Electrochemical Detection Of Nucleic Acids Research Articles

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this "E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

Sequence-specific electrochemical detection of nucleic acids in real samples

This paper reviews past and current developments in the field of electrochemical biosensors with a focus on the sequence-specific detection of nucleic acids in real samples. After electrochemical hybridization sensors had been first described in 1993, it took nearly a decade until some of the many proposed protocols were indeed applied to real samples like blood or tissue. Electrochemical transduction schemes used either rely on electroactive moieties such as intercalators, groove binders, covalently attached labels, and products of enzyme markers or they are completely indicator free like impedance-based detection principles. Most detection schemes require a polymerase chain reaction amplification step to allow for sufficient selectivity and sensitivity. Today, several companies develop electrochemical microarrays able to detect dozens to many thousands of sequences in a single experiment.

Ultrasensitive electrochemical detection of nucleic acid based on the isothermal strand-displacement polymerase reaction and enzyme dual amplification

We describe an ultrasensitive electrochemical detection of DNA protocol based on the isothermal strand-displacement polymerase reaction (ISDPR) and enzyme dual amplifications. Target DNA triggered an ISDPR to produce numerous bi-functionalized duplex DNA complexes. Following an immuno-magnetic collection via an immunoreaction between the attached digoxin on the duplex DNA and the anti-digoxin antibody on the magnetic bead, horseradish (HRP) tracers were bound to the duplex DNA through a biotin–streptavidin interaction. The quantification of DNA was realized by square wave voltammetric detection of the enzymatic products with a screen-printed gold electrode. The voltammetric response was proportional to the concentration of DNA in the range of 0.1 fM–0.5 pM, and the limit of detection was estimated to be 0.06 fM. The new protocol showed great promise for simple, cost-effective, and quantitative gene analysis.

DNA Diagnostics: Nanotechnology-Enhanced Electrochemical Detection of Nucleic Acids

The detection of mismatched base pairs in DNA plays a crucial role in the diagnosis of genetic-related diseases and conditions, especially for early stage treatment. Among the various biosensors that have been used for DNA detection, EC sensors show great promise because they are capable of precise DNA recognition and efficient signal transduction. Advancements in micro- and nanotechnologies, specifically fabrication techniques and new nanomaterials, have enabled for the development of highly sensitive, highly specific sensors making them attractive for the detection of small sequence variations. Furthermore, the integration of sensors with sample preparation and fluidic processes enables for rapid, multiplexed DNA detection essential for POC clinical diagnostics.

Functionalized GaN nanowire-based electrode for direct label-free voltammetric detection of DNA hybridization

This study demonstrates the utility of functionalized GaN nanowires (GaNNWs) for electrochemical detection of nucleic acids, in aqueous solution, using cyclic voltammetry. In order to link probe DNA to the NW surface, we employed an organosulfur compound, 3-mercaptopropyl trimethoxysilane (MPTS), to functionalize the GaNNW surface. Interestingly, the MPTS-modified GaNNWs exhibited a potential window of 4.5 V, the widest reported to date, with very low background current, which provides an advantage for sensing DNA immobilization/hybridization, down to sub-pM concentration, via monitoring adenine and guanine oxidation. The oxidation of guanine was characterized by its peak potential and peak current, where the former serves as a fingerprint for DNA hybridization and the latter as a parameter for the extent of hybridization. Moreover, the GaNNW-based sensor exhibited excellent consistency in hybridization-dehybridization-rehybridization cycles.

Direct DNA Hybridization at Disposable Graphite Electrodes Modified with Carbon Nanotubes

The performance of glassy carbon (GCE) and graphite pencil electrodes (PGE) modified with multiwalled carbon nanotubes (CNTs) are compared, based on the direct electrochemical detection of nucleic acids. This is accomplished by monitoring the differential pulse voltammetry changes of the guanine signal. CNT-modified PGE compares favorably to that of the commonly used CNT-modified GCE owing to the intrinsic improved performance of the supporting PGE. The better intrinsic characteristics of the PGE are related to its composite structure and higher level of porosity compared to GCE. The performance characteristics of the direct DNA hybridization on the disposable CNT-modified PGE are studied in terms of optimum analytical conditions such as probe concentration, target concentration, hybridization time, and selectivity. The new DNA biosensor described here has shown some important advantages such being inexpensive, sensitive, selective, and able to generate reproducible results using a simple and direct electrochemical protocol.

Effect of Tin-Doped Indium Oxide Electrode Preparation Methods on the Mediated Electrochemical Detection of Nucleic Acids

Oxidation of guanine residues in nucleic acids using a metal mediator, tris(bipyridyl) ruthenium(II) (Ru(bpy)32+), provides a sensitive and simple method for detection of unmodified nucleic acids. ...

Flow Detection of Nucleic Acids at a Conducting Polymer-Modified Electrode

A new protocol for electrochemical detection of nucleic acids, based on polypyrrole (PPy)-coated electrodes, is demonstrated using flow injection analysis. The conducting polymer detection scheme offers a fast, sensitive, universal detection of oligonucleotides, DNA, and RNA. The response is attributed to the adsorption/desorption of the nucleic acids onto the PPy film during the passage of the sample plug over the coated electrode. The influence of various experimental parameters of the film preparation and the electrochemical detection has been investigated. Optimal performance has been achieved using the nitrate film dopant, a detection potential of −0.15 V (vs Ag/AgCl), and a flow rate of 0.6 mL/min. Good linearity and reproducibility (RSD = 2.1%) are observed, along with very low detection limits (e.g., 6.1 × 10-16 mol of dsDNA in the 20-μL samples). Compared to common detectors based on the electroactivity of DNA, the conducting polymer detection scheme is not limited to purine-containing nucleic acids or to a strongly alkaline media and is less susceptible to interferences from easily oxidizable species (e.g., ascorbic acid). The attractive behavior of the PPy flow detector holds great promise for monitoring nucleic acids in other flow systems, microscale separations, and on-chip platforms.