DNA-MEDIATED CHARGE TRANSPORT DEVICES FOR PROTEIN DETECTION

Your advice and support have allowed me to take risks in the laboratory, which, as you have taught me, leads to the most interesting discoveries. You have shown me how to be an effective teacher, especially your ability to incorporate real-world examples and demonstrations into your lectures. You have also been a constant source of both scientific and emotional support, especially during difficult periods in the laboratory.

Lynn, Regina and Linda: you have all added humor to my life and provided me with so much insight and guidance. You were there to congratulate me at the highs and pick me up at the lows. Thank you to my mother and Michael for all the help and advice you have given me over the years and for reminding me of the importance of pursuing a career in something that truly inspires me.

You've solved every problem I've had, sat with me in the lab on weekends, read every draft I've written, and listened to all my conversations. With these improved monolayers, significantly more sensitive detection of TATA transcription factor binding protein (TBP) is achieved.

DNA-modified Electrodes Fabricated using Copper- Free Click Chemistry for Enhanced Protein Detection 56

Electrochemical Patterning and Detection of DNA

Arrays on a Two-Electrode Platform 96

Label-Free Electrochemical Detection of Human

A Multiplexed, Two-Electrode Platform for Biosensing based on DNA-Mediated Charge Transport 155

DNA Electrochemistry shows DNMT1

Methyltransferase Hyperactivity in Colorectal Tumors 199

Development of Glassy Carbon Flow-through Cells for

Surface modification of glassy carbon rod electrodes 254 Passivation of glassy carbon rod electrode modified against ferricyanide and.

Thymine Dimers for DNA Nanocircuitry Applications

Thymine Dimer Formation and Repair in Solution 298 Electrochemical Experiments with Thymine Dimer Formation and Repair. Thymine Dimer Formation and Repair in Modified DNA in Solution 308 Rh(phi)2bpy'3+ as a Covalent Redox Probe: Synthesis and Preliminary Studies.

Eight Teflon spacers at different heights were tested for electrochemical signal and mismatch discrimination 178 Figure 5.7 Detection strategy and constant-potential amperometry assay for DNA CT. Nile blue redox reporters 185 Figure 5.10 Titration of transcription factors TATA-binding protein (TBP) and CopG. 305 Figure 8.14 Rh(phi)2bpy3+ signal on multiplexed chips 307 Figure 8.15 Damage to free DNA by irradiation in the presence of methylene blue.

Introduction

Simple chemical methods can also be used to regulate DNA placement on planar electrodes and effectively control the homogeneity and spacing of DNA duplexes within the sensing monolayer. Self-assembled thiolated DNA (top) forms regions of very high DNA density that can prevent access of large biomolecules to probe sequences on the surface, leaving other areas of the electrode surface free of DNA. Due to the deformation of the cyclooctyne ring, the reaction between the azide and the alkyne occurs spontaneously.

A method was therefore developed to covalently attach methylene blue directly to the DNA end. Right: Chronoculometry of the DNA-modified electrode without protein (black) and with bound TBP (red).66 As can be seen, the total charge accumulation in the presence of TBP is significantly less than in its absence. This result was demonstrated at DNA-modified electrodes with DNA containing a T<>T.74 Rather than requiring the incorporation of a redox probe, a signal from the DNA-bound protein cofactor flavin is observed.

If the DNA remains unmethylated, upon addition of the restriction enzyme, the DNA is cleaved (lower path), and the electrochemical signal is turned off (black cyclic voltammogram).80. These low-density monolayers exhibit all the characteristics of DNA-mediated electrochem., including sensitivity to. When a force is no longer applied to the tip, the depth of the resulting hole is measured.

A linear increase in amount of DNA on the surface with increasing percentage of azide indicates that the solution concentration of azide is a valid approximation of the amount of azide assembled in the monolayer, and that the DNA binding to the active headgroups on the surface is essentially quantitative. The preliminary addition of BSA ensures that subsequent signal reductions upon TBP addition are due to the specific binding of the protein. Subsequent readout of the sequences on the surface is accomplished by manual scanning of a microelectrode over the secondary electrode.

Multiple DNA sequences are patterned on the same substrate electrode by successive surface washes and the addition of an alternate DNA sequence with copper catalyst between the substrate and the patterned pads. The strategy for the spatially separated electrochemical activation of the catalyst is shown in Figure 3.3. The bulk electrochemical signal from the substrate pad measurement shows a classic electrocatalytic peak, indicating the presence of well-matched DNA on the electrode.

A DNA strand perfectly complementary to the alkynyl strand that was part of the original mismatch-containing duplex was then incubated on the surface for 1 h, causing previously mismatched sequences to become good matches and vice versa. Rescanning of the substrate electrode showed an almost complete replacement of the signal sites, indicating that most of the DNA helices on the surface had been dehybridized and rehybridized to the alternative complement (Figure 3.7). 2004) Electrochemical recognition of label-free DNA hybridization using reverse current modulation in SECM, Angew.

We use direct detection from the secondary electrode of the conversion of the electrocatalytic partner to methylene blue, ferricyanide, as a measure of the amount of DNA charge transfer occurring at the substrate electrode.

Figure 1.1 Schematic illustrations of the structures of graphene (top) and DNA (bottom)

DNMT1 Lamin A/C

Mixed monolayers were formed on one of the plates using an ethanolic solution of 1 M 12-azidododecane-1-thiol (C12thiolazide) and 1 M 11-mercaptodecyl phosphoric acid (Sigma Aldrich). Specifically, a constant potential of -350 mV was applied to a secondary electrode for 25 minutes, allowing for precise attachment of the appropriate DNA to a primary electrode. The DNA-mediated signal remains fully 'on' after restriction when the electrode is pretreated with a minimum of 65 nM DNMT1 protein on a hemimethylated DNA substrate in the presence of the SAM cofactor, although protein is readily detectable at a 15 nM concentration with 48±3% signal protection.

In addition, the reproducibility of the platform is shown (Figure 4.5) along with the quantification of the 15 individual electrodes in a single assay. Interestingly, high concentrations of lysate were found to decrease the electrochemical signal, probably due to crowding on the DNA-modified electrode, thus limiting access and binding of the methyltransferase of interest. The constant potential amperometry was run for 90 seconds with an applied potential of 320 mV to the secondary electrode and -400 mV to the primary electrode.

The orientation of the tested conditions on the 5x3 array is shown (inset) with circular electrodes colored to correspond to the activity data presented in the bar graph. Next, we tested the ability of the platform to discriminate between lysate from a parental (HCT116 wild-type) colorectal carcinoma cell line and a cell line that does not express DNMT1 (HCT116 DNMT1-/-). As shown in Figure 4.7, the specific detection of DNMT1 activity depends on the methylation state of the substrate and the presence of the SAM cofactor.

Our platform is also sensitive and selective without the use of radioactivity, fluorescence, or antibodies by combining electrocatalytic signal amplification and the sensitivity of DNA CT chemistry to report changes in DNA integrity. Electrochemical approach for DNA methylation detection and methyltransferase activity assay, Chem. AgCl/Ag was deposited on the sensing (top) electrode array to reduce Cu(II) and initiate DNA coupling to the azide-terminated monolayers.

For example, well-matched and mismatched DNA were attached to the same array by the preliminary activation of secondary electrodes one through nine in the presence of well-matched DNA, followed by flushing of the platform and subsequent activation of secondary electrodes ten through fifteen in the presence of DNA containing a single base mismatch. An overview of the process of electrochemical clicking to attach DNA to an electrode, followed by DNA CT-facilitated current measurement, is shown in Figure 5.3. As can be seen from the CVs in Figure 5.4, after copper activation and subsequent rinsing of the surface, no residual copper is visible on the surface.

CVs lack the conventional methylene blue redox couple in DME due to the low DNA coverage and redox probe and electron sink concentrations required for detection at the secondary electrode. The secondary electrode is held at a sufficiently positive potential to oxidize the ferrocyanide that is generated in the solution as a result of the reduction of ferricyanide by LB after CT of DNA.