of CH is smaller for the alkylthiol SAM covered goldelectrode when compared to the electrolyte (or water) covered bare gold electrode. The value of CH isalso smaller for longer alkylthiol SAMs.The value of CD depends on the electrolyte concentration & applied potential. Usually, for an elec-trolyte concentration higher than 10mM, the value of CD is larger than the value of CH. The value of Cd142.5. Self-assembled monolayer (SAM) on electrodescalculated from a series combination of CH and CD, consequently is dominated by the value of CH.47The capacitance for the alkylthiol SAM on Au is on the order of a few (1-2) μF/cm2 while it is on theorder of several 10s of μF/cm2 for bare gold electrode in the same electrolyte.47CH for an electrode which is only partially covered by an alkylthiol SAM can be expressed as aparallel combination of two capacitors: one for the part covered by the alkylthiol and another for thebare gold electrode. If θ represents the fractional surface coverage of the SAM, then mathematicallyCH can be calculated asθCkytho+ (1− θ)Cbre−god where Ckytho and Cbre−god representthe capacitance of an electrode completely covered by an alkylthiol SAM and an electrolyte (or water)covered bare gold electrode respectively.482.5.3 DNA SAM on Au electrodesDNA modified with an alkylthiol at one end can be attached to Au vis -S-Au interactions as described in2.5.1. A two step method is used. The formation of DNA modified thiol (5 ´ -HS-(CH2)6-DNA3´) SAM onAu is the first step. This is followed by a subsequent immersion in 6-mercapto-1-hexanol (MCH) whichis the second step. This was found to result in a mixed SAM with the MCH acting as a spacer betweenthe DNA strands. MCH was found to displace the nonspecifically adsorbed DNA on Au and to formaccurately controlled average DNA surface coverages.49 Nonspecific interactions of DNA on Au occursprimarily with the DNA base pairs interacting with the Au surface while specific adsorption occurs viathe thiol (Fig: 2.10). The extent of non-specific interactions has also been found to be dependent on theDNA sequence.50Figure 2.10: Cartoon depiction of a non-specifically and specifically bound DNA on Au electrode.In-situ fluorescence measurements of these mixed SAMs can be realized by modifying the free (3’)end of the ssDNA with a fluorophore tag. The fluorescence measurements however indicated hetero-geneity in coverages with DNA mono layers on some parts of the surface and large aggregates of DNA152.5. Self-assembled monolayer (SAM) on electrodeson other parts of the surface.51 Reversing the two step method i.e immersion in MCH first and then inalkylthiol modified DNAwas found to result in DNA coverages less than 1% of the theoretical maximawithfewer aggregates and fluorescence modulation response (due to DNA surface re-orientation) to appliedpotential modulation.51 Since the DNA backbone is negatively charged, applying a negative charge tothe solid support repels the DNA to a ’standing’ state (i.e. oriented perpendicular to gold surface) whilea positive charge attracts the DNA to a ’lying’ state (i.e. oriented parallel to gold surface). The decreasein fluorescence intensity due to metal mediated quenching is higher when the DNA is in a ’lying’ state(fluorophore is positioned closer to the electrode surface) than when the DNA is in a ’standing’ state (flu-orophore is positioned farther to the electrode surface). This is shown in Fig: 2.11.52,53 The absolutefluorescence intensity counts and the fluorescence intensity modulation response of the MCH-ssDNASAMs can also be correlated to DNA surface coverage (molecules/cm2) for different crystallographicfacets.54Figure 2.11: Cartoon depiction of ’flipping’ of a bound DNA between a ’standing’ state and a ’lying’ statein response to negative and positive potential applied to the electrode.2.5.4 DNA hybridization on Au electrodesDetection of target DNA surface hybridization with probe ssDNA on the electrode surfaces forms the ba-sis for the development of biological and environmental sensors.55,56 Over the past few years, Plaxcoand group have prepared several probe bound electrochemical biosensors (E-DNA) capable of detect-ing a specific sequence of target nucleic acids. This was by utilizing a redox moiety (like methyleneblue) bound to a hairpin DNA structure that is oriented away from the electrode due to changes in theconformations of the hairpin upon specific target-probe hybridization. The redox moiety when positionedcloser to the electrode surface generates a large faradaic current which decreases in magnitude as the162.5. Self-assembled monolayer (SAM) on electrodesmoiety moves away from the electrode. The electrical current response was used to monitor the fractionof the probe strands in the hybridized duplex state.2,3,57 The hybridization efficiency as a function of thessDNA probe density has also been studied for the E-DNA sensors.58 Apart from electrochemical tech-niques, other works have also focused on the detection of the hybridization step via surface plasmonresonance59, optical60, electrical61 and gravimetric62 based methods.63The actual process of surface hybridization is thought to first involve a nonspecific interaction of thetarget onto the surface which then “searches” across the 2-D layer for an immobilized probe to bind.Initially, the interactions are primarily through long range weak pi− pi interactions. A part of the targetcomplementary strand then undergoes short range base pairing with the corresponding part of the im-mobilized DNA strand (nucleation site). The complete hybridization occurs eventually, annealing theSAM to form a dsDNA.64 A cartoon depiction of the surface hybridization process can be seen in Fig:2.12. Single-molecule FRET experiments to study these interactions have been performed and conclu-sively show only a small fraction to be in a completely hybridized duplex state with several interactionspossibly existing in a transient nonspecifically or a partially hybridized state.65 External stimuli can alsocause a nucleation bubble at a specific part of the dsDNA disrupting the base pairing which eventuallyleads to dehybridization of the ds-DNA.66Figure 2.12: Cartoon depiction of surface mediated DNA hybridization mechanism.Presence of nonspecific interacting probe ssDNA on Au greatly impedes hybridization with the com-172.5. Self-assembled monolayer (SAM) on electrodesplementary target strand as the ssDNA lies horizontally on the electrode surface.49 The choice of thealkylthiol passivator is hence critical as for example, carboxylic acid terminated alkylthiols could poten-tially induce more nonspecific interactions between the the carboxylate end group and ssDNA basepairs through dihedral cationic bridges.67 Hybridization efficiency is also affected to a great extent bythe coupled factors of ssDNA probe surface coverage and the ionic strength of the hybridization solu-tion. A very dense probe layer would electrostatically repel any incoming target DNA strand, however,the ionic strength would dictate the range of this repulsion.4,68 Researchers have found that the surfacehybridization efficiency can be close to a 100% for some of the lowest ssDNA surface coverages with thehybridization obeying Langmuir adsorption kinetics. At high surface coverages, less than 10% hybridiza-tion efficiency may result with very slow hybridization kinetics. The fact that high density probe surfacesresult in less efficient surface hybridization has been confirmed previously using SPR7, Fluorescence69& electrochemical techniques70.For ssDNA probe (25 base pairs) with surface coverages lower than 2X1012 molecules/cm2, heat-ing has been reported to increase the hybridization efficiency (by a factor of 22%) when compared tohybridization efficiency of similar surfaces without heating.71 At these coverages, a sharp increase inhybridization efficiency was also reported in the first 15 minutes after which the hybridization efficiencyseems to plateau. Heating was found to not affect the the ssDNA coverages (no thermal induced desorp-tion) and had no distinct advantages for samples with high probe surface coverages.71 Others that havestudied effects of heated electrodes on hybridization while the bulk of the hybridization electrolyte wasmaintained at room temperature also report higher hybridization efficiencies with heated electrodes.72Previous experiments to study surface DNA hybridization using a rotating disk electrode and heated elec-trodes have also shown that increased temperatures significantly affect hybridization efficiency as op-posed to rotation/ mass transport effects.73 Some other techniques that have been previously employedto increase surface hybridization efficiency include treatment with low-frequency acoustic waves74 & ap-plication of potential75.Melting of the dsDNA on the electrodes is markedly different than corresponding denaturation in thesolution. Themelting parameter on the the solid support is influenced by the interactions with the nearestss/dsDNA density (charge).76 Applying a negative charge to the solid support in an electrolyte where thenegatively charged dsDNA backbone resides within the electrochemical double layer can possibly causedenaturation and uncoiling of the dsDNA because of electrostatic repulsion.77 The negative potentialrequired to induce dsDNA uncoiling is largely sequence dependent.78 Recent studies have also shownpossible DNA denaturing at high ionic strengths (with dsDNA base pairs lying outside of the double layer)182.6. Quantum dots (QDs)and with PDNA (oligomers of peptide nucleic acids not having a negatively charged backbone that mimicDNA structure) at negative potentials which might suggest that electrostatic repulsion is probably onlyone of the many factors that causes dehybridization at these conditions.792.6 Quantum dots (QDs)2.6.1 Properties and surface chemistryQDs are semiconductors in sizes varying from 2nm-10nm.80 QDs can exhibit size dependent opticalproperties, with a smaller size correlated to a larger band gap (blue-shifted emission) due to quantumconfinement effects.81 QDs also exhibit very bright photoluminescence, a wide absorption spectra andsize tailorable narrow emission spectra (full width at half maxima ~30nm) (Fig: 2.13), resistance to pho-tobleaching and longer excited states when compared to organic fluorophores.82 Typically, CdSe/ZnS(Core/Shell) QD is used for bioanalysis with the larger available surface area to volume ratio modified byspecific hydrophillic bifunctional ligands or amphiphilic polymers to detect biomolecules of interest us-ing click chemistry/ affinity interactions or covalent coupling interactions.83 Algar et. al have previouslystudied in depth QD bioconjugation stability and kinetics with the conjugation tailorable proportional toa Poisson distribution depending on the number of bioconjugates used per QD.22 Additionally, QDs/QDbioconjugates can undergo a range of energy transfer mechanisms including FRET84,85, BRET/ CRET,NSET86, charge transfer87 that can be optimized for biosensing applications.Figure 2.13: Broad absorption band and narrow excitation dependent symmetrical emission spectra ofQDs employed in this work.192.6. Quantum dots (QDs)2.6.2 QD SAM on Au electrodesQDs find several in-vitro and in-vivo applications in biological sensing because of their unique opticaland surface properties.1,80,88 However, research towards functionalization of QDs is mostly on surfacebioconjugation and ligand exchange. On the other hand, ssDNA/ dsDNA SAMs immobilization on a vari-ety of substrates (Au particularly) has been extensively researched for the detection of several biologicaltargets like proteins, aptamers etc55,89,90 using many optical91and electrochemical techniques.63Previously, research has been conducted to study the energy transfer mechanisms between Aunanoparticles and QDs. A large triangular DNA origami framework has been employed to preciselyassemble gold nanoparticles and quantum dots at several fixed distances with respect to each otheron the DNA origami framework to study the distance dependent fluorescence quenching response ofQDs to Au nanoparticles.92 For 30nm Au nanoparticles and large QD-Au nanoparticle separations of15nm to 70nm, the fluorescence loss was found to be mainly through non-radiative mechanisms withthe energy transfer being inversely proportional to the power of 2.7 of the separation distance (unliketypical FRET). Energy transfer mechanisms of QDs in close proximity to a comparatively infinitely largemetal surface (when immobilized as a SAM on Au), however, is expected to be very different than theenergy transfer mechanism to small Au nanoparticles, a few nms in diameter.Another report used both gold and aminopropylsiloxane functionalized glass as solid supports to con-struct CdS nanparticle arrays by carrying out extensive hybridization between two different CdS nanopar-ticles with bioconjugation of 20+ ssDNA and complementary ssDNA strands respectively. Pulsed irra-diation to generate photocurrents (aided by using [Ru(NH3)6]+3 electrochemistry) was used for thecharacterization of the 3-dimensional multilayer array.93Interestingly, semiconductor QDs have also been shown to exhibit an electrochemiluminescence(ECL) behavior by Bard et. al.94 ECL involves radiative emission by any species that is capable ofundergoing high energy oxidative or reductive electron transfer mechanisms in response to external ap-plied potentials. This behavior directly depends on the number of unpassivated surface atoms on theQD (or indirectly on the QD capping) which could potentially act as electronic traps for holes or electrons.Organic and metallic semiconductor QDs can also adsorb/desorb or oxidize/reduce onto/from the elec-trode surface depending on the applied external potentials. This quantification, previously researchedby Bard et. al95 can be read from the anodic and cathodic peaks present in the cyclic voltammetrygraph. Complementary target DNA strands with a biotin modification have been hybridized with ssDNAprobe on the electrode surface and avidin-modified QDs were tagged onto the hybridized DNA strandsby biotin-avidin chemistry and the extent of hybridization has been previously studied by both ECL and202.7. Supplementing the cartoonstripping voltammetry.96 QDs ECL has been previously employed in DNA biosensing.97–99 However,the surface interactions of QD bioconjugates (either specific or non-specific), the stability of these com-plex assemblies on electrodes or the potential induced distance dependent quenching mechanisms inproximity to electrodes have not been researched before.2.7 Supplementing the cartoonThe previous sections discussed so far in this chapter provides the theoretical background to under-stand the assembly processes of alkythiol and DNA SAM on Au electrodes, the conditions that mightbe favorable to carry out surface hybridization with QD-DNA bioconjugates and the available spectro-electrochemical characterization techniques. This section aims to supplement the understanding of thesystem by describing in brief the geometrical dimensions of the biomolecules (or the length scales) andhow they are effected by various parameters. This has relevance in estimating the required surfacedensities of the ssDNA probe on the electrode surface and subsequently rationally choosing severalinput parameters to optimize the surface modifications for a successful assembly of quantum dots onthe electrode surface. Concepts of persistence length, effective diameter and hydrodynamic radius hasbeen discussed next.Persistence length of a polymer is a characteristic property used to define it’s flexibility. A singlestranded DNA can be thought to be analogous to “spaghetti”. Tangents drawn at any two points cho-sen at random on this “spaghetti-like” ssDNA can be mathematically correlated to each other using theformula 〈cosΘ〉 = e −LP where θ is the angle between the tangents at the two points, L is the distancebetween the two chosen points and P is the persistence length. If the second point is chosen at a dis-tance 0 and eventually slowly increased to a distance L with respect to the the first point, 〈〉 representsthe average over all points from 0 −→ L along the contour of the ssDNA.100 As can be seen from theformula, the expectation value of the cosine of the angle between the tangents at two random pointson the ssDNA falls quickly as the distance between the two points increases.101 For small distancesbetween two points on the ssDNA, the segment behaves as a rigid rod and one can expect the twopoints to be pointing in the same direction (correlation closer to 1). However, for the system under study,considering a point on the ssDNA near the alkylthiol attached to the Au solid support versus a distantpoint near the fluorophore modification, one can expect the two points to be pointing in very differentdirections (as the correlation falls to zero).Persistence length of a charged polymer also depends on the salt concentration of the solution that212.7. Supplementing the cartoonthe polymer is present in.102 In general, the persistence length is longer for a smaller salt concentra-tion. The ssDNA has a negatively charged phosphate backbone while the surrounding solution used forassembly usually contains some amounts (usually mM concentration) of positive cations like K+ , Mg+2etc. The presence of cations shields the negative charge on the DNA backbone decreasing its persis-tence length.103,104 dsDNA on the other hand, is more rigid and “rod-like” when compared to ssDNAand hence has a higher persistence length. The presence of a higher salt concentration in solution (ora higher ionic strength electrolyte) also does not as significantly affect the dsDNA persistence length asit does for ssDNA persistence length.105 Some analytical techniques that can be used to measure thepersistence length include AFM and optical tweezers.106,107A negatively charged DNA in a given electrolyte can theoretically be modeled as a cylinder with acertain effective diameter.108The effective diameter is defined as the diameter of a neutral moleculethat mimics the conformation of a charged molecule after taking into account the various electrostaticinteractions in a given electrolyte.109 For an electrolyte with a higher salt concentration (or a higher ionicstrength), the negatively charged DNA backbone is shielded and hence, the effective diameter is smaller.DNA in general has a higher affinity for the divalent Mg+2 by a factor of around 100 when compared toit’s affinity for the monovalent Na+ .110 Since the negatively charged DNA backbone is more effectivelyscreened in the presence of Mg+2, the effective diameter of the DNA in 10mM MgCl2 is approximatelysimilar to the effective diameter in 200mMNaCl.109,111 The bending of DNA in an electrolyte as expectedcan also increase its effective diameter.112For quantum dots, it’s important to introduce the idea of a Stoke-Einstein hydrodynamic radius. Thehydrodynamic radius is defined as the equivalent radius of a hard sphere that can effectively accountfor all the stumbling/dancing around of the possibly hydrated molecule.113The solution used for assembly contained about 100mM NaCl and 50mMMgCl2while the DNA usedfor the assembly consisted of 30 base pairs. Under the assumption that the dsDNA is essentially arigid rod, one can estimate a length of approximately 10nm (30 base pairsX0.34nm/base pairs) and aneffective diameter of approximately 2nm.109 The length of ssDNA at high electrolyte concentration can beestimated to be approximately 2nm.103 A rough estimate of 1.5nm and 1nm can bemade for the MUDOLand MCH adsorbates (as the bond lengths for C, S, and O are approximately 120-154pm, 181-225pmand 143-214pm respectively). The geometric radius of the combined QD core-shell was approximately10nm while the hydrodynamic radius of the gluthathione capped QD in the solution can be estimatedto be close to 16nm. A His-tag peptide was used for bioconjugation of the QD with the target DNA andconsisted of approximately 32 amino acids with 6 amino acids penetrating the gluthathione layer. Hence,222.7. Supplementing the cartoonan estimate of 2nm (26 amino acidsX1nm/amino acids) can be made for the His-tag peptide (which alsodoes not stretch out completely in solution). The dimensions for the QD bioconjugates were sourcedfrom Dr. Algar’s lab. A cartoon depiction of the system with the respective estimated dimensions (notdrawn to scale) can be seen in Fig: 2.14Figure 2.14: Cartoon depiction of the system (QD-DNA biconjugates hybridized with ssDNA on Auelectrode) with the dimension estimates (Not drawn to scale).The net end to end (horizontal) diameter of the 10 DNA strands per QD and the 2 DNA strands perQD bioconjugate can hence be estimated as approximately 24nm (i.e. 4nm+16nm+4nm) and 16nm re-spectively. Assuming a hexagonal-packing (densest possible packing) of equal QD spheres monolayerin a regular fashion (packing density of 0.9069)114 and a 1:1 surface hybridization with every ssDNAprobe on the electrode surface, one can estimate the maximum required ssDNA probe surface cover-age as approximately 2*1011molecules/cm2- 4.5*1011molecules/cm2. 1 The coverage of ssDNA proberequired on the Au electrode surface is hence approximately 0.375% - 1% of maximum ssDNA probecoverages reported in literature. Rational choices of input parameters has been made in the subse-quent chapters with an attempt to create an optimized ssDNA probe surface for QD-DNA bioconjugatessurface hybridization.1calculated using 0.9069/(pi*( d2 )2*10−14) molecules/cm223Chapter 3Methods3.1 Glassware and Teflon cleaningA mixture of one part concentrated solution of H2SO4 (95-98% ACS Sigma Aldrich) and one part con-centrated solution of HNO3(68-70% ACS VRW Analytical) was prepared. Dirty glassware and Teflonpieces (after each experiment) were completely immersed in this prepared solution and the solution washeated for three-four hours and was allowed to cool down overnight. The glassware and Teflon pieceswere filled with / immersed in Milli-Q water for at least 12 hours before use. The glassware and theTeflon pieces were rinsed in Milli-Q water once again before the next set of experiment.3.2 Monocrystalline gold bead electrodesMonocrystalline gold bead electrode with many possible (h,k,l) crystallographic facets on the surfacehas been utilized as the solid support to build alkylthiol and DNA SAMs.3.2.1 PreparationApproximately 2cm of Au wire (1mm diameter, 99.999% purity Alfa Aesar) was cut and one end of thewire was subsequently immersed in the prepared aqua regia solution for 10 minutes to chemically etchthe surface. Aqua regia solution was freshly prepared by mixing one part concentrated solution of HNO3(68-70% ACS VRW Analytical) and three parts concentrated solution of HCl (37% ACS Sigma Aldrich).The etched gold was rinsed with ultra-pure Milli-Q water and melted in the flame of a butane torch toform a sphere roughly 2-3 mm in diameter. Slow melting of the sphere and subsequent slow coolingresulted in the formation of different orientations of crystallographic facets on the surface of the goldsphere. Re-melting was carried out till a monocrystalline surface was obtained. Two-three aqua regiaand re-melt cycles were further repeated. Slow melting up-to 3/4th of the sphere and subsequent slowcooling helped to preserve the monocrystalline nature while also forming bigger crystallographic facets243.2. Monocrystalline gold bead electrodeswith fewer defects. The monocrystalline gold bead was then held in the flame of the butane torch till itglowed orange-red (hard annealing) and was rinsed with Milli-Q water. This was repeated thrice.3.2.2 CleaningA three-electrode electrochemical cell with a platinum counter electrode, a standard calomel electrodeas the reference electrode and the monocrystalline gold bead as the working electrode was assembled.Approximately 30mL of 0.1M KOH (99.99% purity semi conductor grade Sigma Aldrich - in Milli-Q water),purged with Argon (>99.998% Praxair) was used as the electrolyte.Potential was cycled from -1.3V vs. SCE to 0.7V vs. SCE over multiple scans encompassing severaloxidation and reduction cycles and potentials negative enough to clean the surface without evolvinghydrogen on the electrode surface. This was followed by hard annealing and rinsing with Milli-Q water(X 3).115 The cyclic voltammetry scan of a Au electrode in KOH can be seen in the Fig: 3.1.Figure 3.1: Cyclic voltammetry scan of a Au electrode in 0.1M KOH (scan rate of 20mV/s) with distinct Auoxidation and reduction peaks. The inset shows an enlarged CV scan of the double layer with hydroxideadsorption and desorption peaks at approximately -0.2V.116To remove any organic contaminants from the electrode surface, either a RCA solution or a basicpiranha solution was used. Five parts Milli-Q water and one part NH4OH (30% ACS Plus Fischer Sci-entific) was mixed together and heated to approximately 70oC on a hot plate for 5 minutes. One part253.2. Monocrystalline gold bead electrodesH2O2(30% ACS Sigma Aldrich) was subsequently added and the resultant solution was allowed to re-act for about 2 minutes to prepare the RCA solution. After removing it off heat, the Au bead electrodewas immersed in the prepared solution for 15-20 minutes. The electrode was rinsed subsequently un-der flowing water and the process was repeated again using another batch of freshly prepared RCAsolution. A similar procedure was repeated using a basic piranha solution instead of a RCA solution inother cases. To prepare the basic piranha solution, three parts NH4OH and one part H2O2 was mixedtogether and the solution was heated to approximately 60oC to initiate the reaction process.117 An aquaregia etch and re-melt process (3.2.1) was repeated once after every four-six sets of experiments toremove any metallic impurities present in the electrode.115,117 A CV scan of a dirty/contaminated Auelectrode can be seen in Fig: 3.2.As a final step, several potential cycling scans from -0.35V vs. SCE to 1.5V vs. SCE were repeatedusing the above mentioned three-electrode electrochemical set-up but with ultra-pure 0.1M H2SO4(93-98% Fischer Scientific) as the electrolyte.115 The cyclic voltammetry scan of a clean Au electrode inH2SO4 can be seen in Fig: 3.3. This was followed by hard annealing and rinsing with Milli-Q water (X3).Figure 3.2: Cyclic voltammetry scan of a Au electrode in 0.1M H2SO4 (scan rate of 20mV/s) with distinctAu oxidation and reduction peaks. The inset shows additional set of peaks at 0.4V and 0.3V which couldprobably be due to the adsorption and desorption of the impurity in Au respectively.263.3. Differential capacitance measurement of Alkylthiol SAMsFigure 3.3: Cyclic voltammetry scan of a clean Au electrode in 0.1M H2SO4 (scan rate of 20mV/s) withdistinct Au oxidation and reduction peaks. The inset shows an enlarged CV scan of the double layerwith sulphate adsorption and desorption peaks at approximately 0V.1183.3 Differential capacitance measurement of Alkylthiol SAMsA three-electrode electrochemical cell with a platinum counter electrode, a standard calomel electrodeas the reference electrode and the monocrystalline gold bead with requisite alkylthiol mono layer asthe working electrode was assembled. Approximately 30mL of 50mM Phosphate buffer solution of pH8.2 (Na2HPO4.2H2O (99.5% Sigma Aldrich) & NaH2PO4(99% FisherBiotech) in Milli-Q water), purgedwith Argon (>99.998% Praxair) was used as the electrolyte. An autolab PG-STAT 12 potentiostat wasused to apply a decreasing potential step profile with a potential step of -50mV with each potential beingapplied for 15s (as shown in Fig: 3.4). A sinusoidal potential perturbation (200Hz, 4.5mV RMS) was alsoadded during the application of this potential profile using an EG&G 5208 lock-in amplifier to measurethe differential capacitance values as discussed in 2.2.1.273.4. DNAFigure 3.4: A decreasing potential step profile with a potential step of -50mV and each potential beingapplied for 15s.3.4 DNAA custom 30 base pair DNA oligonucleotide with the sequence 5’-CTG-TAT-TGA-GTT-GTA-TCG-TGT-GGT-GTA-TTT-3’ modified further with an AlexaFluor 488 fluorophore at the 3’ end (Fig: 3.5(a)) and ahexyl disulfide fuctionalized group at the 5’ end (Fig: 3.5(b)) was purchased from IDT. This was workedup in a Tris-NaCl buffer of pH 7.5 to a concentration of 25μM and stored in the freezer. Prior to use,small batches were reduced using Tris(2-carboxyethyl) phosphine hydrochloride (or TCEP-HCl) andwere subsequently purified using a column as described below.(a) (b)Figure 3.5: (a) AlexaFlur 488 fluorophore functionalization and (b) C6alkyl disulfide functionalization atthe 3’ and 5’ end respectively of the custom synthesized DNA oligonucleotide.283.5. DNA SAM on Au(111) facets3.4.1 DNA reductionThe disulfide fuctionalized protecting group at the 5’ end had to be reduced to a free thiol functionalizedgroup before further use. To do this, 32μ of the 25μM DNA previously worked up in Tris-NaCl wasreacted with 4μ of Tris-HCl buffer of pH 7.5 and 4μ of 0.1M TCEP-KOH (prepared by dissolving TCEP-HCl ½98% Sigma Aldrich in 100mM KOH to neutralize the acid) for a minimum of 3 hours at roomtemperature with periodic vortexing.3.4.2 DNA purification post reductionThe resultant reduced 20μM DNA-TCEP 40μ solution was further purified to remove the excess TCEP& the free protecting group from the solution. An illustra-Microspin G-50 column (GE Healthcare) wasfilled and drained at 3000 rpm for 30 seconds followed by washing with 300μ of Tris-HCl and drainingfurther at 3000 rpm for 1 minute each time (X3). The 20μM DNA-TCEP 40μ solution was added tothe column and the column was drained at 3000 rpm for 2 minutes. The resultant (approximately 10μMreduced and purified C6thiol modified DNA AF488 40μ solution) was stored in the freezer for up-to 2weeks. At times, lower DNA concentrations (approximately 3-5μM C6 thiol modified DNA AF488 40μsolution) were also prepared instead by following a similar procedure but using a smaller concentrationof initial DNA worked up in Tris NaCl.3.4.3 DNA quantification post reduction and purificationThe exact concentration of the reduced and purified DNA stock was determined by measuring an ab-sorbance spectra using an Ocean Optics USB2000 spectrometer with a deuterium lamp and recordingthe peak value at 260nm (ε=3.08 X 105M−1cm−1). The DNA stock was further diluted to the desiredconcentration using an immobilization buffer of pH 7.5. The immobolization buffer (or IB) was pre-pared as 10mM Tris Buffer (Tris(hydroxymethyl) aminomethane & Tris(hydroxymethyl) aminomethanehydrochloride ½99.9(bio-performance) Sigma Aldrich), 100mM NaCl (¾99.5(BioXtra) Sigma Aldrich)and 50mM MgCl2 (¾99.5(BioXtra) Sigma Aldrich).3.5 DNA SAM on Au(111) facetsDNA SAMs were prepared selectively on the (111) facets of Au monocrystalline bead electrodes by twodifferent approaches as explained below. A flowchart with the cartoon depiction of the two preparation293.5. DNA SAM on Au(111) facetsapproaches can be seen in Fig: 3.6. These approaches are a slight modification of a previous procedurereported in literature.46Figure 3.6: Flowchart depicting the two preparation approaches to prepare selectively modified ssDNAprobe surfaces.Approach 1 (Without intermediate MCH back-fill)The electrodes were initially immersed in 11-Mercapto-1-undecanol solutions (or MUDOL) to form astable SAM. Following this, a negative potential (referred henceforth as Etret) was applied to selectivelyreductively desorb some or all of the MUDOL from the (111) facets. The regions on the electrode surfacewhere MUDOL was desorbed is referred to as ’defective’ compared to parts covered with MUDOL.C6thiol modified DNA AF488 was allowed to specifically adsorb onto these defective regions (aka. back-fill) on the (111) facets.Approach 2 (With intermediate MCH back-fill)An alternate approach was also utilized. The defects formed on the (111) facets after Etret applica-tion to a MUDOL SAM were first back-filled with a shorter alkyl thiol, 6-Mercapto-1-hexanol (or MCH).C6thiol modified DNA AF488 was subsequently allowed to undergo a place exchange reaction with theMCH forming well spaced out DNA SAMs on the (111) facets. Certain experimental (input) parametersnamely- the Etret potentials applied, the DNA concentrations, and the electrode immersion times in303.5. DNA SAM on Au(111) facetsDNA solutions were varied across samples.3.5.1 MUDOL SAM100μ, 1mM MUDOL solutions (97% Sigma Aldrich) were prepared in EtOH (90% HPLC grade & de-natured: 5% MeOH & 5% C3H8O Sigma Aldrich) from a 25mM stock solution (prepared and stored inthe freezer for no longer than 2 months). Clean monocrystalline gold bead electrodes were immersedin the 100μ, 1mM MUDOL solutions for 90 minutes at room temperature. Thereafter, the electrodeswere rinsed with EtOH and stored in 100μ EtOH for 0-30 minutes before the next step of the experimentwas performed.3.5.2 Etret potential to selectively desorb MUDOL SAM off Au(111)sA three-electrode electrochemical set-up with a platinum counter electrode, a standard calomel elec-trode as the reference electrode and the monocrystalline gold bead with a MUDOL SAM as the workingelectrode was assembled. Approximately 30mL of a 50mM Phosphate buffer solution of pH 8.2, purgedwith Argon (>99.998% Praxair) was used as the electrolyte. An autolab PG-STAT 12 potentiostat wasused to measure the open-circuit potential (or OCP) which was also applied thereafter to the workingelectrode for 15s. This was followed by an application of an Etret potential for 400s. A sinusoidal poten-tial perturbation (200Hz, 4.5mV RMS) was applied during both the OCP & Etret potential applicationsusing an EG&G 5208 lock-in amplifier to enable measurement of the differential capacitance changeduring the course of the experiment. Etret potentials typically used were either -0.950V, -0.925V, -0.900V, -0.875V or OCP (control) for any given experiment. An inverted-bubbler mechanism was alsoused during the experiment to fill the electrochemical cell with an atmosphere of humidified argon. Thishelped to prevent drying of phosphate salts on any bare gold surfaces (formed as a result of Etret ap-plication) as the working electrode was disconnected from the electrochemical set-up. The electrodeswere then rinsed thoroughly with Milli-Q water. Chapter 4 discusses the relevant electrochemical resultsfor these layers.3.5.3 MCH SAM100μ, 1mM MCH solutions (99% Sigma Aldrich) were prepared in MeOH (HPLC grade Fisher Chem-ical) from a 50mM stock solution (prepared and stored in the freezer for no longer than 2 months).Monocrystalline gold bead electrodes were immersed in the 100μ, 1mM MCH solutions for 90 minutes313.6. QD-DNA bioconjugatesat room temperature. Thereafter, the electrodes were rinsed with MeOH and Milli-Q water before thenext step of the experiment was performed.3.5.4 DNA SAMMonocrystalline gold bead electrodes were immersed in 40μ solutions of the desired concentrations ofpreviously reduced and purified C6thiol modified DNA AF488 in IB for fixed amounts of time. After DNAassembly, the electrodes were immersed in a solution of IB overnight to remove any DNA that mightbe bound by non-specific interactions to the gold surface. The DNA concentrations typically used wereeither 1μM, 0.4μM, 0.2μM or 0.1μM while immersion times in DNA solutions were either 10minutes,6hours or 24hours for any given experiment.3.6 QD-DNA bioconjugatesDifferent QD-DNA bioconjugates were sourced from Dr. Russ Algar’s lab at UBC (prepared by Hyungki(David) Kim). A His-tag peptide (CSGPPPPPGSGHHHHHH) was used in the bioconjugation step. Thesix Histidines at one end of this peptide were inserted into the Gluthathione (or GSH, Fig: 3.7) cappingof the CdSe/ZnS (core/shell) Quantum dots. Sulphur of the cysteine group at the other end of thispeptide was allowed to form a disulfide bond with the the sulphur at the 5’ end of the complementaryDNA strand (5’-HS-C6-AAA-TAC-ACC-ACA-CGA-TAC- AAC-TCA-ATA-CAG-3’).119,120 0.2 DNA strandsper QD, 2 DNA strands per QD, 10 DNA strands per QD and 50 DNA strands per QD bioconjugateswere synthesized by varying the ratio of DNA used per QD. The probability of precisely modifying theQDs with DNA is expected to follow a Poisson distribution. Gel-electrophoresis experiment was runon the four different QD-DNA bioconjugates and unmodified QDs. The results are shown in Fig: 3.8(data supplied by HD Kim). Addition of negatively charged DNA is expected to impart some increasedelectrophoretic mobility to the QD-DNA bioconjugates in comparison to the unmodified QDs. However,as the size of the QD-DNA bioconjugate is increased, a decrease in mobility can be expected.119 A clearevidence of bioconjugate modification is realized for a higher ratio of DNA per QD in Fig: 3.8.Figure 3.7: Gluthathione structure.323.7. QD-DNA bioconjugates hybridization with ssDNA SAM on Au(111)sFigure 3.8: Gel electrophoresis data (1% TBE Agarose,100 V, 30 minutes in 1X pH 8.3 TBE buffer) for:Well 1: QD Only (10 uL), Well 2: 0.2 DNA strands per QD, Well 3: 2 DNA strands per QD, Well 4: 10DNA strands per QD, Well 5: 50 DNA strands per QD, Well 6: QD Only (~3 uL).3.7 QD-DNA bioconjugates hybridization with ssDNA SAM onAu(111)s3.7.1 Control samplesControl experiments were performed so as to studyQD-DNA bioconjugates and unmodifiedGSH cappedQDs interactions with alkylthiol SAMs (MUDOL and MCH) and defects on the electrode surface. For thecontrol experiments, three different types of SAMs were initially prepared on different monocrystallineAu bead electrodes as detailed below:(1) A MUDOL SAM was prepared on the electrode (Fig: 3.9)Figure 3.9: Schematic of the first control experiment to study QD-DNA bioconjugates interactions withMUDOL SAM.(2) A MUDOL SAM was prepared on the electrode and subsequently an Etret potential of -0.925Vwas applied to selectively desorb the MUDOL SAM off the Au(111) facets and the defects created wereback filled with MCH (Fig: 3.10)333.7. QD-DNA bioconjugates hybridization with ssDNA SAM on Au(111)sFigure 3.10: Schematic of the second control experiment to study QD-DNA bioconjugates interactionswith MCH SAM on the (111) facets.(3) A MUDOL SAM was prepared on the electrode and an Etret potential equivalent to the opencircuit potential (OCP) was applied. The defects created (if any) were back-filled by immersing theelectrode in a MCH solution first and subsequently in a 40μ, 0.2μM C6thiol modified DNA AF488solution for 6 hours. Applying a potential equivalent to OCP is analogous to not applying any potentialat all and hence, this serves as a control for the Etret application step.3.7.2 Preliminary hybridization samplesThe electrodes were initially modified by preparing a MUDOL SAM, applying an Etret potential of -0.925V, back filling the defects created on the (111) facets with MCH and subsequently carrying out aplace exchange reaction with 40μ, 0.4μM or 0.2μM C6thiol modified DNA AF488 for 6hours (Fig: 3.11).Figure 3.11: Schematic of the preliminary hybridization experiments to study specific adsorption of QD-DNA bioconjugates.TheQD-DNA bioconjugates sourced fromDr. Russ Algar’s lab were initially present in a TBS (100mMTris(hydroxymethyl) aminomethane, 150mM NaCl) buffer of pH:7.5. Initial hybridization experimentswere attempted with the QD-DNA bioconjugates in TBS at room temperature. Later hybridization exper-iments were however attempted with the QD-DNA bioconjugates being diluted to the IB solutions and at343.8. Fluorescence microscopy spectro-electrochemical set-upan elevated temperature of 45oC in order to improve the hybridization efficiencies.Since the 0.2 DNA strands per QD bioconjugates is expected on average to contain a majority ofQDs with no DNA strands attached to them and a minority of QDs with only one DNA strand attachedto them, this solution has been primarily used to study non-specific adsorptions of QDs in the controlexperiments. For the preliminary hybridization experiments, the 2 DNA strands per QD and 10 DNAstrands per QD solutions have been used since they contain a majority of QDs that have been modifiedby DNA strands.3.7.3 Hybridization at room temperatureFollowing the initial SAM preparations (3.7.1 and 3.7.2), the electrodes were rinsed with TBS. Theywere subsequently immersed in 40μ QD-DNA bioconjugates solution (in TBS) at room temperature fora time between 20-24 hours. QD-DNA bioconjugates solution of concentrations either 2.5nM, 5nM or10nM was used for the hybridization step. In general, to remove any QDs/ QD-DNA bioconjugates thatmight be bound by non-specific interactions to the surface, the electrodes were immersed in TBS for3-5 hours.3.7.4 Hybridization at 45oCFollowing the initial SAM preparations (3.7.1 and 3.7.2), the electrodes were rinsed with IB. They weresubsequently immersed in 40μ QD-DNA bioconjugates solution (in IB) at a temperature of 45oC for2 hours. QD-DNA bioconjugates solution of concentrations either 5nM or 10nM was used for the hy-bridization step. The electrodes were then immersed in IB overnight for more than 20 hours to removeany QDs/ QD-DNA bioconjugates that might be bound by non-specific interactions to the surface.3.8 Fluorescence microscopy spectro-electrochemical set-upA spectro-electrochemical cell (similar to the three electrode electrochemical cell) used had a 250μmthick optical window at the base enabling the use of an inverted fluorescence microscope to capture im-ages of the working electrode while also performing electrochemical measurements. A three-electrodespectroelectrochemical cell with a platinum counter electrode, a standard calomel electrode as the refer-ence electrode and the monocrystalline gold bead with the requisite self-assembled mono layers as theworking electrode was assembled. A small volume (approximately 5mL) of Tris-KNO3(imaging buffer) of353.8. Fluorescence microscopy spectro-electrochemical set-uppH of 7.5, purged with Argon (>99.998%, Praxair) was used as the electrolyte. The imaging buffer wasprepared as 10mM Tris (Tris(hydroxymethyl) aminomethane, ½99.9(bio-performance), Sigma Aldrich)and 10mM KNO3 (¾99.5(BioXtra), Sigma Aldrich). The required potential profiles were applied to theworking electrode using a HEKA potentiostat. An EG&G 5210 lock-in amplifier was used to add a sinu-soidal perturbation to enable recording of the differential capacitance values.An inverted fluorescence microscope (Olympus IX70) equipped with a 5X magnification objective, aX-Cite exacte fluorescence light source (Hg arc lamp), & a Photometrics Evolve CCD camera (imagingsize 512 X 512 pixels) was used to record the fluorescence images. Appropriate filter cube sets to detectAF488 emission i.e. GF filter cube set (Excitation: 450-490nm, Dichroic: 495nm, Emission: 500-550nm)and to detect quantum-dots emission i.e. DIM filter cube set (Excitation: 450-490nm, Dichroic: 585nm,Emission: 600-680nm) were also employed. A schematic of the excitation and emission filter wavelengthband of the GF and DIM filter cube sets can be seen in Fig: 3.12. Standard imaging conditions had thelamp intensity set at 30%, the exposure time set at either 3s or 0.5s and the EM (electron multiplying)gain set at a value of 300.The next section focuses on the potential profiles that were applied to the working electrode during thefluorescence microscopy spectro-electrochemical measurements and the data collection stage. Finally,the data and image analysis steps have been described.Figure 3.12: Absorption and emission spectra of AF 488 and QD and the Chroma filters used as part ofthe spectroscopic instrumentation for their detection. QD spectra was measured using an Agilent CaryEclipse fluorimeter. AF488 spectra was generated from the manufacturer’s website. 121363.9. Potential profiles3.9 Potential profilesTwo different types of potential profiles namely: potential modulation profiles & reductive potential stepprofiles were applied to the modified electrode surfaces during the fluorescence microscopy spectro-electrochemical measurements. For each modified electrode surface characterization, in general, thesequence of :(1) at-least three potential modulation profiles (2) at-least three reductive potential stepprofiles and (3) at-least two potential modulation profiles (again) were applied to the electrodes and oneor more fluorescence images were taken at each potential of these potential profiles.3.9.1 Potential modulation profilesPotential modulation profiles were used to study potential induced re-orientation of DNA on the electrodesurface and to irreversibly remove any physiosorbed (or non-specifically adsorbed) target QD-DNA bio-conjugates from the electrode surface.In the potential modulation profile, potentials were changed in steps as shown in Fig: 3.13. It startsat a base potential of +350mV with each step potential differing by -25mV until a final step potentialof -400mV was reached. The potential was also stepped back to the base potential after each steppotential. The base as well as the step potentials were applied for 3s each and one fluorescence imagecorresponding to each potential was captured by the camera.Figure 3.13: Potential modulation profile. ’+’ corresponds to a fluorescence image captured by thecamera.373.9. Potential profilesThe potential modulation profile was slightly modified to improve the characterization of the QD layers(to account for the much slower response of the bulky QD tagged ds-DNA). One such modification ofthe potential modulation profile can be seen in Fig: 3.14. It starts at a base potential of +350mV witheach step potential differing by -100mV until a final step potential of -450mV was reached. The baseas well as the step potentials were applied for approximately 15s each and five fluorescence imagescorresponding to each potential (in 3s intervals) were captured by the camera.Figure 3.14: Modified potential modulation profile-1. ’+’ corresponds to a fluorescence image capturedby the camera.Another version of the modified potential modulation profile can be seen in Fig: 3.15. It starts ata base potential of +300mV with each step potential differing by -100mV until a final step potential of-300mV was reached. The base as well as the step potentials were applied for approximately 30s eachand ten fluorescence images corresponding to each potential (in 3s intervals) were captured by thecamera.383.9. Potential profilesFigure 3.15: Modified potential modulation profile-2. ’+’ corresponds to a fluorescence image capturedby the camera.3.9.2 Reductive potential step profilesThe reductive potential step profiles helped to reductively remove chemisorbed (specifically adsorbed)SAMs from the electrode surface. It starts at a potential of 0V, with each step potential differing by -25mVuntil a final step potential of -1.2V was reached. The potentials were applied for approximately 0.5seach and one fluorescence image corresponding to each potential was captured by the camera. Thereductive potential step profile was slightly modified to improve the characterization of the QD layers.In this, the potentials were applied for approximately 1s each and two fluorescence images (in 0.5sintervals) corresponding to each potential were captured by the camera. The reductive potential stepprofile and the modified reductive potential step profile can be seen in Fig: 3.16.Potential modulation profile was applied again to the bare electrode after the SAM was reductivelyremoved using reductive potential step profile. The fluorescence images collected during this potentialmodulation profile were subsequently used to correct for any background reflected/ scattered or straylight. This is explained in detail in the next section.393.10. Image analysisFigure 3.16: Reductive potential step profiles correlating the fluorescence image numbers and the po-tentials applied to the working electrode.3.10 Image analysisThe collected fluorescence images were analyzed using an open source image processing package,Fiji. The outline of the generic image analysis used is discussed below.3.10.1 Background correction procedureThe procedure to correct for reflected/ scattered or stray light is discussed. After reductively removingthe SAMs using reductive potential step profiles, the fluorescence images of the bare gold electrodewere collected using the potential modulation profile. These fluorescence images were averaged andsubsequently subtracted from the fluorescence images of the SAMs modified electrode which werecollected using the potential modulation profile before reductive removal of the SAMs. This was used togenerate background corrected fluorescence potential modulation images. An example of this procedurefor an image collected at step potential of -0.4V can be seen in Fig: 3.17.403.10. Image analysisFigure 3.17: Background correction steps for a fluorescence image taken at a step potential of -0.4V.In a similar fashion, the fluorescence images of the bare gold electrode collected using the reductivepotential step profile were also averaged and subsequently subtracted from the fluorescence images ofthe SAMs modified electrode collected using the first reductive potential step profile. This was used togenerate background corrected reductive potential step images.Background correction was performed for data collected with both GF filter cube set (used for AF488detection) and the DIM filter cube set (used for QD detection). The background corrected images werefurther processed using a look-up table to identify between these two data sets in this thesis. A “fire”look-up table was used for images collected with a GF filter cube set while a “red” look-up table wasused for images collected with the DIM filter cube set. Finally, appropriate scale bar and a calibrationbar were added to these background corrected images.3.10.2 Normalized ΔFS calculation procedureThis calculation was performed to draw conclusions about the surface density of the probe ssDNA on theelectrode surface (results have been discussed and explained in Chapter 5). The fluorescence imagesof the SAMs modified electrode surface collected using the potential modulation profile (similar to Fig:3.13) were split into two groups. The fluorescence images which were collected when the base potentials(’B’) were applied were sequentially sorted in the first group while the fluorescence images which werecollected when the step potentials (’S’) were applied were sequentially sorted in the second group. Adifference of the ’S’ group with respect to the B ’group’ was calculated to generate an image-stack withthe appropriate ΔFS images for each respective step potential i.e.ΔFS = FS− FBAn example of ΔFS calculation for three different step potentials (0.35V, 0V, and -0.40V) can be413.10. Image analysisseen in Fig: 3.18. The fluorescence image from the base potential value of +0.35V was used for thecalculation.Figure 3.18: ΔFS calculation steps for step potentials (S)=+0.35V, 0V and -0.40V with base potentials(B)=+0.35V.The ΔFS image stack was further divided by the the background corrected fluorescence image at themost negative step potential (represented as max.FS,b.c) to generate the normalized ΔFS image-stack.NormzedΔFS =ΔFSm.FS,b.c=(FS− FB)m.FS,b.cAn example of FS−FBm.FS,b.c calculation for three different step potentials (0.35V, 0V, and -0.40V) canbe seen in Fig: 3.19. The fluorescence image at the most negative step potential of -0.40V was usedfor the calculation.423.10. Image analysisFigure 3.19: Normalized ΔFS calculation steps for step potentials (S) =+0.35V, 0V and -0.40V with themost negative step potential=-0.40V.Appropriate scale bar and a calibration bar [0-1] were added to the images in the normalized ΔFSstack. A combination of despeckle filter and Gaussian blur filter of different radii were also used at differ-ent stages of calculation to optimize image processing (Fig: 3.20). More detailed and descriptive imageoptimization steps can be found in the Appendix 1. The white pixels in Fig: 3.20 are correlated to regionsof very low fluorescence signals due to the absence of DNA modification in these regions. The normal-ization approach used will make these pixels have a large positive value (which shows up as a whitepixel when using this look up table). Since we are interested in the modulation of the adsorbed DNA, itis clear that these regions, which do not have adsorbed DNA, do not contain any useful information.433.10. Image analysisFigure 3.20: Optimization of normalized ΔFS.3.10.3 Correcting for spectral bleed-throughThe emission spectra for AF488 has a tail that extends into the emission filter wavelength band used todetect the luminescence from the QDs (see Fig: 3.12). A surface with only ssDNA AF488 without QDmodification (prepared by a fellow lab mate, Tianxao Ma) was imaged using both the GF as well as theDIM filter cube set. A small amount of signal was detected for this surface even with the DIM filter cubeset due to the spectral bleed-through of AF488 emission. An example can be seen in Fig: 3.21. Thisspectral bleed-through will be connected when analyzing the QD fluorescence images.Initially, background correction was performed on the images collected with the GF and the DIM filtercube sets. Average fluorescence intensity within a ROI (“region of interest”) was calculated for the DIMand GF background corrected fluorescence images. The former was divided by the later to calculate thespectral bleed-through factor. A similar analysis was performed with multiple different ROIs to calculatethe average and the standard deviation of the spectral bleed-through factor. This calculation has beenshown in Appendix 2.443.10. Image analysisFigure 3.21: Fluorescence images of a surface modified with ssDNA AF488 without QD modificationcaptured using GF and DIM filter cube sets.45Chapter 4Electrochemical characterization ofalkylthiol SAMsThis chapter focuses on rationally determining the negative treatment potentials (aka. Etret) neededto be applied to the MUDOL coated monocrystalline Au bead electrodes to partially reductively desorbMUDOLSAMoff the (111) facets.This has been achieved by directly comparing the electrochemical layerstability (SAM reductive desorption response to an applied decreasing potential step profile) for MUDOLand MUDA SAMs (MUDA i.e. 11-Mercaptoundecanoic acid) as such an approach has been previouslyreported for MUDA SAMs in literature.46 Specifically in this chapter, the area normalized double layercapacitance has been measured to draw inferences about the alkythiol SAM reproducibility, stability andsurface coverages.4.1 MUDOL and MUDA SAMsTo prepare the MUDOL SAMs and the MUDA SAMs, the monocrystalline Au bead electrodes wereimmersed in either 100μ, 1mM MUDOL solutions or 100μ, 1mM MUDA solutions (in EtOH) at roomtemperature for either 30 minutes, 90 minutes or 240 minutes. A minimum of 3 MUDOL and 3 MUDAcoated electrode samples for each of the immersion times were prepared and studied. These layerswere tested for (1) reproducibility by comparing the capacitance values at 0V. Theses layers were furthertested for (2) electrochemical layer stability by measuring the capacitance values as a function of adecreasing potential step profile (Fig: 3.4).4.1.1 Alkylthiol SAM reproducibilityThe capacitance values at 0V measured for MUDOL and MUDA SAM coated electrodes for differentimmersion times can be seen in Fig: 4.1.464.1. MUDOL and MUDA SAMsFigure 4.1: Capacitance values (measured at 0V) for MUDOL and MUDA coated electrodes for threedifferent immersion times (30 minutes, 90minutes, 240minutes).For an alkylthiol SAM coated electrode, a lower measured capacitance value indicates a higher sur-face coverage (as discussed in 2.5.2). As evident in Fig: 4.1, a higher surface coverage of alkylthiolSAM is obtained for longer immersion times for both MUDOL & the MUDA SAMs. The percent error inthe capacitance values measured for the MUDOL SAMs across different samples for the different immer-sion times (30 minutes, 90 minutes and 240 minutes) was approximately 13%, 12% & 6% respectivelywhile that for the MUDA SAMs was approximately 8%, 4% and 2.5% respectively. This corroboratesthe idea that a higher immersion time results in a more reproducible layer. Small differences in the un-derlying surface features across different electrodes can also influence the SAM packing contributingto measured variance. Alternately, small changes to the area of the working electrode immersed in theelectrolyte (due to wetting/evaporation) during the data collection time might influence the calculatedcapacitance values.Previous contact angle measurements reported in literature confirm that for the -OH functional-ized alkylthiols, the end chain undergoes time-dependent re-organization, annealing the SAM layer.122Hence, the immersion time is expected to influence the packing of the SAM monolayer (and hence themeasured capacitance values) for the MUDOL SAMs. From the values in Fig: 4.1, it is evident that forthe different sets of immersion times, larger differences in the capacitance values were observed forMUDOL SAMs when compared to similar sets of immersion times for MUDA SAMs. A MUDOL SAMprepared using 90 minutes immersion time was similar to a MUDA SAM prepared using 240 minutesimmersion time. Considering multiple factors like: time for the SAM formation, a high SAM surfacecoverage and reproducibility, a 90 minutes MUDOL immersion time was found to be favorable.474.1. MUDOL and MUDA SAMs4.1.2 Electrochemical alkythiol SAM stabilityAn attempt has been made here to compare the response of MUDOL vs. MUDA SAM coated electrodes(for different immersion times of 30 minutes, 90minutes and 240minutes) to a decreasing potential stepprofile to investigate the differences in MUDOL vs. MUDA reductive desorption characteristics. To doso, a decreasing potential step profile similar to Fig:3.4 was applied and the changes in the capacitancevalues were plotted. These results (measured in an alkaline phosphate buffer electrolyte of approximatepH:8.2) are shown in Fig: 4.2.Figure 4.2: Capacitance values (uF/cm2) plotted as a function of a decreasing potential step profile forMUDA and MUDOL coated electrodes for three different immersion times (30 minutes, 90 minutes &240 minutes).As previously explained in 2.5.2, the double layer capacitance for a bare gold electrode is larger whencompared to a partially alkylthiol coated electrode which in turn is larger when compared to a completelyalkylthiol coated electrode surface. For the different immersion times, both MUDOL and MUDA SAMscapacitance values starts somewhere between 1.5-2.5uF/cm2 around 0V. As the potential is slowlydecreased to more negative values, the capacitance starts to increase in magnitude suggesting thatsome of the alkylthiol is reductively desorbed from the electrode surface.484.2. Selective reductive desorption of MUDOL SAM from the (111) facetsAs previously discussed in 2.5.1, Au(111) has the least negative alkylthiol reductive desorption po-tential value compared to other crystallographic facets. Thus, the shoulder (indicated by black arrows)in the alkylthiol desorption curves in Fig: 4.2, corresponds to the desorption from the (111) facets. Fromthe figure, it is evident that the shoulder in the reductive desorption curve is broader (spread over a widerpotential range of 0.325V i.e. from -0.875V to -1.2V ) for MUDOL SAMs while it is narrower (spread overa smaller potential range of 0.075V i.e. from -0.775V to -0.850V) for MUDA SAMs.The differences in the shape of the desorption curves could be in part due to differences in packinginteractions of MUDA and MUDOL layers on the (111) facets and also due to differences in how theterminal functionalizations (ω: -OH and -COOH ) affect interactions at the SAM-electrolyte interface.The terminal hydrophilic functionalizations of both MUDOL and MUDA SAMs do not effectively blockcations from the electrolyte from entering in between the SAM chains especially in the early stages ofthe desorption process. The blocking characteristics is poorer for MUDA SAMs than MUDOL SAMs onaccount of higher hydrophilicity of ω: -COOH terminal group than of ω: -OH terminal group. This tendsto stabilize the the thiolates (excessive unbalanced negative charges) formed after reductive desorptionof alkylthiols thus favoring a faster desorption kinetics.123As can be seen from the Fig: 4.2, the potential corresponding to the onset of reductive desorptionis comparatively more negative for MUDOL SAMs than for MUDA SAMs (-0.875V/SCE as opposed to-0.775V/SCE). This is probably due to inter-molecular repulsion between the deprotanated carboxylicacid groups (expected in a pH~8.2 electrolyte67) of MUDA which decreases SAM stability and promotesan earlier desorption in response to less negative potentials.124To account for these differences in reductive desorption characteristics, more negative treatmentpotentials (aka. Etret) were chosen and applied for a longer time to selectively reductively desorbMUDOL from the (111) facets than the values reported in literature46 for MUDA SAMs.4.2 Selective reductive desorption of MUDOL SAM from the (111)facetsIt is clear that the reductive desorption forMUDOLSAMs starts upon application of potential of -0.875V/SCE.The potential value corresponding to the onset of reductive desorption is correlated to the underlyingsurface energetics of the different crystallographic facets. This value is least negative for the (111)crystallographic facets which is reasonably separated from the values corresponding to even other low494.2. Selective reductive desorption of MUDOL SAM from the (111) facetsindex crystallographic facets like the (100)s or the (110)s.125 Hence, a few different Etret potentialswere chosen starting from -0.875V (onset of desorption potential) with more negative potentials differingby at least 25mV each to partially selectively reductively desorb MUDOL SAM from the (111) facets.The fraction of MUDOL reductively removed from the (111) facets is expected to be a function of thevalue and the time of application of the different Etret potentials.Capacitance change as a function of the time of application of Etret potential on MUDOL coatedelectrodes (prepared by immersion for 90 minutes) can be seen in Fig: 4.3.Figure 4.3: Capacitance change as a function of the time of application of different Etret potentials onMUDOL coated electrodes (prepared by immersion for 90 minutes).It is apparent that more negative Etret potentials applied for the same amount of time results inmeasured capacitance values that are higher than those for less negative Etret potentials. Therefore,more defects are created in the MUDOL SAMs per unit time for more negative Etret potentials. In otherwords, the kinetics or the rate of reductive desorption of MUDOL SAM is faster for more negative Etretpotentials. The shape of the reductive desorption curve is steeper initially and flattens out with time.This is consistent with the idea that desorption starts off as multiple small nucleation patches initiallyand subsequently as slow outward growth of these patches (as discussed in 2.5.1).Reproducibility of these results (Fig: 4.4) is affected by several experimental factors including differ-ences in the fractional areas of the (111)s with respect to other crystallographic facets across different504.2. Selective reductive desorption of MUDOL SAM from the (111) facetselectrodes, slight differences in the electrode surface features resulting in differences in the initial SAMstability, small changes in the pH of the electrolyte or due to the wetting of the bead electrode during thecourse of the measurement.Figure 4.4: (a) Capacitance change as a function of the time of application of different Etret potentials(multiple experiment replicates to test reproducibility of results in Fig: 4.3) (b) Average capacitancevalues measured after Etret potential application for 400s across replicates.As previously discussed in 2.5.2, the fraction of the defects created as a result of Etret application(due to removal of the MUDOL SAM) can be expressed as514.2. Selective reductive desorption of MUDOL SAM from the (111) facets(1− θ) = Cθ=1−CCθ=1−Cθ=0where θ and C are the fractional surface coverage and the capacitance value of MUDOL coatedelectrode post Etret application. Cθ=1 and Cθ=0 are the capacitance values of a completely MUDOLcoated electrode (1.910 μF/cm2 for 90 minutes immersion) and a completely clean, bare electrode(18μF/cm2).Thus from the formula, we can calculate that applying Etret potentials of -0.950V, -0.925V, -0.900Vor -0.875V (for 400s) on a MUDOL coated Au bead electrode (prepared by immersion for 90 minutes)creates defects on about 16%, 12%, 7% and 3% of the total surface area of the electrode. This is shownin Fig: 4.5.Figure 4.5: Fraction of MUDOL SAM desorbed as a function of Etret potential (applied for 400s).52Chapter 5Spectro-electrochemicalcharacterization of selectivelymodified alkylthiol-DNA SAMsIn Chapter 4, it was found that applying different negative treatment potentials (aka. Etret) to MUDOLSAM coated monocrystalline Au bead electrodes can create defects in the SAM on the (111) facets. Twodifferent approaches for preparing ssDNA probes on the electrodes have been employed in this work(previously detailed in 3.5) - by (1) back-filling the defects with C6thiol-modified DNA AF488 or by (2)back-filling the defects with MCH and subsequently displacing some of the MCH with C6thiol-modifiedDNA AF488 (place-exchange reaction). An in-situ inverted fluorescence microscope coupled with anelectrochemical set-up was used to characterize the SAMs on the electrodes by capturing fluorescenceimages of the electrode surfaces while also applying certain potential profiles to the electrodes (refer3.8 and 3.9). The characterization was performed for multiple different SAMs formed by varying threeinput parameters like the Etret potential value, the DNA concentration and the immersion time in theDNA solution. The results have been discussed in this chapter with the goal of rationally identifying thepreparation conditions to create an ideal ssDNA probe layer on the electrode surface that can be furtherused to carry out successful hybridization with the target QD-DNA bioconjugates.5.1 Approach1: Without intermediate MCH back-fillTwo of the three experimental factors were varied to form the SAMs using this approach. The Etretpotentials used for sample preparations were either -0.875V or -0.900V or -0.925V or -0.950V while theDNA deposition time was chosen as either 10 minutes or 24 hours. The DNA concentration was keptconstant at 1μM. The collected spectro-electrochemical data was analyzed by two different methods by535.1. Approach1: Without intermediate MCH back-fill- (1) Comparing the background corrected fluorescence intensities (at -0.4V) and by - (2) Comparingthe normalized ΔFS calculations. These methods were previously discussed in 3.10.1 and 3.10.2.A potential of -0.4V is not negative enough to reductively desorb the chemically adsorbed C6thiol-modified DNA AF488. Thus, the ssDNA SAM is expected to be stable at this potential for extendedperiods of time. As was previously discussed in 2.5.3, applying a negative potential to the electrodeelectrostatically repels the negatively charged DNA (backbone) away from the electrode surface to a“standing” state.52 As discussed in 2.3.1.1, a higher fluorescence signal can also be expected the fartherthe fluorophore stands from the electrode surface. Hence, background corrected fluorescence imagesof the electrode surfaces at -0.4V was used for comparison.5.1.1 Background corrected fluorescence intensities (at -0.4V)Analyzing these electrode surfaces after only modifying the (111) facets with DNA should result in flu-orescence signals only from these regions. As expected, the fluorescence images from the differentsamples showed fluorescence intensities only on the (111) facets. In these images, since more thanone (111) facet is visible, an average and standard deviation can be calculated. The average (111)fluorescence intensity (counts) and the standard deviation (error bar) for different samples as a functionof Etret potential is shown in Fig: 5.1.For the different Etret potentials, average (111) fluorescence intensities for the samples that wereprepared with a 10 minutes DNA immersion time have been joined together using a curve. This curveactually depicts the functional dependence of ssDNA probe surface coverage with respect to the Etretused because the magnitude of fluorescence intensities is directly proportional to the amount of fluo-rophore tagged DNA on the (111) facets. One other sample was prepared using an Etret potential of-0.875V and a 24 hours DNA immersion time.545.1. Approach1: Without intermediate MCH back-fillFigure 5.1: Average (111) fluorescence intensities (counts) as a function of Etret potentials for surfacesprepared using Approach1: Without intermediate MCH back-fill.The (111) ssDNA surface coverage was found to be higher for surfaces prepared with more negativeEtret potentials since this creates more defects which are then subsequently labeled by the DNA. Abigger error bar was found for the samples prepared with less negative Etret potentials indicating thatthese potentials do not not modify all the (111) facets in a consistent fashion. This could be due tovariation in the sample preparation of the facets which may influence the kinetics of thiol desorption. Asource of error that was unaccounted for in these measurements relates to the orientation angle of thedifferent (111) facets on the spherical bead electrodes with respect to the camera (farther from- or closerto-) which could introduce slight errors in the measured fluorescence signals.From the figure, it is also noticeable that for a surface prepared with an Etret potential of -0.875V,the number of defects (open sites for DNA adsorption) is probably too low for any appreciable DNAassembly for a DNA immersion time of 10 minutes. However, a DNA immersion time of 24 hours resultsin (111) ssDNA probe surface coverage comparable to a surface prepared with an Etret potential of-0.900V and a DNA immersion time of 10minutes. This is consistent with the idea that a higher assemblytime or a higher number of open adsorption sites results in a higher probability of adsorption.Furthermore, for all images, no fluorescence signals were observed from any other regions indicatingthat there is virtually no non-specific adsorption of DNA on other parts of the electrode or any appreciable555.1. Approach1: Without intermediate MCH back-fillplace-exchange reaction between DNA (C6thiol-terminated) and MUDOL(C11thiol-terminated).5.1.2 Normalized ΔFS calculationsA potential modulation profile identical to Fig: 3.4 was applied to the modified electrode surfaces in whichfluorescence images corresponding to each potential (base potentials as well as the step potentials)was captured by the camera. As already discussed previously in 3.10.2, the normalized fluorescencemodulation response for a step potential S was calculated as (FS- F+0.35V )/F−0.40V .Increasing magnitudes of negative step potentials when applied to the electrodes pushed the DNAstrands to a position farther away from the electrode surface producing images with higher (111) flu-orescence signals. However, when the base potentials (+0.35Vs) were applied to the electrodes, themeasured fluorescence signals were different depending on the local ssDNA surface packing densities.The extent of inter-DNA repulsion with the neighbouring DNA strands pushed the fluorophore taggedDNA strands away from the electrode surface counteracting the attraction to the electrode surface.Hence, at base potentials, higher (111) fluorescence signals result for higher ssDNA surface packingdensities. However, a high fluorescence intensity measured at the positive base potential may be a con-sequence of a higher surface coverage of DNA. To enable comparison of different regions and differentexperiments, a normalized modulation response was calculated. This was done by dividing the fluores-cence intensities measured at any applied potential by that measured at -0.4V (which is correlated tossDNA surface coverage). This will scale or normalize the fluorescence signal detected during applica-tion of potentials between +0.35V and -0.40V. The normalized fluorescence modulation (F S- F+0.35V)/F−0.40V response should be smaller for densely packed ssDNA surfaces and closer to 1 for packingdensities that are small enough to allow the DNA to move freely.The normalized ΔFS images i.e. (FS-F+0.35V )/F−0.40V images for three different step potentials (S=-0.40V, 0V and +0.35V) for a surface prepared with an Etret potential of -0.925V and a 10 minutesDNA immersion time can be seen in Fig: 5.2. The exact image analysis steps as discussed in 3.10.2were followed.565.1. Approach1: Without intermediate MCH back-fillFigure 5.2: (FS- F+0.35V )/F−0.40V images for three different step potentials (S=-0.40V, 0V and +0.35V)for a surface prepared using Approach1: Without intermediate MCH back-fill with an Etret potential of-0.925V and 10 minutes DNA immersion time.A similar image analysis was performed for all modified electrode surfaces discussed in the previoussub-section (prepared using Approach1: Without intermediate MCH back-fill) at all step potentials. Forthese images, the average (111) normalized fluorescence modulation response as a function of theapplied step potentials (S) was plotted and can be seen in Fig: 5.3. The normalized ΔFS images i.e.(FS- F+0.35V )/F−0.40V images for a step potential (S) of -0.40 V for all modified electrode surfaces hasalso been indicated in the figure.Figure 5.3: Average (111)ΔFS/F values as a function of the step potentials (S) for surfaces preparedusing Approach1: Without intermediate MCH back-fill.The average (111) (FS-F+0.35V)/F−0.40V values (for S = -0.40V) for the surfaces prepared using575.2. Approach2: With intermediate MCH back-filldifferent Etret potentials (of -0.900V, -0.925V and -0.950V) and for a DNA immersion time of 10 minuteswas found to be approximately similar in magnitude (0.25 ±0.05). Since more negative Etret potentialsdo not significantly affect how the DNA strands pack on the surface especially for short DNA assemblytime of 10 minutes, it can be concluded that the ssDNA is in a constrained environment.The average (111) (FS-F+0.35V )/F−0.40V values (for S = -0.40V) for the surface prepared with anEtret potential of -0.875V and a DNA immersion time of 24 hours was, however, found to be almostnegligible (0.061 ±0.01) indicating that the ssDNA is quite highly packed together on the surface. TheDNA assembly time was found to affect the local DNA surface packing densities. An attempt to formlayers with low ssDNA surface packing densities by decreasing the DNA immersion time below 10 min-utes is also not an ideal solution, since, the layers formed cannot be expected to be consistent. It is alsohighly likely, that a 1μM DNA deposition for 10minutes is probably not adequate to back-fill all the defectscreated in the MUDOL SAMs.46 Hence, decreasing the concentration of the DNA deposition solutionbelow 1μM with the goal of preparing low ssDNA surface packing densities, can also result in a finallayer with more unoccupied defects which could act as sites for subsequent non-specific interactions oftarget QD-DNA bioconjugates (during surface hybridization step).Since very sparsely packed ssDNA probe surfaces is ideal for the target QD-DNA bioconjugates todiffuse between the probe strands and initiate hybridization (from the discussion in 2.7), this approachof surface preparations was found to be not useful.5.2 Approach2: With intermediate MCH back-fillIn this approach, the electrodes were prepared by an intermediate immersion in a MCH solution betweenthe Etret potential application and DNA immersion steps. All of the three experimental factors werevaried to form the SAMs using this approach. The Etret potentials used for sample preparations wereeither OCP, -0.875V or -0.900V or -0.925V, the DNA deposition time was chosen as either 6 hours or24 hours while the DNA concentration was chosen as either 0.1μM, 0.2μM or 0.4μM. The results fromthe characterization of these layers are discussed below.5.2.1 Background corrected fluorescence intensities (at -0.4V)The average (111) fluorescence intensity (counts) for different modified electrode surfaces preparedusing this approach as a function of Etret potential is plotted in Fig: 5.4(a).585.2. Approach2: With intermediate MCH back-fillFor the different Etret potentials (-0.875V, -0.900V and -0.925V), the intensities for the surfacesthat were prepared using a 0.1μM DNA solution, a 0.2μM DNA solution, and a 0.4μM DNA solutionhave been indicated using red points, blue points and green points respectively. These surfaces wereprepared by immersing the electrodes in the respective DNA solutions for a constant time of 6 hours. Asexpected, fluorescence images of these surfaces (a few selective ones shown in the figure) -capturedwhile applying -0.40V, showed fluorescence intensities only from the (111) facets.One sample was prepared using an Etret potential of -0.900V, a DNA concentration of 0.1μM and a24 hours DNA immersion time and has been marked on the same plot using black points. Interestingly,the fluorescence image of this surface (shown in the figure) had fluorescence on both the (111) and(100) crystallographic facets.Fluorescence images for two control experiments prepared using an Etret potential applicationequivalent to the OCP (approximately -0.005V or analogous to no potential application), a DNA concen-tration of 0.1μM and for DNA immersion times of 6 hours and 24 hours are shown in Fig: 5.4(b).The average (111) fluorescence intensities for the surfaces prepared with this approach (i.e. withintermediate MCH back-fill) is lower approximately by a factor of 10 when compared with surfaces thatwere prepared with the previous approach (i.e. without MCH back-fill). The surfaces prepared with thisapproach have 10 times less DNA.595.2. Approach2: With intermediate MCH back-fill(a)(b)Figure 5.4: Average (111) fluorescence intensities (counts) as a function of Etret potentials for surfacesprepared using Approach2: With intermediate MCH back-fill.Fig: 5.4(a) shows that samples prepared with more negative Etret potentials have higher (111) ss-DNA surface coverages. Also surface coverages increases more dramatically for more negative Etretpotentials. In other words, samples prepared with different DNA concentrations (0.4μM, 0.2μM,0.1μM)for the same DNA immersion time (6hours) were found to result in (1) negligible differences in surfacecoverage for a comparatively less negative Etret potential of -0.875V or (2) a significant difference insurface coverage for a more negative Etret potential of -0.925V. The variance in the fluorescence in-tensities is highest for surfaces prepared with an Etret potential of -0.900V, similar to the findings forthe samples prepared using the previous approach.The surface prepared with an Etret potential of -0.900V and 0.1μM DNA for 24 hours immersionwas found to have the maximum average (111) fluorescence intensities. It is likely that an Etret of605.2. Approach2: With intermediate MCH back-fill-0.900V creates many defects only on the (111) facets and the very few defects on the (100) facets areaccessed by the DNA only after an assembly time of 24 hours. Again, the data seems consistent withthe idea that a higher assembly time, a higher concentration of adsorbate or a higher number of openadsorption sites results in a higher probability of adsorption.The control surface prepared with an Etret potential equivalent to the OCP also shows fluorescencearound the edges of (111) facets for a 24 hours DNA immersion time and not for a 6 hours immersiontime (Fig: 5.4(b)). The edges around the (111) facets are inherently similar to the large (111) terraces in-terspersed with several step edges and are some of the least well formed features on themonocrystallineelectrodes. Hence, it is quite likely that some amount of thiol exchange happens with the MCH spon-taneoulsy displacing some of the MUDOL in these regions. The DNA then undergoes place exchangewith the MCH selectively labeling the hexagonal edges around the (111) facets which only occurs aftera longer DNA immersion time.5.2.2 Normalized ΔFS calculationsThe normalized ΔFS images i.e. (FS- F+0.35V )/F−0.40V images for three different step potentials (S=-0.40V, 0V and +0.35V) for some of the surfaces prepared using this approach (i.e. With intermediateMCH back-fill) can be seen in Fig: 5.5.615.2. Approach2: With intermediate MCH back-fillFigure 5.5: (FS- F+0.35V )/F−0.40V images for three different step potentials (S=-0.40V, 0V and +0.35V)for surfaces prepared using Approach2: With intermediate MCH back-fill.For these images, the average (111) normalized fluorescence modulation response as a function ofthe applied step potentials (S) can be seen in Fig: 5.6.625.2. Approach2: With intermediate MCH back-fillFigure 5.6: Average (111)ΔFS/F values as a function of the step potentials (S) for surfaces preparedusing Approach2: With intermediate MCH back-fill.The average (111) (FS-F+0.35V)/F−0.40V values (for S = -0.40V) for the surfaces prepared using anEtret potential of -0.925V, a DNA immersion time of 6 hours and different DNA concentrations (0.1μM,0.2μM, 0.4μM) was found to be approximately (0.8 ±0.2). The high value of the normalized modulationresponse indicates that these layers are sparsely packed with any chosen ssDNA surrounded by onlya few ssDNA neighbors. Sparsely packed ssDNA and low ssDNA surface coverages for these threesurfaces indicate that possibly any of these could be utilized to carry out successful hybridization withthe target QD-DNA bioconjugates.The average (111) (FS- F+0.35V)/F−0.40V value (for S= -0.40V) for the surface prepared with anEtret potential of -0.875V is only 0.2, a consequence of very low fluorescence intensities complicatingthe analysis.The surface prepared with an Etret potential of -0.900V appears to have different packing densitieson the (111) and the (100) crystallographic facets. The average (111) (FS- F+0.35V )/F−0.40V values (forS = -0.40V) for this layer is close to 0.6 and represents medium packing ssDNA densities.Together, the surfaces prepared using the two approaches detailed in this chapter spans a widerange of ssDNA surface packing densities. The next sub-section compares the capacitance values635.2. Approach2: With intermediate MCH back-fillduring reductive desorption for the layers prepared by the two approaches. This is important to bothquantify the layer stability and to compare the amount of unoccupied open defects.5.2.3 Characterization using capacitance measurementsFig: 5.7 shows the capacitance values during a reductive potential step profile (Fig: 3.16) for two layersprepared without- (Approach1) and with- (Approach2) an intermediate MCH backfill between the Etretpotential application and DNA immersion steps. Capacitance value (at 0V) for the layer prepared withthe second approach is lower than that for a layer prepared with the first approach even though boththe layers were prepared with an Etret potential of -0.925V. Hence, this indicates that there are feweropen, unoccupied sites for the final layer prepared with the second approach. This is beneficial sincelesser number of unoccupied defects considerably reduces the possibility of non-specific interactions oftarget QD-DNA bioconjugates during the subsequent hybridization step. Also, the layer prepared withthe second approach seems to be more electrochemically stable as the capacitance values starts toincrease at a more negative potential (-0.70V vs -0.50V).Figure 5.7: Capacitance (uF/cm2) as a function of the reductive potential step profile (Fig: 3.16) forsurfaces prepared using the two approaches: Without- (red points) andWith- (black points) intermediateMCH back-fill.645.3. Conclusion5.3 ConclusionSurfaces prepared with an intermediate MCH back-fill between the Etret potential application and DNAassembly steps were found to have smaller average background corrected fluorescence intensity countsat -0.4V, higher average (FS-F+0.35V)/F−0.40V values at negative step potentials, smaller average ca-pacitance values at 0V than surfaces that were prepared without the intermediate MCH back-fill step.Hence, these surfaces have the perfect combination of low ssDNA probe surface coverages, sparselypacked ssDNA probes on the surface and few unoccupied-defective sites on the surface which as dis-cussed are perfect for carrying out hybridization with target QD-DNA bioconjugates. Furthermore, anEtret potential of -0.925V was found to be the best treatment potential for preparing consistent & se-lectively modified (111) SAMs. For the preliminary experiment to study hybridization of target QD-DNAbioconjugates with the ssDNA probes on the surface, initial preparation conditions of an Etret potentialof -0.925V, an intermediate MCH back-fill step, a DNA concentration of either 0.2μM or 0.4μM and aDNA immersion time of 6 hours will be used.65Chapter 6Spectro-electrochemicalcharacterization of QD SAMs on AuelectrodesIn Chapter 5, it was found that preparation conditions of an Etret potential of -0.925V, a MCH back-fillstep, a DNA concentration of either 0.2μM or 0.4μM and a DNA immersion time of 6 hours are rationalchoices to form ssDNA probe layers with the right selectivity, surface coverage and surface density, andleast number of unoccupied defects. In this chapter, results from the control and preliminary surfacehybridization experiments with the target QD-DNA bioconjugates either at room temperature or at anelevated temperature of 45oC has been discussed. As was previously mentioned in 3.8, DIM & GF filtercube sets were used in the spectro-electrochemical set-up to detect QD (tagged onto the target DNAstrands) fluorescence and AF488 (tagged onto surface probe DNA strands) fluorescence respectively.The fluorescence images collected with the DIM filter cube set were modified with a red look-up tableto distinguish them from fluorescence images collected with the GF filter cube set which were modifiedwith a fire look-up table.6.1 Surface hybridization at room temperature (with targets inTBS)6.1.1 Control experimentsThe experimental procedures for these control sample preparations have been previously discussedin depth in Chapter 3 (refer 3.7.1). These controls were immersed in either a 2.5nM or 5nM 0.2 DNAstrands per QD bioconjugate solution (in TBS) for over 20+ hours at room temperature. The beads were666.1. Surface hybridization at room temperature (with targets in TBS)thoroughly rinsed with TBS before imaging.To recap, the first control involved preparation of a MUDOL coated monocrystalline bead electrode.The DIM fluorescence image after hybridization step is shown in Fig: 6.1. No appreciable fluorescenceintensities were observed either on the (111) facets or on other regions even after applications of poten-tial modulation profiles similar to Fig: 3.13. This shows that QD-DNA bioconjugates do not undergo anynon-specific or specific interactions with the MUDOL SAM that has not previously experienced Etretpotential.Figure 6.1: DIM fluorescence image of the first control after hybridization with 0.2 DNA strands per QDbioconjugates (in TBS) at room temperature.The second control involved preparation of a MUDOL coated monocrystalline bead electrode mod-ified with an Etret potential of -0.925V and MCH back-fill step. The DIM fluorescence image afterhybridization step is shown in Fig: 6.2(a). During applications of several potential modulation profilessimilar to Fig: 3.13, gradual increase in intensities only on the (111) facets was observed (Fig: 6.2(b)).676.1. Surface hybridization at room temperature (with targets in TBS)Figure 6.2: DIM fluorescence images of the second control after hybridization with 0.2 DNA strands perQD bioconjugates (in TBS) at room temperature.The intensities measured from the (111) as function of step potentials (S) (& at base potentials (B)of +0.35V before stepping to the corresponding step potentials) during application of several potentialmodulation profiles can be seen in Fig: 6.3 & 6.4. Fluorescence images (uncorrected for backgroundscattering) corresponding to the first base potential and the last step potential for each potential modu-lation profile is also shown.The intensities on the (111) facets continues to rise gradually during the application of the secondpotential modulation profile (Fig: 6.3(#2)). Intensities seem to stabilize over the next set of applications ofpotential modulation profiles (Fig: 6.3(#4 & #6)). Interestingly, there is also some fluorescence responseto potential modulation. When the negative step potentials and the positive base potentials of +0.35V(before stepping to the corresponding step potentials) are applied, the local (111) intensities seem to behigher and lower in magnitude. The behavior also seems consistent across the different (111) facets.DIM fluorescence image following immersion in TBS solution for 2+ hours, shows no intensities eitheron the (111) facets or any other regions even in response to applications of a second set of potentialmodulation profiles (Fig: 6.2(c)).686.1. Surface hybridization at room temperature (with targets in TBS)Figure 6.3: Intensities from the (111) facets as a function of step potentials (S) (& at base potentials (B)of +0.35V before stepping to the corresponding step potentials) for several potential modulation profiles(similar to Fig: 3.13). DIM fluorescence images (uncorrected for background scattering) correspond-ing to the first base potential and the last step potential for each potential modulation profile is alsoshown. These results are of the second control hybridization experiment with 0.2 DNA strands per QDbioconjugates (in TBS) at room temperature.696.1. Surface hybridization at room temperature (with targets in TBS)Figure 6.4: Intensities from the (111) facets as a function of several potential modulation profiles (fromFig: 6.3) on a time axis.One possible explanation is that the QD-DNA bioconjugates interact non-specifically with any defectson the (111) facets. Since the QD-DNA bioconjugates lie on the gold electrode surface, their fluores-cence is strongly quenched by the electrode surface. Applied potentials repels the non-specificallybound QD-DNA bioconjugates away from the electrode surface and the fluorescence is quenched toa lesser extent. Hence, some fluorescence intensities are detected. Since the zeta potential of theQD-DNA bioconjugates is a small negative number (around -80mV), it is likely that they also experiencesmall attractive and repulsive electrostatic forces to the surface upon application of positive and negativepotentials resulting in minor fluorescence modulations.Non-specifically adsorbedQD-DNA bioconjugates which are repeled away from the electrode surfaceafter the applications of potential modulation profiles still interact very weekly with the surface defects.However, they are able to diffuse away from the electrode surface once immersed in the TBS solution.Therefore following this, no intensities were observed on the (111) facets or other regions.The third control was a MUDOL coated monocrystalline bead electrode modified with an Etretpotential equivalent to the OCP, a MCH back-fill step, and a 0.2μM DNA immersion step for 6 hours.The DIM fluorescence image after hybridization step is shown in Fig: 6.5(a). Maximum intensities wereobserved around the edges of the (111) facets which as was previously mentioned in Chapter 5 is themost ’defective’ feature of the monocrystalline gold bead electrode. Low intensities were also observedon the (111) facets.706.1. Surface hybridization at room temperature (with targets in TBS)Figure 6.5: DIM fluorescence images of the third control after hybridization with 0.2 DNA strands perQD bioconjugates (in TBS) at room temperature.It is possible that some small amounts of thiol exchange occurs with MCH displacing some of theMUDOL in these regions during the preparation of this surface. Since MCH does not pack as wellas MUDOL (van der Waals forces are weaker for -C6 than -C11chains), a few surface defects might bepresent. The QD-DNA bioconjugates are able to interact only weakly with these types of surface defectsand hence are present at distances farther from the electrode surface. Therefore, the fluorescence ofthe QD-DNA bioconjugates is not strongly quenched and some signals are detected. However, duringthe course of an application of a potential modulation profile similar to Fig: 3.13, no intensities weredetected (Fig: 6.5(b)). This indicates that applications of potential modulation profiles is adequate torepel these kinds of weakly bound non-specific adsorptions of QD-DNA bioconjugates.6.1.2 Preliminary hybridization experimentThe experimental procedure for this sample preparation has been previously discussed in depth in Chap-ter 3 (3.7.2). To recap, the electrode was prepared as a MUDOL coated monocrystalline bead modifiedwith an Etret potential of -0.925V, a MCH back-fill step, and a 0.2 μM DNA immersion step for 6 hours.This was immersed in a 10nM 10 DNA strands per QD solution (in TBS) for over 20+ hours at room tem-716.1. Surface hybridization at room temperature (with targets in TBS)perature and was rinsed with TBS before imaging. GF and DIM fluorescence images after hybridizationstep are shown in Fig: 6.6(a).Figure 6.6: GF and DIM fluorescence images of the preliminary hybridization experiment with 10 DNAstrands per QD bioconjugates (in TBS) at room temperature.Intensities from the Left(111) facet of the DIM and GF fluorescence images as a function of steppotentials (S) (& at base potentials (B) of +0.35V before stepping to the corresponding step potentials)of a potential modulation profile (similar to Fig: 3.13) are shown in Fig: 6.7. The DIM & GF fluorescenceimages (uncorrected for background scattering) corresponding to the first base potential and the laststep potential of the potential modulation profile are also shown.The fluorescence intensity of the bound QD-DNA bioconjugates while applying the different basepotentials of +0.35V (before stepping to the consecutive step potentials) is not steady and continuouslyincreases in magnitude. This rise in the measured intensities is a clear indication of non-specificallybound QD-DNA bioconjugates leaving the surface- repelled from the surface in response to the appliedpotential modulation profile. The QD-DNA bioconjugates non-specific adsorption is however localizedselectively on the (111) facets. The extent of QD-DNA bioconjugates interaction appears to be lessconsistent across the two different (111) facets on this surface. A difference in consistency of targetinteractions across different (111) facets can be influenced by several factors including differences inlocal surface features and local probe ssDNA SAM assembly.726.2. Surface hybridization at 45oC (with targets in IB)Figure 6.7: Intensities from the Left(111) facet of DIM & GF fluorescence images as a function of steppotentials (S) (& at base potentials (B) of +0.35V before stepping to the corresponding step potentials) ofa potential modulation profile. DIM & GF fluorescence images (uncorrected for background scattering)corresponding to the first base potential and the last step potential of the potential modulation profileare also shown. The results are of preliminary hybridization experiment with 10 DNA strands per QDbioconjugates (in TBS) at room temperature.DIM fluorescence image following immersion in TBS for 2+ hours showed almost negligible intensities(except for a few QD-DNA bioconjugates hotspots) on the (111)s even in response to a second set ofapplied potential modulation profiles thereby indicating that all of the interactions previously observedwere non-specific only (Fig: 6.6(c)).Other possibilities to enhance surface hybridization efficiency such as hybridization at elevated tem-peratures or by introducing divalent cations in the hybridization solution have been explored in the nextsection.6.2 Surface hybridization at 45oC (with targets in IB)As discussed in Chapter 2, elevated temperatures have been shown to increase hybridization efficienciesand decrease hybridization time (2.5.4). An attempt has beenmade in this work to carry out hybridizationat a chosen elevated temperature of 45oC.The previous control experiments to study non-specific interactions were performed using 0.2 DNAstrands per QD solutions (in TBS). For the hybridization experiments, 2 DNA strands per QD or 10 DNAstrands per QD solutions is preferred. This is because unlike 0.2 DNA strands per QD solution whichcontains a majority of unmodified QDs, these solutions have a majority of QD-DNA bioconjugates. Thenature of surface interaction is expected to be different with these solutions. The elevated temperaturecan also be expected to influence the nature of surface interactions of the QD-DNA bioconjugates.736.2. Surface hybridization at 45oC (with targets in IB)Hence, a control experiment with one of these solutions (either the 10 DNA strands per QD or the 2DNA strands per QD solution) being hybridized at 45oC is necessary.The QD-DNA bioconjugates sourced from Dr. Russ Algar’s lab were initially in a TBS solution whichcontains TRIS salts and Na+ ions. The bioconjugates were now diluted to an IB solution containingMg+2 ions. This is because Mg+2 helps to form dihedral bridges between the ssDNA probes and thethe target complementary DNA strands on the QD-DNA bioconjugates thereby aiding the hybridizationprocess.In order to study the fluorescence response to potential modulation of the target QD-DNA bioconju-gates, modified potential modulation profiles- 1 and 2 (similar to Fig: 3.14 and Fig: 3.15) have also beenused henceforth. In these profiles, the base potentials and the step potentials were applied for a longerduration and multiple images were captured at each base and step potentials to study the fluorescencemodulation response more in depth.6.2.1 Control experimentFor the fourth control, a MUDOL coated monocrystalline bead electrode modified with an Etret poten-tial of -0.925V and MCH back-fill step was subsequently immersed in a 5nM 2 DNA strands per QDbioconjugate solution (in IB) at 45oC for 2 hours. The bead was rinsed with IB before imaging.The DIM fluorescence images (uncorrected for background scattering) in response to modified po-tential modulation profiles (Fig: 3.14 and Fig: 3.15) are shown in Fig: 6.8(a). The 2 DNA strands perQD interactions were localized as unhomogeneous distributed spots only on the (111) facets and havebeen highlighted as yellow ROIs (’regions of interest’).The applied potentials repels the non-specifically interacting QD-DNA bioconjugates off the elec-trode surface thereby decreasing the distance dependent metal mediated quenching and more non-homogeneous spots with higher intensity signals are observed initially. These intensities eventuallystabilize over time. Once far from the electrode surface, they appear to diffuse rapidly away from theelectrode into the electrolyte (-and hence rapidly away from the field of view) thereby causing a decreasein the measured fluorescence signals. The behavior is however not consistent across the two (111)facets that were imaged with one of the facets experiencing a stronger target QD-DNA bioconjugatesinteraction.746.2. Surface hybridization at 45oC (with targets in IB)Figure 6.8: (a) Assemblage of DIM fluorescence images (uncorrected for background scattering) withQD-DNA bioconjugates interactions highlighted as yellow ROI’s (regions of interest) as a response tomodified potential modulation profiles. (b)Subsequent DIM fluorescence image after overnight immer-sion in IB. The results are of a control surface after hybridization with 2 DNA strands per QD bioconju-gates (in IB) at 45oC.The bead electrode was then disconnected from the set-up and immersed in IB solution overnight.During the course of an application of a second set of modified potential modulation profiles, no fluo-rescence intensities were observed either on the (111) facets or any other regions (Fig: 6.8(b)). Similarto all the other previous examples, immersion in buffer following applications of negative potentials orpotential pulse profiles seems to have helped to remove all the non-specific adsorptions.6.2.2 Preliminary hybridization experimentsIn summary, the electrodes were prepared as a MUDOL coated monocrystalline bead modified with anEtret potential of -0.925V, a MCH back-fill step, and a 0.4 μM DNA immersion step for 6 hours. Theywere subsequently immersed in either 10nM or 5nM of either 0.2 DNA strands per QD, 2 DNA strandsper QD or 10 DNA strands per QD bioconjugates solution (in IB) for 2 hours at 45oC. The beads wererinsed with IB before imaging.DIM fluorescence images were collected at OCP (i.e. without potential application) and in response756.2. Surface hybridization at 45oC (with targets in IB)to modified potential modulation profiles (similar to Fig: 3.14 and Fig: 3.15). DIM fluorescence imagesfor one of the experiments (hybridization with 2 DNA strands per QD solution) is shown in Fig: 6.9(a).In Fig: 6.9(a), the interaction of the QD-DNA bioconjugates is specific to the (111) facets. DIM intensi-ties measured from the (111) facets at OCP seem to fluctuate indicating that some of the non-specificallyadsorbed QD-DNA bioconjugates is leaving the electrode surface even without the application of a po-tential. In response to the application of modified potential modulation profiles, the intensities increasegradually, stabilize and eventually decrease in magnitude. This is consistent with the results for removalof non-specifically bound QD-DNA bioconjugates as discussed in previous sections. From the images,it is also evident that the extent of non-specific adsorption on one of the (111) facets (Top-Left) is largerwhen compared to the other (111) facet (i.e. Bottom). Applied potential profiles were able to removemost of the non-specifically bound QD-DNA bioconjugates from the bottom(111) facet while only somefrom the top-left(111) facet. The bead electrodes were subsequently immersed in IB overnight andsome of the target QD-DNA bioconjugates were removed showing these were non-specifically bound.The DIM fluorescence image collected following overnight IB immersion is shown in Fig: 6.9(b).Figure 6.9: (a) Assemblage of DIM fluorescence images (uncorrected for background scattering) afterpreliminary hybridization with 2 DNA strands per QD bioconjugates (in IB) at 45oC as a response toopen-circuit potentials and modified potential modulation profiles. (b) Subsequent DIM fluorescenceimage after overnight immersion in IB.766.2. Surface hybridization at 45oC (with targets in IB)DIM fluorescence images collected after removing non-specific adsorption by application of modifiedpotential modulation profiles and subsequent overnight immersion in IB for the following cases are shownin Fig: 6.10:(I) Control hybridization with 5nM 2 DNA strands per QD(II) Preliminary hybridization with 10nM 0.2 DNA strands per QD(III) Preliminary hybridization with 10nM 2 DNA strands per QD(IV) Preliminary hybridization with 5nM 10 DNA strands per QDFigure 6.10: DIM fluorescence images collected after applications of modified potential modulation pro-files and subsequent overnight IB immersion for control and preliminary hybridization experiment withQD-DNA bioconjugates (in IB) at 45oC.Intensities from the (111) facets for these cases as a function of an application of another set ofmodified potential modulation profiles is shown in Fig: 6.11.The control or (I) did not show any intensities on either the (111) facets or on other regions. Intensitiesfrom (111) facet for (II) i.e. preliminary hybridization with 0.2 DNA strands per QD decreased consistentlyin response to applications of modified potential modulation profiles. The intensities eventually drop tozero. This is expected since 0.2 DNA strands per QD solution contains a majority of QDs that areunmodified and surface interactions can be expected to be mostly non-specific in nature.Intensities from (111) facet for (III) i.e. preliminary hybridization with 2 DNA strands per QD showsfluorescence response to potential modulation for negative step potentials (between 0V to -0.2V) andat the positive base potentials of +0.35V (before stepping to these negative step potentials). At steppotentials of -0.35V or -0.45 V, an inverse fluorescence response is observed. The intensities seem todecrease when these potentials are applied but has a higher magnitude when positive base potentialsof +0.35V are subsequently applied to the electrode (Fig: 6.11).776.2. Surface hybridization at 45oC (with targets in IB)Figure 6.11: (a) A sequence of modified potential modulation profiles -1 & -2 (b) DIM intensities from the(111) facets as a function of the applied sequence of modified potential modulation profiles for cases inFig: 6.10.Intensities from (111) facet for (IV) i.e. preliminary hybridization with 10 DNA strands per QD shows786.2. Surface hybridization at 45oC (with targets in IB)comparatively negligible fluorescence response to potential modulation. This might be due to multiple(more than one) DNA strands on the QD-DNA bioconjugates interacting with multiple ssDNA probeswhich restricts QD movement. However, intensities still show the inverse fluorescence response aroundnegative step potentials of -0.35V and -0.45V.Fluorescence response can be explained as an overlay of two different processes. Negative steppotentials of -0.35V and -0.45V might be aiding the removal of any strongly interacting non-specificallybound QD-DNA bioconjugates, thereby decreasing the fluorescence intensities that are measured. Al-ternatively, these potentials might probably also cause a partial/ full electrochemical melting (or dehy-bridization)6,77 thereby resulting in an orientational change of the duplexes or removal of some of thespecifically bound QD-DNA bioconjugates. Since the hydrodynamic diameter of the QD-DNA biocon-jugates (approximately 16-20nm) is much larger than the physical dimensions of the ds-DNA system,it is likely that the hybridized duplex state is not very stable to applied negative potential pulses. Anypartially/ completely dehybridized QD-DNA biconjugates either interacts non-specifically with surfacedefects or diffuses into the electrolyte decreasing the fluorescence intensities that are detected. Thesubsequent rise in intensities at positive base potentials could be due to a re-organization/ annealingof the duplex hybridized state wherein the system recovers a part of the lost fluorescence intensities bypromoting re-hybridization of some of these QD-DNA bioconjugates. Since, the fluorescence responseof QDs in the vicinity of a large metallic electrode is not very well understood, the data is also hard tointerpret.When the potential window is decreased to (+0.30V to -0.30V) from (+0.35V to -0.45V), bound QD-DNA bioconjugates seem to be comparatively more stable showing both smaller fluorescence responseto potential modulation and smaller loss in intensities. To conclude, hybridization at an elevated tem-perature of 45oC with targets in IB seems to have resulted in strong target-probe interactions which arestable despite immersion in buffer for an extended period of time and also within a particular potentialpulse window.In order to further study the stability of the bound QD-DNA bioconjugates, a modified reductive po-tential step profile similar to Fig: 3.16 was subsequently applied. DIM intensities from the (111) facetsfor these cases as a response of the modified reductive potential step profile can be seen in Fig: 6.12.For case (III) i.e. preliminary hybridization with 2 DNA strands per QD, as expected the intensityis stable up to -0.3V. The intensity decreases in a straightforward manner from -0.3V till -0.6 V. Thiscould be in part due to the removal of non-specifically bound QD-DNA bioconjugates and in part due topossible electrochemical melting (dehybridization) of the hybridized duplex. Intensity shows a maximum796.2. Surface hybridization at 45oC (with targets in IB)value at -0.8V which can be attributed to spectral bleed-through of fluorophore AF 488.Figure 6.12: DIM intensities from the (111) facets as a function of modified reductive potential step profilefor cases previously discussed in Fig: 6.10 and Fig: 6.11.The ssDNA probe is reductively desorbed from the electrode surface starting at a potential of -0.6Vand as the fluorophore tagged ssDNA is pushed further from the electrode surface, an increase in AF488fuorescence intensity is expected. Once the fluorophore tagged ssDNA slowly diffuses away from thefield of view, a decrease in AF488 fluorescence intensity can be expected. The amount of spectralbleed-through is directly proportional to the AF488 fluorescence intensity. A complete analysis of thespectral bleed-through characteristics has been performed in Appendix 2.Case (II) i.e. preliminary hybridization with 0.2 DNA strands per QD did not have any bound QD-DNA bioconjugates after the application of the second set of modified potential modulation profiles andtherefore has an initial zero signal. Similar AF488 fluorescence spectral bleed-through characteristicsfrom -0.6V to -1.2V is however observed (as expected).The control shows a zero signal at all potentials since it neither has bound QD-DNA bioconjugatesnor fluorophore AF488 tagged DNA.It is also important to mention that the QD-DNA bioconjugates (in TBS) which were used for hy-bridization experiments at room temperature (Section: 6.1) were diluted to an IB solution and re-usedfor hybridization experiments at 45oC (Section: 6.2). From the results that were previously discussedin this Chapter, it becomes clear that the QD-DNA bioconjugate solutions are stable at room tempera-ture for extended time periods and can be subsequently re-used for several experiments. Hybridizationat an elevated temperature of 45oC was found to favor an increased flux of QD-DNA bioconjugates to806.3. Conclusionthe electrode surface, possibly promoting some specific surface hybridization. However, the QD-DNAbioconjugate solutions could not be subsequently re-used. The fluorescence and gel electrophoresisresults (data supplied by HD Kim) for 2 DNA strands per QD and 10 DNA strands per QD bioconjugatesolutions following hybridization experiments at room temperature (for 20+hours) and at 45oC (for 2hours) can be seen in Fig: 6.13. The absence of detectable signals indicates that either most of theQD-DNA bioconjugates were used up during the previous experiments or probably that the heating &subsequent cooling cycle possibly decreases the photoluminescence of the QD-DNA bioconjugate solu-tions, indirectly affecting their stability. The temperature (between 22oC (room temperature) and 45oC)and time of heating hence, needs to be optimized for better results.Also, the His-tag peptide attached to the complementary DNA strand was prepared almost two yearsago. This was dried and stored in the freezer before it was attached to the GSH capped QDs. Onceprepared, the QD-DNA bioconjugate solutions were re-used over several months. It is also possible thatthese factors might have also partially contributed to the instability of the bioconjugates and thereforemay have influenced the results as seen in Fig: 6.11.Figure 6.13: (a) Fluorescence spectra of 2 DNA strands per QD and 10 DNA strands per QD bioconju-gates (in IB), of the IB solution and equivalent unmodified QDs (b) Gel electrophoresis data (1% TBEAgarose,100 V, 30 minutes in 1X pH 8.3 TBE buffer) for: Well 1: 2 DNA strands per QD, Well 2: 10 DNAstrands per QD, Well 3: unmodified QDs. The results are of QD-DNA bioconjugate solutions followinghybridization experiments at room temperature (for 20+hours) and at 45oC (for 2 hours).6.3 ConclusionNon-specific adsorptions of QD-DNA bioconjugates contributes significantly to the final measured fluo-rescence signals in both the control and the preliminary hybridization experiments. Non-specific interac-816.3. Conclusiontions persist despite rinsing the electrode with buffer and applications of several potential pulse profiles.Specific adsorptions of target QD-DNA bioconjugates was possibly detected for selectively modified ss-DNA probe surfaces after hybridization with the 2 DNA strands per QD and 10 DNA strands per QDbioconjugates solutions (in IB) at 45oC for 2 hours. The change in fluorescence was monitored duringthe change in the applied potentials and an interesting result was observed. Typically, the fluorescenceintensity increases when the potential is made more negative. This is what was observed for potentialsteps to -0.2V. But for more negative potentials steps, the change in fluorescence was found to behavein an opposite fashion. Namely, the fluorescence decreased when stepping to potentials more negativethan -0.3V (an inverse fluorescence response). The inverse fluorescence response has been attributedin part due to a partial/ complete electrochemical melting (dehybridization) at negative step potentials of-0.35V and -0.45V and to duplex annealing/ re-hybridization at subsequent base potentials of +0.35V.The hybridized duplex state is probably not very stable due to the large dimensions of QD-DNA biocon-jugate in comparison to the dimensions of ds-DNA. The hybridized duplex was however found to stablewithin a potential pulse window of +0.30V to -0.30V.82Chapter 7Summary & future work7.1 SummaryThe initial half of this thesis deals with site-selectively modifying only the (111) facets of MUDOL coatedmonocrystalline gold bead electrodes with an optimum surface coverage and packing density of ssDNAprobes tagged with a fluorophore AF488. An electrochemical desorption procedure involving applica-tion of selective negative treatment potentials (Etret) to MUDA coated monocrystalline bead electrodeshas previously been reported to achieve reductive removal of MUDA SAMs only from the (111) facets.46This procedure had to be suitably modified to achieve similar results with MUDOL SAMs instead asthe carboxylate terminal group of MUDA might promote significant non-specific interactions of probessDNA67 resulting in poor surface mediated hybridization with complementary targets.49 An electro-chemical technique i.e. differential capacitance measurement was used to compare MUDOL and MUDASAMs reproducibility and electrochemical stability. A rational conclusion of these experiments was thatdifferent Etret potentials of -0.875V, -0.900V, -0.925V and -0.950V could be applied for approximately400s to MUDOL coated monocrytsalline bead electrodes (prepared using 90 minutes assembly time) topartially (albeit to different extents) reductively desorb MUDOL SAMs off (111) facets. The mathemat-ical quantification of defects created on the (111) facets as a result of MUDOL removal has also beenestimated using the change in the measured capacitance values following the Etret application step.C6thiol modified ssDNA tagged with fluorophore AF488 was assembled on these defect sites eitherdirectly or following an intermediate assembly of MCH spacer molecules. An in-situ inverted fluores-cence microscope coupled with an electrochemical control was used to characterize the different sur-faces prepared with or without the intermediate MCH back-fill step by varying several input parameterslike the Etret potential value, the DNA concentration (0.1 μM or 0.2μM or 0.4μM or 1μM) and the elec-trode immersion time in the DNA solution (10 minutes or 6 hours or 24 hours). Background corrected(111) fluorescence intensity (counts) at -0.4V, normalized fluorescence modulation response and differ-ential capacitance value measured at OCP was used as a measure of ssDNA surface coverages, local837.1. SummaryssDNA packing densities and to relatively quantify unoccupied defect sites. A MUDOL coated beadmodified with a potential of -0.925V in which the defects were back-filled with MCH and then with AF488tagged DNA (by immersing the electrode in a 0.2μM or a 0.4μM DNA solution for 6 hours) was found tobe optimum in terms of having a favorable low ssDNA surface coverage, a low ssDNA surface densityand containing few unoccupied defect sites.The later half of this thesis deals with studying the interactions of target QD-DNA bioconjugates af-ter surface mediated hybridization step with the goal to develop specifically bound QD SAMs on Auelectrodes. Control experiments were performed to study non-specific interactions of QD-DNA biocon-jugates on MUDOL SAMs, MUDOL SAMs with some MCH SAMs on the (111) facets and MUDOL SAMswith few unoccupied defects. Preliminary hybridization of optimized surfaces (with ssDNA SAMs on the(111) facets) with 0.2 DNA strands per QD, 2 DNA strands per QD, and 10 DNA strands per QD biocon-jugates was also studied. The hybridization experiments were either performed at room temperature(with the targets in the TBS containing TRIS and Na+ ions) or at 45oC (with the targets in IB containingTRIS, Na+and Mg+2 ions).Significant fluorescence signals corresponding to non-specific adsorptions of target QD-DNA biocon-jugates was detected selectively only on the (111) facets. In response to applied negative potentials/potential pulse profiles, the (111) DIM intensities increased gradually in magnitude, stabilized and even-tually decreased in magnitude indicating that the non-specifically interacting targets were repelled off theelectrode surface. Rinsing with buffer and applications of several potential pulse profiles was found tobe unable to completely remove all the non-specific adsorptions although overnight immersion in bufferwas effective in this regard.Possible specific adsorption (hybridization) of targets with ssDNA probes was observed only afterhybridization with 2 DNA strands per QD and 10 DNA strands per QD bioconjugates (in IB) at 45oC.The former showed some (111) DIM fluorescence response to potential modulation at negative steppotentials between 0V and -0.2V and at the preceding positive base potentials of +0.35V. For the later,this was not observed since multiple (more than one) DNA strands on the target could be hybridizedwith the ssDNA probes restricting the QD’s movements in response to applied potentials. However, aninverse (111) DIM fluorescence response was observed for both at negative step potentials of -0.35Vand -0.45V and at the succeeding base potentials of +0.35V. This has been attributed in part due toelectrochemical melting (dehybridization) of the targets at these negative step potentials to the electrodesurface/ electrolyte and the subsequent re-annealing/ re-hybridization of the duplex at the positive basepotentials. The instability of the duplex could possibly be due to the large dimensions of the QDs in847.2. Future workcomparison to the length scale of the dsDNA. These duplexes were found to be stable within a potentialpulse window of +0.3V to -0.3V.7.2 Future workAs was previously mentioned in Chapter 1, using QD SAMs on Au electrodes as probes to developcommercial electrochemical & luminescent biosensors is a relatively novel idea and very little scientificinformation has been previously published in this regard. More experiments need to be performed inorder to develop a complete understanding of the system studied in this thesis.The instability of the duplex state causing an electrochemical melting (dehybridization) at negativepotentials of -0.35V and -0.45V vs. SCE needs to be addressed first. One possible way to achievethis could be to employ linker DNA strands that can successfully hybridize with the upper-half segmentof the probe DNA strands on the electrode surface and with the lower-half segment of the target DNAstrands tailored on to the QDs (see Fig: 7.1). Such a system is expected to be stable across a widerpotential pulse window in theory since the hybridized DNA base pairs lie further from the dimensionallylarge QD sphere and also further in-to the electrochemical (electrode-electrolyte) double layer (therebyexperiencing lesser influences of the applied potentials). Since the QDs are positioned farther awayfrom the electrode surface in this scenario, the fluorescence signals detected will also be higher. Sucha system (QDs on Au electrode) can be then used to detect BOIs (biomolecules of interest) building onpossibilities of QD’s surface bioconjugation, FRET and multiplexing capabilities.Influence of temperature on the DNA hybridization efficiency and stability of the QD-DNA bioconju-gates also needs to be explored. Temperature and concentration of the target QD-DNA bioconjugatesolutions can influence the possibility of aggregate formation (specifically or non-specifically interactingdimers, trimers, tetramers etc), enhancing or disrupting the possibility of formation of completely hy-bridized DNA duplexes.126,127 Hence, a systematic investigation by repeating the hybridization experi-ment at other different temperatures (e.g. 30oC, 35oC, 40oC, 50oC etc) for longer or shorter duration(e.g. 15 minutes, 45 minutes, 4 hours etc) with different concentrations of target QD-DNA bioconjugatesmight be useful. Similarly, the influence of the experimental condition like ssDNA probe surface cov-erage and density on hybridization efficiency can be studied by employing any of the other selectivelymodified DNA probe surfaces discussed in Chapter 5 in the hybridization experiment.857.2. Future workFigure 7.1: Cartoon depiction of the system: QD-DNA biconjugates (targets) indirectly hybridized withssDNA (probes) on the electrode surface via linker DNA strands (not drawn to scale) - a different ap-proach to assemble QDs on Au electrode.Time gating the signal acquisition i.e. discriminating amongst the data generated in a typical fluores-cence excitation-emission experiment based on fluorescence lifetime values can also provide significantinsights on whether the target QD-DNA bioconjugates interact specifically or non-specifically with thessDNA probes and the surface defects. This has been previously employed in experiments where thelocal electrostatic interactions such as presence of DNA probe affected (increased) the QD fluorescencelifetime value when compared to the fluorescence lifetime values of QDs in solution or only QD hotspotson surfaces.65,128Finally, the nature of exciting QD emission needs to be studied as well. QD-DNA bioconjugatesexcitation could be direct or due to FRET pairing with AF488 fluorophore tagged DNA. In this thesis, thefluorophore tagged DNA was necessary to measure selectivity, surface coverage and surface densitiesof probe DNA layers using a coupled spectro-electrochemical approach. A repeat of the experiments inthis thesis with probe DNA without fluorophore modification is also required.86References[1] Wegner, K. D.; Hildebrandt, N. Chemical Society Reviews 2015, 44, 4792–4834.[2] Ricci, F.; Plaxco, K. W. Microchimica Acta 2008, 163, 149–155.[3] Ricci, F.; Adornetto, G.; Moscone, D.; Plaxco, K. W.; Palleschi, G. Chemical communications2010, 46, 1742–1744.[4] Gooding, J. J. Electroanalysis: An International Journal Devoted to Fundamental and PracticalAspects of Electroanalysis 2002, 14, 1149–1156.[5] Chan, V.; Graves, D. J.; McKenzie, S. E. Biophysical journal 1995, 69, 2243–2255.[6] Monserud, J. H.; Schwartz, D. K. Acs Nano 2014, 8, 4488–4499.[7] Peterson, A. W.; Heaton, R. 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International journal of molecular sciences 2009, 10, 1930–1941.94Appendix ANormalized ΔFS calculationprocedure (Image optimization)As was previously mentioned in Chapter 3, a combination of Accurate Gaussian Blur filters and De-speckle filters were used at various stages during normalized ΔFS calculations to optimize the imagequality. This section dwells a little deeper into the specifics of this process. The images shown in thisthesis contains 512X512 pixels. Applying a Despeckle filter (or a median filter) replaces each pixel in-tensity value with the median intensity value of pixels in the immediate 3X3 neighborhood. This helpsto remove salt-and-pepper noise i.e. very high and very low intensities. Applying an Accurate Gaus-sian Blur filter (of certain input radius r) achieves smoothing thus reducing the image noise. It is alsoimportant to mention that the improvement in the quality of the images does not alter or modify the finalresults. This is proved next.To recap, the normalized fluorescence modulation response for a step potential S was calculated as(refer: 3.10.2):NormzedΔFS =(FS− FB)m.FS,b.cThe images corresponding to (FS− FB) and m.FS,b.c contains negligible intensities (i.e. in therange (−ε, ε) where ε≈0) on parts of the electrode surface that does not contain fluorophore taggedDNA. For these parts, the resultant images corresponding to (FS−FB)m.FS,b.c contains intensities that are intheory not well defined. Applying a series of despeckle filters and gaussian blur filters helps to bring thenormalized intensity counts that are not well defined within the acceptable range of [0-1].Fig: A.1 shows how applying either Accurate Gaussian Blur filters of different radii (i.e. AGB’R’) to(FS−FB) andm.FS,b.c (Fig: A.1(Top)) or a combination of both Despeckle and AGB’R’ filters to (FS−FB) and m.FS,b.c (Fig: A.1(Bottom)) results in higher quality images corresponding to (FS−FB)m.FS,b.c forthree different step potentials (S) of +0.35V, 0V, -0.40V (also the most negative step potential) & basepotential (B) of +0.35V.95Appendix A. Normalized ΔFS calculation procedure (Image optimization)Figure A.1: Summary of normalized ΔFS fluorescence images at step potentials (S) of +0.35V, 0V and-0.40V after modification with Accurate Gaussian Blur filters of different radii and Despeckle filters.A plot of average (111) normalized fluorescence response as a function of step potentials (S) for thefour extreme cases (corresponding to the edge corners of the two matrices in Fig: A.1) can be seen inFig: A.2. The figure shows an overlap of results across the four extreme cases for two different (111)facets. This proves that utilizing a combination of Accurate Gaussian Blur filters and Despeckle filtersat different stages does not alter or modify the results on parts of the electrode which contains DNA96Appendix A. Normalized ΔFS calculation procedure (Image optimization)tagged fluorophore ( >0 intensity values for m.FS,b.c). Utilizing these filters improves the overallimage quality resulting in sharper images with reduced noise.Figure A.2: Average (111) normalized fluorescence response as a function of step potentials (S) forchosen cases (corresponding to the edge corners of matrices in Fig: A.1).97Appendix BSpectral bleed-through correction (forAF488)As was previously discussed in 3.10.3, the emission spectra for fluorophore AF488 has a narrow tail thatextends into the emission wavelength band of the DIM filter (used to detect QDs). The fraction of AF488intensity that is detected using the DIM filter cube set with respect to the AF488 intensity detected usingthe GF filter cube set has been referred to as the AF488 spectral bleed-through factor. Fig: B.1 shows abackground-corrected GF and a DIM fluorescence image of a surface with ssDNA probes tagged withAF488. For the 6 different ROIs (’Regions of interest’) marked on these images, the intensities measuredon the DIM fluorescence image (ROI 1- 375.36 counts, ROI 2- 323.28 counts, ROI 3- 244.59 counts,ROI 4- 286.06 counts, ROI 5- 309.51 counts, ROI 6- 301.85 counts) were approximately 3.9 ±0.63 %of the corresponding intensities measured on the GF fluorescence image (ROI 1- 8424.59 counts, ROI2- 7899.91 counts, ROI 3- 8715.56 counts, ROI 4- 8059.337 counts, ROI 5- 7289.472 counts, ROI 6-7146.38 counts). Since these images were captured under the same imaging conditions of EM gainas the conditions used for the samples relevant to our work, an average AF488 spectral bleed-throughfactor of 0.039 ±0.0063 can be assumed for further analysis.Figure B.1: Fluorescence images captured using a GF filter cube set (Left) and a DIM filter cube set(Right) of AF488 tagged ssDNA probe layer - with 6 ’ROIs’ marked on the images for calculation ofspectral bleed-through factor.98Appendix B. Spectral bleed-through correction (for AF488)In Chapter 6, strong target QD-DNA bioconjugate interactions were detected on the (111) facets usinga DIM filter cube set after the hybridization step. Since the (111) facets had been modified previouslyusing a 0.4μM C6thiol modified DNA AF488, some amount of the detected signal could be in part dueto contributions from spectral bleed-through of AF488. Hence, it is important to quantify this amount.The fluorescence response of the (111) facets as a function of the reductive potential step profile(similar to Fig: 3.16) for the surface: MUDOL coated monocrystalline bead modified with an Etretpotential of -0.925V, a MCH back-fill step, and a 0.4μM and 6 hours C6thiol modified DNA AF488, canbe seen in Fig: B.2.Figure B.2: Intensity response of the (111) facets from background corrected GF fluorescence imagesin response to a reductive potential step profile for the surface (MUDOL, Etret=-0.925V, MCH, 0.4μMand 6hours DNA AF488).It is evident from the figure that the amount of GF fluorescence intensity (counts) detected is signif-icantly dependent on the potential applied to the electrode surface. The measured average (111) GFfluorescence intensity from 0V to -0.6V is approximately 100 counts, at -0.8V is approximately 3519.842± 1521.70 counts, and at -1.2V is approximately 0 counts. Differences in the measured intensity valuesacross two (111) facets is in part due to orientation of the (111) facets on the spherical bead elec-trode with respect to the camera and also on the direction of diffusion of the desorbed AF488 fluo-rophore tagged DNA after reductive desorption at negative potentials (above -0.6V). The correspondingAF488 spectral bleed-through to the DIM filter cube from 0V to -0.6V is approximately 3.9 ±0.63 counts(100*(0.039 ±0.0063)), at -0.8V is between 78-200 counts ±9.5 counts and at -1.2V is 0 counts. Thesevalues can be suitably subtracted from DIM fluorescence images collected in response to the reductive99Appendix B. Spectral bleed-through correction (for AF488)potential step profile to measure only QD signals.All the images in this work collected in response to the reductive potential step profile were with anexposure time of 0.5s while those in response to the potential modulation profile were with an exposuretime of 3s (6 times more intense). Hence, to correct for spectral bleed-through of AF488 to the DIMfilter cube in the images corresponding to the potential modulation profile (in which potential profiles areusually <-0.6V), approximately 23.4±3.78 counts (i.e. (3.9 ±0.63) * 6) needs to subtracted.100

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