Electroanalytical Chemistry

A Series of Advances, Volume 27

Allen J. Bard, Cynthia G. Zoski

• Publisher: CRC Press
• Year: 2017
• eBook ISBN:
  9781351981668
• Topic: Physical Sciences
• Subtopic: Biotechnology

This volume is part of a continuing series that provides authoritative reviews on recent developments and applications of well-established techniques in the field of electroanalytical chemistry. Each volume provides the necessary background and starting point for graduate students undertaking related research projects and is of special interest to practicing analytical chemists concerned with electroanalytical techniques. Volume 27 continues this tradition with innovative contributions from internationally respected scientists who highlight new technologies and trends in Protein Biosensing, Bipolar Electrochemistry, and X-ray Absorption Spectroscopy in Electrochemistry.

1	Two-Electrode Platforms for Protein Biosensing Based on Charge Transport through the DNA Double Helix Ariel L. Furst, Michael G. Hill, and Jacqueline K. Barton

CONTENTS

1.1Introduction

1.2Methyltransferase Detection Using an Electrochemistry Platform Based on DNA CT

1.2.1Assay for Methyltransferases

1.2.2Testing for SssI and DNMT1

1.3Controlling Duplex Loading and Spacing in Self-Assembled Monolayers

1.3.1Nanostructured Microelectrodes

1.3.2Controlled Self-Assembly onto Flat Surfaces

1.3.3Addressable Low-Density DNA Monolayers via Cu(I)-Catalyzed Coupling

1.3.4Two-Electrode Platform for Grafting Addressable DNA Arrays

1.4Two-Electrode Detection Platform for DNA CT

1.4.1Detection at the Secondary Electrode

1.4.2Detection Targets

1.5A Thin-Layer, Two-Electrode Multiplexed Platform

1.5.1Thin-Layer Patterning

1.5.2Thin-Layer Detection

1.6DNMT1 Activity Assay from Unprocessed Tumor Samples

1.6.1DNMT1 Activity in Crude Cell Lysates

1.6.2A Measure of DNMT1 Hyperactivity in Tumors

1.7Conclusions

References

1.1INTRODUCTION

The ability to detect biologically relevant targets is critical in fundamental research as well as for the development of clinical diagnostics. Nucleic acid platforms, in particular, represent an especially robust and flexible class of sensors, capable of detecting small molecule, protein, and DNA/RNA targets [1]. Among the many different readout strategies employed for DNA-based sensing, such as fluorescence [2–5], conductivity [6,7], or mass [8–12] changes that accompany hybridization, we have focused on electrochemical methods [13,14]. Electrochemical instrumentation is both low cost and portable, making this method of detection ideal for clinical diagnostics.

Early DNA electrochemical detection strategies involved the direct reduction of nucleic acid bases adsorbed onto mercury electrodes. Such electrodes facilitated facile detection of both DNA hybridization and DNA damage. Hybridization of a target sequence increases the amount of adsorbed DNA, resulting in larger signals. Additionally, signals obtained from single-stranded and double-stranded DNA differ significantly, enabling detection of small single-stranded contaminants within a double-stranded DNA sample [15]. DNA is generally detected at mercury drop electrodes using signals resulting from the reduction of adenine and cytosine residues, although the guanine reduction product is also detectable at very negative potentials. However, hybridization experiments are difficult to perform at a mercury electrode, likely due to interactions between the hydrophobic DNA bases and the similarly hydrophobic electrode surface.

Sinusoidal voltammetry has also been used to measure the direct oxidation of amine-containing nucleosides as well as the sugar-phosphate backbone of nucleotides [16]. DNA detection using the sugar-phosphate backbone is advantageous, as the measurement of the nucleobase electroactivity can be limited in a DNA duplex owing to the accessibility of the bases. Oxidation of the sugar-phosphate backbone is not similarly restricted. While this platform is potentially capable of detecting zeptomoles of DNA, the lack of a high degree of differentiation among sequences, however, is not favored for biosensor applications.

Other systems typically have relied on indirect detection schemes, in which a redox-active mediator is employed to report on the composition of target DNA or to induce redox reactions on the bases themselves [17]. Often, the target DNA is labeled with a small, electrochemically active moiety; hybridization then is detected by the appearance of an electrochemical signal. This technique mimics common fluorescence-based methods in that the target, rather than the probe, is labeled [18]. Target labeling has the advantage of presenting a “signal-on” method of detection (i.e., a hybridization event must successfully occur for an electrochemical signal to appear), yet detection is ultimately limited by the thermodynamic stability of the DNA duplexes formed. Nonspecific hybridization can result in false positive signals, making the identification of subtle sequence variants, such as occur with single-nucleotide polymorphisms, difficult.

Detection systems in which the probe sequence instead is labeled have also been explored. One common approach involves the application of a hairpin DNA construct as the probe molecule [19]. Composed of a stem region that features a self-complementary sequence and a disordered loop region containing the target sequence, the construct is labeled with a redox probe at the stem terminus. In the hairpin form, the probe is close to the electrode surface where it is redox-active. Upon hybridization of the loop to a complementary target, the hairpin opens up, forcing the probe away from the electrode surface, resulting in an attenuated electrochemical signal [20]. Similarly, detection schemes based on DNA “sandwich” assemblies have been investigated. These structures involve three sequences of DNA: (1) a target molecule; (2) a probe molecule tethered to the surface; and (3) a reporter sequence [21–24]. The reporter sequence binds to an overhang of the probe-target duplex and can either directly generate an electrochemical signal or can be a component of an ancillary redox cycle. This strategy negates the need for target labeling and still maintains a “signal-on” detection scheme.

Electrochemical DNA-based protein detection methods have generally been limited. The majority of such platforms depend on DNA aptamers for sensitive and specific detection. DNA aptamers, single-stranded DNA sequences that form unique secondary structures, are evolved to bind specifically to a single protein. The conformational changes that occur upon target protein binding to DNA aptamers are then transduced into a change in the electrochemical signal. DNA aptamer-based electrochemical platforms have been used for the detection of a variety of proteins, including the tumor markers carcinoembryonic antigen (CEA) and α-fetoprotein (AFP) [25]. Because of their specificity, DNA aptamers lend themselves to multiplexing and arrays. DNA aptamers have been incorporated into multielectrode arrays for the simultaneous monitoring of multiple tumor markers [25]. However, in these systems, there is no guarantee that a significant conformational change will occur upon target binding. To overcome this problem, a neutralizer displacement is often used. With neutralizer displacement, a weakly interacting “neutralizer” strand initially hybridizes with the DNA aptamer until the target protein binds; upon protein binding, the neutralizer strand is displaced [26]. Despite the advantages of DNA aptamers with respect to specificity, their evolution remains difficult and time-consuming, making their applications for electrochemical platforms limited.

Many of the DNA-based electrochemical detection methods are therefore essentially electrochemical hybridization assays that rely on the thermodynamic stability of specific base pairings. Pursuing an alternative strategy, our laboratory has focused on the intrinsic electronic properties of the DNA double helix as the signaling element for electrochemical DNA detection. We rely on DNA-mediated charge transport (DNA CT), chemistry that is exquisitely sensitive to and reports on the integrity of the DNA duplex [27]. DNA CT can proceed over long molecular distances but is inhibited by intervening base lesions, mismatches, DNA-binding proteins, anything that perturbs the stacking of the DNA base pairs. First reported for long-range, excited-state quenching reactions that occurred through double-stranded DNA [28,29], we have, since that time, explored ground-state DNA CT electrochemically using alkanethiol-modified DNA duplexes self-assembled as DNA monolayers on gold [30–32]. Heterogeneous ET rates through these monolayers to intercalators bound at specific sites along the individual helices are limited by tunneling through the aliphatic thiol linker and not through the much longer DNA π-stack [33]. Remarkably, the presence of just a single intervening base mismatch can be sufficient to cut off DNA CT, even while electrochemical signals to nonintercalative probe molecules (e.g.,

) remain unaffected [31].

These findings signaled to us that we could use the sensitivity of CT through DNA films as the basis for biosensing that did not depend on differential hybridization; any perturbation of π-stacking, even for fully hybridized surfaces, is detected through an attenuated electroc...

Table of contents

• Cover
• Half-Title
• Title
• Copyright
• Contents
• Introduction to the Series
• Contributors to Volume 27
• Contents of Other Series Volumes
• 1 Two-Electrode Platforms for Protein Biosensing Based on Charge Transport through the DNA Double Helix
• 2 Recent Advances in Bipolar Electrochemistry
• 3 X-Ray Absorption Spectroscopy in Electrochemistry: From Fundamentals to Fixed Energy X-Ray Absorption Voltammetry  
• Index