Electrochemical Detection Of Nucleic Acids Research Articles

Exonuclease III–assisted cascade signal amplification strategy for label-free and ultrasensitive electrochemical detection of nucleic acids

In this work, a simple, signal-on and label-free electrochemical biosensor for ultrasensitive DNA detection is reported on the basis of an autocatalytic and exonuclease III (Exo III)-assisted cascade signal amplification strategy. In the presence of target DNA (T-DNA), the hybridization between the 3′-protruding DNA fragment of hairpin DNA probe (HP1) and T-DNA triggered the Exo III cleavage process, accompanied by the releasing of T-DNA and autonomous generation of new DNA fragment which was used for the successive hybridization with the another hairpin DNA (HP2) on the electrode. After the Exo III cleavage process, numerous quadruplex-forming oligomers which caged in HP2 were liberated on the electrode surface and folded into G-quadruplex-hemin complexes with the help of K+ and hemin to give a remarkable electrochemical response. As a result, a low detection limit of 4.83fM with an excellent selectivity toward T-DNA was achieved. The developed electrochemical biosensor should be further extended for the detection of a wide spectrum of analytes and has great potential for the development of ultrasensitive biosensing platform for early diagnosis in gene-related diseases.

Electrochemical detection of nucleic acids, proteins, small molecules and cells using a DNA-nanostructure-based universal biosensing platform.

The occurrence and prognosis of many complex diseases, such as cancers, is associated with the variation of various molecules, including DNA at the genetic level, RNA at the regulatory level, proteins at the functional level and small molecules at the metabolic level (defined collectively as multilevel molecules). Thus it is highly desirable to develop a single platform for detecting multilevel biomarkers for early-stage diagnosis. Here we report a protocol on DNA-nanostructure-based programmable engineering of the biomolecular recognition interface, which provides a universal electrochemical biosensing platform for the ultrasensitive detection of nucleic acids (DNA/RNA), proteins, small molecules and whole cells. The protocol starts with the synthesis of a series of differentially sized, self-assembled tetrahedral DNA nanostructures (TDNs) with site-specifically modified thiol groups that can be readily anchored on the surface of a gold electrode with high reproducibility. By exploiting the rigid structure, nanoscale addressability and versatile functionality of TDNs, one can tailor the type of biomolecular probes appended on individual TDNs for the detection of specific molecules of interest. Target binding occurring on the gold surface patterned with TDNs is quantitatively translated into electrochemical signals via a coupled enzyme-based catalytic process. This uses a sandwich assay strategy in which biotinylated reporter probes recognize TDN-bound target biomolecules, which then allow binding of horseradish-peroxidase-conjugated avidin (avidin-HRP). Hydrogen peroxide (H2O2) is then reduced by avidin-HRP in the presence of TMB (3,3',5,5'-tetramethylbenzidine) to generate a quantitative electrochemical signal. The time range for the entire protocol is ∼1 d, whereas the detection process takes ∼30 min to 3 h.

Biofouling-Resilient Nanoporous Gold Electrochemical Sensors for DNA Detection

Introduction: Rapid detection of biomolecular markers is essential for early diagnosis and management of diseases as well as for fundamental biological studies. Electrochemical sensors have been powerful tools for addressing this need, where nanostructured electrodes have shown significant promise in enhancing the performance of these sensors. In order to develop electrochemical biosensors with rationally-designed nanostructures, it is imperative to have a fundamental understanding of the structure-property relationships that dictate sensor performance. In this study, microfabrication-compatible nanoporous gold (np-Au) thin films with tunable pore morphology are used as a model electrode to investigate the effect of electrode morphology on sensor performance.Specifically, by modifying np-Au pore morphology (i.e., pore size, effective surface area) and employing electrochemical characterization, we studied the effect of morphology on (i) grafting density of 26mer DNA probes, (ii) limit of detection in hybridization to a DNA target, and (iii) dynamic range in detecting different target concentrations1. We subsequently assessed the performance of np-Au electrodes for nucleic acid detection in complex biological media, with the hypothesis that the optimal pore geometry should allow permeation of nucleic acids into the porous electrode while sieving out biofouling molecules. Results and Discussion: Np-Au samples were fabricated by sputtering gold/silver alloy on glass coverslips and subsequent etching of silver to self-assemble the np-Au films (un-annealed np-Au). To obtain samples with different pore morphology (annealed np-Au), the films were thermally-treated at 225 °C. The np-Au sensor displayed a 100-fold shift in dynamic range of detection towards lower concentrations of DNA (10 nm – 200 nM) as compared to a planar gold electrode. Annealed np-Au films showed improved accessibility and a further 10-fold shift in dynamic range (Figure 1). The hybridization on np-Au films can be viewed as a combination of two events: (i) target DNA entering the pores and (ii) target DNA binding with probe upon entering the pore. Geometric accessibility determines the efficiency of a target entering the porous structure. Once inside the porous structure, the probability of a target binding to the probe depends on probe density. Target molecule encounters a significantly higher number of probes within a pore compared to a planar surface. These two factors together (i.e., enhanced accessibility and higher probe grafting density in porous films) led to the detection of lower concentrations of target and resulted in a shift in dynamic range of detection with morphology (Figure 1). A major obstacle to nucleic acid detection in biological fluids is electrode fouling by constituents that obscure the transport of analytes to the active electrode surfaces. Np-Au electrodes circumvent this by enabling DNA detection in physiologically relevant complex media (bovine serum albumin and fetal bovine serum (FBS))2. In contrast, sensor performance was compromised for planar gold electrodes in the same conditions. Un-annealed np-Au electrodes produced a signal suppression (hybridization efficiency) of ~38% with a dynamic range of 25 nM to 200 nM (Figure 2) in FBS. Hybridization efficiency decreased by 10% for np-Au with coarser pores (annealed np-Au) revealing a pore size dependence of sensor performance in biofouling conditions. This nanostructure dependent functionality in complex media suggests that pores with the optimal size act as sieves for blocking the biomolecules from inhibiting the surfaces within the porous volume while allowing the transport of nucleic acids analytes and redox molecules Conclusions: In summary,we demonstrated that np-Au electrodes exhibit high electrochemical sensing performance in the presence of common biofouling media and allow for sensitive electrochemical detection of nucleic acids. The un-annealed np-Au electrodes exhibited the highest signal suppression followed by annealed np-Au and planar Au electrodes in presence of FBS. This indicates that pore size plays a significant role in dictating the extent of biomolecule adsorption and transport. This novel technology for detecting target DNA concentrations in tunable dynamic ranges, coupled with sustained functionality in complex biological media, should assist in the development of robust point-of-care diagnostic platforms.

Ultrasensitive electrochemical detection of nucleic acid by coupling an autonomous cascade target replication and enzyme/gold nanoparticle-based post-amplification

Owing to the intrinsic importance of nucleic acid as bio-targets, the development of isothermal and ultrasensitive electrochemical DNA biosensor is very essential for biological studies and medical diagnostics. Herein, the autonomous cascade DNA replication strategy was effectively married with the enzyme/gold nanoparticle-based post-amplification strategy to promote the detection performance toward target DNA. A hairpin DNA probe (HP) is designed that consists of an overhang at 3′-end as the recognition unit for target DNA, a recognition site for nicking endonuclease, and an alkane spacer to terminate polymerization reaction. The autonomous DNA replication–scission–displacement reaction operated by the nicking endonuclease/KF polymerase induced the autocatalytic opening of HP, which was then specifically bound by the enzyme/gold nanoparticles for further dual-signal amplification toward target-related sensing events. A low detection limit of 0.065fM with an excellent selectivity toward target DNA could be achieved. The proposed biosensor could be also easily regenerated for target detection. The developed biosensor creates an opportunity for the effective coupling of the target replication with post-amplification strategies and thus opens a promising avenue for the detection of nucleic acid with low abundance in bioanalysis and clinical biomedicine.

Label-free and Electrochemical Detection of Nucleic Acids Based on Isothermal Amplification in Combination with Solid-state pH Sensor

Label-free quantification methods for nucleic acids are attracting attention, since they provide potential tools in clinical applications such as disease diagnosis or prognosis observation. In this research, we developed a simpler and cost-effective electrical monitoring device of nucleic acid amplification, combining an isothermal amplification method and an Iridium/Iridium oxide (Ir/IrOx) electrode without labeling. The fabricated Ir/IrOx electrode showed ideal Nernstian response under the various pH buffer solutions, and its potential response was less affected even in the presence of charged proteins. Moreover, we successfully real-time monitored primer generation-rolling circle amplification (PG-RCA) at the 0, 10, 100, 1000 pM of target DNA by detection of released proton during amplification reaction at the constant reaction temperature. This label-free and portable device might be received considerable attention as a useful platform for a point-of-care testing in clinical use.

Ultrasensitive Electrochemical Detection of Nucleic Acids Based on the Dual-Signaling Electrochemical Ratiometric Method and Exonuclease III-Assisted Target Recycling Amplification Strategy.

Because of the intrinsic importance of nucleic acids as biotargets, the simple and sensitive detection of nucleic acids is very essential for biological studies and medical diagnostics. In this work, a novel, simple, and selective electrochemical DNA biosensor for the sensitive detection of target DNA (T-DNA) has been developed based on the dual-signaling electrochemical ratiometric method and exonuclease III (Exo III)-assisted target recycling amplification strategy. The assay strategy includes both "signal-on" and "signal-off" elements. The stem-loop (hairpin) DNA capture probe (HP), which was labeled by thiolated methylene blue (MB) at the 3'-protruding termini and ferrocene (Fc) in the middle of the loop, first self-assembled on the gold electrode surface via a Au-S bond. In the presence of T-DNA, the T-DNA hybridized with HP, which triggered the Exo III cleavage process and accompanied the release of T-DNA. As a result, the MB tags were away from and the Fc tags close to the gold electrode surface, leading to the decrease of the oxidation peak current of MB (I(MB)) and the increase of that of Fc (I(Fc)). The value of ΔI(Fc)/|ΔI(MB)| (ΔI(Fc) and ΔI(MB) are the change values of the oxidation peak currents of Fc and MB, respectively) is linear with the concentration of T-DNA from 0.01 pM to 0.8 pM. The detection limit (4.16 fM) of the developed method is much lower than that of the most reported electrochemical DNA biosensors. This strategy provides a simple and sensitive approach for the detection of T-DNA and has promising applications in bioanalysis, disease diagnostics, and clinical biomedicine.

Developing Fully-Integrated Biosensing Systems on the Laboratory Benchtop

Fully-integrated biosensing systems that detect the presence, absence, and quantity of biomolecules (nucleic acids and proteins) in clinical samples have a wide range of applications in clinical diagnosis of cancers, infectious diseases, and hereditary diseases. Furthermore, handheld and inexpensive biosensors that can be operated with a high degree of automation by novice users make it possible for these systems to be applied to point-of-care (POC) settings where the current state-of-the-art diagnostic tests, designed for centralized laboratories, are not applicable. In order to develop handheld, automated, and inexpensive biosensing systems applicable to the point-of-care, it is essential to combine multiple devices for sample preparation and biomolecular detection on the same platform. This requires multiple materials and structures having a wide range of properties and length-scales to be integrated on the same platform. Furthermore, translation of such multi-material systems from the laboratory to the market indicates the need for inexpensive, rapid, and scalable fabrication methods for creating tunable materials and structure. We have developed an integrated fabrication process for creating all-electrical biosensing platforms using methods available on the laboratory benchtop. In order to create electrodes that are structurally tunable from the 10 nm to the 10 mm length-scale and meet the conductivity demands of sample preparation and biosensing devices, we have combined methods including craft cutting, ink-jet printing, self-assembly, electroless deposition, and electrodeposition to deposit and pattern application-specific materials on polymer substrates. In addition, we have used shape memory polymers to further tune the properties of the patterned electrodes such as surface texture, film thickness, and conductivity. Materials characterization results obtained using techniques including scanning electron microscopy, atomic force microscopy, and four-point-probe method indicate that the fabrication method results in electrodes of the appropriate configuration (micro-coils, interdigitated electrodes, electrode arrays), surface texture (tunable from tens of nanometer to tens of microns), and conductivity (>5% of the bulk) for use in an all-electrical biosensing platform. Using the abovementioned fabrication methods, we have developed three classes of devices for bacterial lysis, magnetic manipulation of cells and biomolecules, and detection of nucleic acids. Bacterial lysis devices that include an interdigitated array of microelectrodes decorated with electrodeposited three-dimensional nano-pillars offer high efficiency lysis (> 95%) at low applied potentials (4 V). The magnetic manipulation devices containing on-polymer magnetic coils coupled with a ferromagnetic capture layer generate forces required for manipulating super-paramagnetic microbeads (1-10 pN) at low applied currents (< 50 mA). Furthermore, we have created a fully-integrated electro-fluidic device for electrochemical detection of nucleic acids. Given that all of these devices are created using the rapid, inexpensive and benchtop-based integrated fabrication process presented here, we expect this method to be applicable to the rapid prototyping and fabrication of several fully-integrated biosensing systems for POC diagnostic applications.

Alkaline-Phosphatase-Based Nanostructure Assemblies for Electrochemical Detection of microRNAs.

Different nanoarchitectures, rich in enzyme labels, are herein investigated for signal amplification in the electrochemical detection of nucleic acids and in particular of microRNAs. Dendritic amplification, accomplished by the use of streptavidin and biotinylated alkaline phosphatase, and enzyme-decorated liposomes are used as labels to amplify the microRNA-sensing, by their association to the probe-microRNA hybrid generated onto a gold transducer. Differential pulse voltammetry and faradaic impedance spectroscopy were employed to characterize these different amplification routes.

Effect of microelectrode structure on electrocatalysis at nucleic acid-modified sensors.

The electrochemical detection of nucleic acids using an electrocatalytic reporter system and nanostructured microelectrodes is a powerful approach to ultrasensitive biosensing. In this report we systematically study for the first time the behavior of an electrocatalytic reporter system at nucleic acid-modified electrodes with varying structures and sizes. [Ru(NH3)6](3+) is used as a primary electron acceptor that is electrostatically attracted to nucleic acid-modified electrodes, and [Fe(CN)6](3-) is introduced into the redox system as a secondary electron acceptor to regenerate Ru(3+) after electrochemical reduction. We found that the electrode structure has strong impact on mass transport and electron-transfer kinetics, with structures of specific dimensions yielding much higher electrochemical signals and catalytic efficiencies. The electrocatalytic signals obtained when gold sensors were electrodeposited in both circular and linear apertures were studied, and the smallest structures plated in linear apertures were found to exhibit the best performance with high current densities and turnover rates. This study provides important information for optimal assay performance and insights for the future design and fabrication of high performance biomolecular assays.

Zinc Oxide Nanowire Decorated Single‐Use Electrodes for Electrochemical DNA Detection

The surfaces of pencil graphite electrodes (PGEs) were decorated with zinc oxide nanowires (ZnO NWs) for the electrochemical detection of nucleic acids. ZnO NWs were synthesized through simple hydrothermal method. PGEs decorated with ZnO NWs (ZnO NW/PGEs) were electrochemically characterized through cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) following morphological characterization through transmission (TEM) and scanning electron microscopy (SEM). Enhanced sensor response obtained using ZnO NW/PGEs contrary to the bare PGE (control) samples. Our preliminary results simply reveal the potential of combining ZnO NWs with disposable sensor technology for the electrochemical detection of DNA.

Immobilization-free programmable hairpin probe for ultrasensitive electronic monitoring of nucleic acid based on a biphasic reaction mode.

This work designs a novel programmable hairpin probe (PHP) for the immobilization-free electrochemical detection of nucleic acid by coupling polymerase/nicking-induced isothermal signal amplification strategy with a biphasic reaction mode for the first time. The designed PHP (including a target-recognition region, a template sequence for enzymatic reaction and an inactivated anti-streptavidin aptamer) could program multiple isothermal reactions in the solution phase accompanying in situ amplified detectable signal at the electrode surface by the labeled ferrocene tag on the PHP. Upon addition of target analyte into the detection solution, target DNA initially hybridized with the recognition region on the PHP. Replication-induced strand-displacement generated an activated anti-streptavidin aptamer with the assistance of polymerase. Then, the polymerase/nicking enzymes could cleave and polymerize repeatedly the replication product, thus resulting in the formation of numerous template-complementary DNA initiator strands. The released initiator strands could retrigger the programmable hairpin probe to produce a large number of activated anti-streptavidin aptamers, which could be captured by the immobilized streptavidin on the electrode, thus activating the electrical contact between the labeled ferrocene and the electrode. Going after the aptamers, the carried ferrocene could produce the in situ amplified electronic signal within the applied potentials. Under optimal conditions, the electrochemical signal increased with the increasing target DNA concentration in the dynamic range from 5 fM to 10 pM with a detection limit (LOD) of 2.56 fM at the 3sblank criterion. Importantly, the methodology with high specificity was also validated and evaluated by assaying 6 target DNA-spiked human serum and calf thymus DNA samples, and the recoveries were 95-110%. Further work for the programmable hairpin probe could be even utilized in a real world sample to detect miRNA-21 at femtomol level.

Exonuclease III-Aided Autocatalytic DNA Biosensing Platform for Immobilization-Free and Ultrasensitive Electrochemical Detection of Nucleic Acid and Protein

Homogenous electrochemical biosensor has attracted substantial attention owing to its simplicity, rapid response, and improved recognition efficiency compared with heterogeneous biosensor, but the relatively low detection sensitivity and the limited detection analytes prohibit its potential applications. To address these issues, herein, a simple, rapid, isothermal, and ultrasensitive homogeneous electrochemical DNA biosensing platform for target DNA and protein detection has been developed on the basis of an exonuclease III (Exo III)-aided autocatalytic target recycling strategy. A ferrocene-labeled hairpin probe (HP1) is ingeniously designed, which contains a protruding DNA fragment at 3'-termini as the recognition unit for target DNA. Also, the DNA fragment that could be used as secondary target analogue was introduced, but it was caged in the stem region of HP1. In the presence of target DNA, its recognition with the protruding fragment of HP1 triggered the Exo III cleavage process, accompanied with the target recycling and autonomous generation of secondary target analogues. This accordingly resulted into the autonomous accumulation of ferrocene-labeled mononucleotide, inducing a distinct increase in the electrochemical signal owing to its elevated diffusivity toward indium tin oxide (ITO) electrode surface. The autocatalytic biosensing system was further extended for protein detection by advising an aptamer hairpin switch with the use of thrombin as a model analyte. The current developed autocatalytic and homogeneous strategy provided an ultrasensitive electrochemical detection of DNA and thrombin down to the 0.1 and 5 pM level, respectively, with a high selectivity. It should be further used as a general autocatalytic and homogeneous strategy toward the detection of a wide spectrum of analytes and may be associated with more analytical techniques. Thus, it holds great potential for the development of ultrasensitive biosensing platform for the applications in bioanalysis, disease diagnostics, and clinical biomedicine.

DNAzyme-guided polymerization of aniline for ultrasensitive electrochemical detection of nucleic acid with bio-bar codes-initiated rolling circle amplification

A novel and versatile amplified electrochemical strategy for ultrasensitive detection of nucleic acid has been developed on the basis of polyaniline (PANI) as the redox label which was guided and deposited by multiple HRP-mimicking DNAzyme as a powerful catalyst and template generated by bio-bar codes-initiated rolling circle amplification (RCA). The “sandwich-type” DNA biosensor was fabricated with the thiol-functionalized capture DNA first immobilized on an Au electrode and hybridized with one end of target DNA, the other end of which was recognized with a signal DNA probe that on the bio-bar codes, respectively. After the RCA reaction, multiple HRP-mimicking DNAzyme were successfully formed, which subsequently catalyzed the oxidation of aniline to PANI and lead to a readily measurable “turn-on” electrochemical signal. As a result of multiple amplification effects, the constructed platform exhibited a good linear response toward nucleic acid over a wide range of concentration from 6.0fM to 1.0nM.

Enzyme-free and ultrasensitive electrochemical detection of nucleic acids by target catalyzed hairpin assembly followed with hybridization chain reaction

An isothermal, enzyme-free and ultrasensitive protocol for electrochemical detection of DNA is proposed based on the ingenious combination of target catalyzed hairpin assembly and hybridization chain reaction (HCR) strategies for two-step signal amplification. The DNA hairpin assembly on the electrode is triggered by target DNA, accompanied by the release of target DNA for the successive hybridization and assembly process. The newly emerging DNA fragment on the electrode after hairpin assembly is further used to propagate the HCR between methylene blue-labeled signal probe and auxiliary probe, inducing a remarkably amplified electrochemical signal. The current dual signal amplification strategy is relatively simple and inexpensive owing to avoid the use of any kind of enzyme or sophisticated equipment. It can achieve a sensitivity of 0.1fM with a wide linear dynamic range from 1×10−15 to 1×10−10M and discriminate mismatched DNA from perfect matched target DNA with a high selectivity. The high sensitivity and selectivity make this method a great potential for early diagnosis in gene-related diseases.

Label-Free and Ultrasensitive Electrochemical Detection of Nucleic Acids Based on Autocatalytic and Exonuclease III-Assisted Target Recycling Strategy

In this work, a very simple, label-free, isothermal, and ultrasensitive electrochemical DNA biosensor has been developed on the basis of an autocatalytic and exonuclease III (Exo III)-assisted target recycling amplification strategy. A duplex DNA probe constructed by the hybridization of a quadruplex-forming oligomer with a molecular beacon is ingeniously designed and assembled on the electrode as recognition element. Upon sensing of the analyte nucleic acid, the strand of molecular beacon in the duplex DNA probe could be stepwise removed by Exo III accompanied by the releasing of target DNA and autonomous generation of new secondary target DNA fragment for the successive hybridization and cleavage process. Simultaneously, numerous quadruplex-forming oligomers are liberated and folded into G-quadruplex-hemin complexes with the help of K(+) and hemin on the electrode surface to give a remarkable electrochemical response. Because of this autocatalytic target recycling amplification and the specifically catalyzed formation of G-quadruplex-hemin complexes, this newly designed protocol provides an ultrasensitive electrochemical detection of DNA down to the 10 fM level, can discriminate mismatched DNA from perfectly matched target DNA, and holds a great potential for early diagnosis in gene-related diseases. It further could be developed as a universal protocol for the detection of various DNA sequences and may be extended for the detection of aptamer-binding molecules.

Bionic catalysis of porphyrin for electrochemical detection of nucleic acids

A novel electrochemical strategy was designed for the detection of DNA based on the bionic catalysis of porphyrin. The detection probe was prepared via the assembly of thiolated double strand DNA (dsDNA) with gold nanoparticles (AuNPs), and then interacted with cationic iron (III) meso-tetrakis (N-methylphyridinum-4-yl) porphyrin (FeTMPyP) via groove binding along the dsDNA surface. The resulting nanocomplex was characterized with transmission electron microscopy, UV–vis absorption and circular dichroism spectroscopy. The FeTMPyP–DNA–AuNPs probe on gold electrode demonstrated the excellent electrocatalytic behaviors toward the reduction of O2 due to the largely loading of FeTMPyP and good conductivity. Based on bionic catalysis of porphyrin for the reduction of O2, the resulting biosensor exhibited a good performance for the detection of DNA with a wide linear range from 1×10−12 to 1×10−8molL−1 and detection limit of 2.5×10−13molL−1 at the signal/noise of 3. More importantly, the biosensor presented excellent ability to discriminate the perfectly complementary target and the mismatched stand. This strategy could be conveniently extended for detection of other biomolecules. To the best of our knowledge, this is the first application of bionic catalysis of porphyrin as detection probe and opens new opportunities for sensitive detection of biorecognition events.

Ultrasensitive Electrochemical Detection of Nucleic Acids by Template Enhanced Hybridization Followed with Rolling Circle Amplification

An ultrasensitive protocol for electrochemical detection of DNA is designed with quantum dots (QDs) as a signal tag by combining the template enhanced hybridization process (TEHP) and rolling circle amplification (RCA). Upon the recognition of the molecular beacon (MB) to target DNA, the MB hybridizes with assistants and target DNA to form a ternary ''Y-junction''. The target DNA can be dissociated from the structure under the reaction of nicking endonuclease to initiate the next hybridization process. The template enhanced MB fragments further act as the primers of the RCA reaction to produce thousands of repeated oligonucleotide sequences, which can bind with oligonucleotide functionalized QDs. The attached signal tags can be easily read out by square-wave voltammetry after dissolving with acid. Because of the cascade signal amplification and the specific TEHP and RCA reaction, this newly designed protocol provides an ultrasensitive electrochemical detection of DNA down to the attomolar level (11 aM) with a linear range of 6 orders of magnitude (from 1 × 10(-17) to 1 × 10(-11) M) and can discriminate mismatched DNA from perfect matched target DNA with high selectivity. The high sensitivity and specificity make this method a great potential for early diagnosis in gene-related diseases.

Nanowires array modified electrode for enhanced electrochemical detection of nucleic acid

The gold nanowires array electrode (AuNWsA) was synthesized by two step electrodeposition, which provided well oriented vertically aligned nanowires. The dimensions of the nanowires were determined by scanning electron micrograph and found to be around 1.5μm in length with 200nm diameter. Each nanowire was separated by a distance of 2–3 times the diameter of the nanowire itself. The electrochemical performance of the AuNWsA electrode was evaluated using cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). Using these analytical tools, this AuNWsA electrode was shown to have a high effective surface area and excellent electron transfer surfaces compared with flat bare Au electrode. The AuNWsA electrode was then used as an electrochemical biosensor electrode by immobilizing probe DNA and analyzed by CV, EIS and Fourier transform infrared measurements. The results of this analysis suggested that the AuNWsA electrode provides good surfaces for the immobilization and hybridization of DNA. The selectivity of the probe DNA immobilized AuNWsA electrode was tested using non-complementary and one base pair mismatching DNA. The detection limit of the AuNWsA electrode was determined to be 6.78×10−9M, which is two times smaller than the bare Au electrode.

Signal-on electrochemical Y or junction probe detection of nucleic acid

A methylene-blue (MB)-labeled molecular beacon junction probe allows for a signal-on electrochemical detection of nucleic acids via target recycling using endonucleases. Electron transfer is reduced when the MB is intercalated in the stem of the molecular beacon, but then electron transfer from MB to a gold electrode is enhanced upon cleavage of the junction probe due to increased probability of MB approaching the electrode when attached to the more flexible ssDNA.

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.