Electrochemical Detection Of Nucleic Acids Research Articles

Label-free and ultrasensitive electrochemical detection of nucleic acids based on an exonuclease III-assisted target recycling amplification strategy using a heated gold disk electrode.

The present work demonstrates a label-free, rapid and ultrasensitive electrochemical sensor for specific DNA detection with an exonuclease III (Exo III)-assisted target recycling amplification strategy and elevated electrode temperature at a heated gold disk electrode (HAuDE). The proposed electrochemical DNA (E-DNA) sensor was designed such that in the presence of the target DNA, the electrode self-assembled capture probe hybridizes with the target DNA to form a duplex structure, which triggers Exo III to specifically recognize this structure and selectively digest the capture probe, while the released target DNA underwent recycling to hybridize with a new capture probe, leading to the gradual digestion of a large amount of capture probes. It was found that during the digestion period, the activity of Exo III could be significantly improved by elevating electrode temperature, thus promoting the digestion reaction and improving the sensitivity for target DNA detection. Furthermore, an electrochemical indicator ([Ru(NH3)6]3+) was electrostatically bound to the capture probe, leading to a significant square wave voltammetry (SWV) response, which directly related to the amount and length of the capture probes remaining in the electrode and provided a quantitative measure for target DNA detection. The proposed strategy realized the highly sensitive detection of the target DNA with a detection limit of 26 aM (S/N = 3) at an electrode temperature of 40 °C during the digestion period, which was about two magnitudes lower than that at 24 °C.

Pseudouridine-modified RNA probe for label-free electrochemical detection of nucleic acids on 2D MoS2 nanosheets.

RNA modification, particularly pseudouridine (Ψ), has played an important role in the development of the mRNA-based COVID-19 vaccine. This is because Ψ enhances RNA stability against nuclease activity and decreases the anti-RNA immune response. Ψ also provides structural flexibility to RNA by enhancing base stacking compared with canonical nucleobases. In this report, we demonstrate the first application of pseudouridine-modified RNA as a probe (Ψ-RNA) for label-free nucleic acid biosensing. It is known that MoS2 has a differential affinity for nucleic acids, which may be translated into a unique electronic signal. Herein, the Ψ-RNA probe interacts with the pristine MoS2 surface and causes a change in interfacial electrochemical charge transfer in the MoS2 nanosheets. Compared with an unmodified RNA probe, Ψ-RNA exhibited faster adsorption and higher affinity for MoS2. Moreover, Ψ-RNA could bind to complementary RNA and DNA targets with almost equal affinity when engaged with the MoS2 surface. Ψ-RNA maintained robust interactions with the MoS2 surface following the hybridization event, perhaps through its extra amino group. The detection sensitivity of the Ψ-RNA/MoS2 platform was as low as 500 attomoles, while the results also indicate that the probe can distinguish between complementary targets, single mismatches, and non-complementary nucleic acid sequences with statistical significance. This proof-of-concept study shows that the Ψ-RNA probe may solve numerous problems of adsorption-based biosensing platforms due to its stability and structural flexibility.

Biocompatible Surface Modification with DNA and Proteins for Carbon Electrode-Based Biosensing

Electrochemical biosensors, which combine biorecognition elements with electrochemical readout to enable sensitive and specific sensing using inexpensive, simple equipment, are a major area of research for the development of “point-of-care” (POC) diagnostics that can be utilized in low-resource settings. Commercially available screen-printed carbon electrodes (SPCEs) are often used for electrochemical biosensors due to its low cost and fabrication from abundant resources. Forming a high quality, stable, and homogenous layer of biomolecules on the carbon surface is crucial for efficient sensing; however, the chemistries used to modify carbon electrodes with biomolecules are either insufficiently specific, susceptible to stripping, or unapproachable due to over-engineering. In this talk, I describe a new, simple chemical strategy to functionalize carbon electrodes with biomolecules for the electrochemical detection of nucleic acids, enzymes, and whole cells.

Electrokinetic Enrichment and Label-Free Electrochemical Detection of Nucleic Acids by Conduction of Ions along the Surface of Bioconjugated Beads.

In this paper, we report a method to integrate the electrokinetic pre-enrichment of nucleic acids within a bed of probe-modified microbeads with their label-free electrochemical detection. In this detection scheme, hybridization of locally enriched target nucleic acids to the beads modulates the conduction of ions along the bead surfaces. This is a fundamental advancement in that this mechanism is similar to that observed in nanopore sensors, yet occurs in a bed of microbeads with microscale interstices. In application, this approach has several distinct advantages. First, electrokinetic enrichment requires only a simple DC power supply, and in combination with nonoptical detection, it makes this method amenable to point-of-care applications. Second, the sensor is easy to fabricate and comprises a packed bed of commercially available microbeads, which can be readily modified with a wide range of probe types, thereby making this a versatile platform. Finally, the sensor is highly sensitive (picomolar) despite the modest 100-fold pre-enrichment we employ here by faradaic ion concentration polarization (fICP). Further gains are anticipated under conditions for fICP focusing that are known to yield higher enrichment factors (up to 100,000-fold enrichment). Here, we demonstrate the detection of 3.7 pM single-stranded DNA complementary to the bead-bound oligoprobe, following a 30 min single step of enrichment and hybridization. Our results indicate that a shift in the slope of a current-voltage curve occurs upon hybridization and that this shift is proportional to the logarithm of the concentration of target DNA. Finally, we investigate the proposed mechanism of sensing by developing a numerical simulation that shows an increase in ion flux through the bed of insulating beads, given the changes in surface charge and zeta potential, consistent with our experimental conditions.

Low-Cost High-Resolution Potentiostat for Electrochemical Detection of Nucleic Acids and Biomolecular Interactions.

A handheld USB-powered instrument developed for the electrochemical detection of nucleic acids and biomolecular interactions is presented. The proposed instrument is capable of scanning ± 2.25 V while measuring currents up to ±10 mA, with a minimum current resolution of 6.87 pA. Therefore, it is suitable for nucleic acid sensors, which have high background currents. A low-cost microcontroller with an on-chip 16-bit analog-to-digital converter, 12-bit digital-to-analog converter, and a built-in USB controller were used to miniaturize the system. The offset voltages and gain errors of the analog peripherals were calibrated to obtain a superior performance. Thus, a similar performance to those of the market-leader potentiostats was achieved, but at a fraction of their cost and size. The performance of the application of this proposed architecture was tested successfully and was found to be similar to a leading commercial device through a clinical application in the aspects of the detection of nucleic acids, such as calf thymus ssDNA and dsDNA, and their interactions with a protein (BSA) by using single-use graphite electrodes in combination with the differential pulse voltammetry technique.

Diagnostic kit based on halloysite nanoclay-ionic liquid nanocomposite modified electrode for electrochemical determination of cancer biomarker

Nucleic acid hybridization is occurred between the selective single-stranded nucleic acid sequence and its target sequence, which is one of the essential procedure for electrochemical detection of nucleic acid. microRNA-21 (miRNA-21) is known as a biomarker in various cancers. The determination of miRNA-21 was attained through by hybridization of inosine substituted miRNA-21 specific DNA probe (Pinosine) with its target miRNA-21. In this study, the surface of pencil graphite electrode (PGE) was firstly modified with halloysite nanoclay-ionic liquid (HNT/IL) nanocomposite. The characterization of surface was performed by Scanning Electron Microscope (SEM) images and Energy Dispersive X-Ray Analysis (EDX) analysis, and the differences at surface modifications were also shown by electrochemical methods with electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). For sensitive and selective determination of miRNA-21, Pinosine and target miRNA concentration, immobilization and hybridization time were optimized by using HNT/IL modified PGE in combination with differential pulse voltammetry (DPV). The detection limit was achieved as 0.17 μg/mL (equals to 23.69 nM) in the linear range of 0.25–2 μg/mL miRNA-21. The selectivity of voltammetric method based on HNT/IL-PGE developed for miRNA-21 was examined in the presence of mismatch (MM) and non-complementary (NC) sequences. Because miRNA-21 is over-expressed in cancer cells, it has been tested in total RNA samples isolated from cancer cell line (breast cancer cell line, MCF-7). In the total RNA samples obtained from MCF-7, the detection limit was calculated as 0.28 μg/mL in the linear range of 1–4 μg/mL. Besides, the healthy cell line (human embryonic kidney cell line, HEK-293) was used as a control group and the results obtained by MCF-7 total RNA samples were compared to the results using HEK-293 total RNA samples in terms of miRNA-21 level.

Antifouling lipid membrane coupled with silver nanoparticles for electrochemical detection of nucleic acids in biological fluids

Electrochemical method capable of detecting specific nucleic acids in complex fluid will undoubtedly advance the diagnosis of many kinds of diseases. Herein, by coupling lipid membrane with silver nanoparticles (AgNPs), we develop a new electrochemical method for sensitive and reliable detection of nucleic acids in biological fluids. The advantages of lipid membrane especially its excellent antifouling ability is employed to enhance the applicability of the method in complex environment; while the significant solid-state Ag/AgCl response of AgNPs is used to ensure the detection sensitivity of the method. The core of this method's workflow is the target-induced Y-shape structure formation, which results in the recruitment of AgNPs to the electrode surface, producing considerable electrochemical responses used for target nucleic acid detection. Taking highly upregulated in liver cancer (HULC), a liver cancer-related long non-coding RNA as a model target, the method exhibits high sensitivity, specificity, and reproducibility with a detection limit of 0.42 fM. Moreover, the method displays desirable usability in biological fluids such as serum, which will be of great potential in clinical diagnosis.

Sensitive Electrochemical Detection of Nucleic Acids Following Electrokinetic Enrichment in a Bed of Bioconjugated Beads for Point-of-Care Testing

In this presentation, we demonstrate that ion concentration polarization (ICP) in the presence of fluid flow drives focusing and efficient capture of target nucleic acids within a bed of oligoprobe-conjugated beads. A key finding is that ion conduction along the surface of the bioconjugated beads is the dominant contributor to current to the electrode that drives ICP, under the condition of ICP-induced depletion of ions from the bulk solution. Therefore, hybridization of a target nucleic acid (a polyanion) to the bead surface leads to a shift in the slope of the current-voltage curve. This approach is versatile in that a target nucleic acid can be detected electrically, in the absence of a label, or electrochemically via a label that generates an electroactive product. The resulting approach allows for a plug-and-play nucleic acid sensor using off-the-shelf bioconjugated beads and simple electronics, making it advantageous for point-of-care testing.

Fuel strand-powered self-propelled electrochemical DNA machine for enzyme-free and distinctly amplified detection of nucleic acid with tunable performance

In this work, a self-propelled DNA machine was proposed for enzyme-free and ultrasensitive electrochemical detection of nucleic acid, which was based on a new strategy of the fuel strand-powered target recycling and successive proximity-based inter-strand displacement for distinct signal amplification. A three-strand duplex DNA (TSD) probe was immobilized onto electrode for target DNA recognition via a fuel strand-powered strand displacement circuit, accompanied with the target recycling and the association of fuel strand onto electrode for signal amplification and readout. The associated fuel strand contained a protruding tail sequence that could be used as a target analogy to activate the neighboring TSD probe based on the proximity-based inter-strand displacement effect, inducing the self-propelled association of fuel strand onto electrode for further signal amplification. The detection performance (dynamic response range, linear interval and detection limit) of current electrochemical DNA machine could be interestingly tuned by changing the tail sequence length of fuel strand, showing the potential for different analysis requirements. The lowest detection limit of 0.1 fM could be achieved, which was lower about two orders of magnitude than that by the typical fuel strand-powered target recycling strategy. Therefore, the developed sensing system exhibits a new, simple and powerful means for amplified detection of nucleic acid and may hold great potentials in bioanalysis, disease diagnosis and biomedicine.

Ultrasensitive detection of microRNA based on a homogeneous label-free electrochemical platform using G-triplex/methylene blue as a signal generator

The electrochemical methods for microRNA (miRNA) detection have received increasing attention because high portability and affordability of electrochemical biosensors may facilitate point-of-care quantitative detection of miRNAs. Among these biosensors, the homogenous label-free electrochemical biosensors for miRNAs are rarely reported due to the lack of a universal and efficient signal read-out-mode. A newly discovered G-triplex, 5′-CTGGGAGGGAGGGA-3’ (denoted as G3), can specifically bind with methylene blue (MB), leading to a significant decrease of the diffusion current of MB. By using miRNAs as a driving force, a two-stage isothermal exponential amplification reaction was proposed to generate G3 through miRNAs. The generated G3 can combine with MB and produce observable current changes, which depend on the concentration of miRNAs. Therefore, a novel homogeneous label-free electrochemical biosensor for miRNA detection was successfully constructed. By choosing let-7a, the down-regulation of which is possibly associated with the over-expression of RAS and HMGA2 oncogenes, as a model, we discovered that this biosensor demonstrated excellent analytical performance in detecting let-7a, with an ultralow limit of detection (0.45 fM) and high specificity (discriminating one nucleotide variation). Moreover, the proposed biosensor was successfully applied in monitoring the expression levels of the low-abundant miRNAs in the human lung adenocarcinoma cell lines. This assay successfully verified the feasibility of G-triplex/MB as an efficient and sensitive probe for immobilization-free and label-free electrochemical detection of nucleic acids, which would greatly promote the rapid development of homogeneous label-free electrochemical biosensors.

Exonuclease III-assisted positive feedback signal amplification strategy for ultrasensitive electrochemical detection of nucleic acids

Diffusion-mediated homogeneous electrochemical assay has attracted substantial attention owing to its simplicity, rapid response, and high recognition efficiency, however, lack of inherent connection between signal reporter and electrode surface restricts the improvement of detection sensitivity and further limits its potential applications. Herein, a facile electrochemical strategy is proposed through integrating the advantages of traditional heterogeneous assay and homogenous assay. In this strategy, a small amount of target DNA can trigger the release of assist DNA from the electrode surface. Then, the assist DNA can induce the homogeneous amplification reaction, which in turn promotes the amplification on the electrode surface by generating numerous target DNA analogues, thereby realizing positive feedback signal amplification. As a result, ultrasensitive electrochemical detection of nucleic acids was achieved in the dynamic range from 5 aM to 5 fM with a detection limit down to 1.2 aM. There is about 2.5 orders of magnitude improvement in sensitivity compared with the control strategy without homogeneous amplification. Moreover, the proposed strategy was demonstrated with high selectivity, reproducibility, and capability of detecting nucleic acids in serum samples, displaying its promising potential in the development of ultrasensitive electrochemical biosensing platform for disease diagnosis applications.

Dual Signal Amplification by eATRP and DNA-Templated Silver Nanoparticles for Ultrasensitive Electrochemical Detection of Nucleic Acids

Herein, an ultrasensitive and novel platform for DNA detection is reported, which combines DNA-templated silver nanoparticles (AgNPs) with electrochemical atom transfer radical polymerization signal amplification. Peptide nucleic acid (PNA) functionalized with thiol was modified to the Au electrode surface as a probe to specifically capture target DNA (T-DNA). After Zr4+ binds to phosphate on DNA, the initiator [α-bromophenylacetic acid (BPAA)] of ATRP is attached to PNA/DNA heteroduplexes based on the phosphate groups of T-DNA and carboxylate groups of BPAA via zirconium-phosphate-carboxylate chemistries. A large number of glyco-syloxyethyl methacrylates (GEMA) were captured on the formed PNA/DNA duplex via ATRP. Afterwards, the polysaccharides were oxidized to polymerized aldehydes with sodium periodate (NaIO4). In addition, AgNPs were deposited on the electrode surface by silver mirror reaction. The results indicate that the amount of AgNPs proportional to the T-DNA was quantified through differential pulse voltammetry. Furthermore, it proves that the modified electrode has good performance in DNA detection, indicating that the DNA sensor has high selectivity, high sensitivity, and stable repeatability. Under the optimal conditions, a good linear relationship is obtained in the range of 10 aM to 10 pM with the correlation coefficient of 0.992, and the detection limit is calculated to be as low as 4.725 aM. In addition, the sensor is successfully used to detect DNA in actual serum samples with satisfactory results, which indicates huge promise for detecting gene biomarkers and clinical analysis.

Electrochemical impedance spectroscopic analysis of nucleic acids through DNA tetrahedron self-walking machine

The design of artificial molecular machines has attracted great interest in recent years. Herein, a DNA tetrahedron self-walking machine is constructed for the application of label-free electrochemical detection of nucleic acids. After target DNA initiated conformation change, autonomous walking is achieved around the DNA tetrahedron, which is powered by nicking endonuclease (NEase). Further hybridization with thiolated auxiliary probe facilitates the immobilization of the DNA nanostructure on the gold electrode interface. After a simple electrochemical impedance spectroscopic analysis, the calculated interfacial resistance (Rct) is found to be linearly related to logarithmic concentration of target nucleic acids in the range from 10 fM to 10 pM with a limit of detection at 3.7 fM. Furthermore, the proposed method has been successfully applied in human blood serum samples, demonstrating great potential practical utility.

A Nuclease Protection ELISA Assay for Colorimetric and Electrochemical Detection of Nucleic Acids.

Early and accurate diagnosis is crucial to monitor infection outcomes and provide timely interventions. However, gold standard polymerase chain reaction assays (PCR) are labor-intensive and require expensive reagents and instrumentation. Nuclease protection has been used for decades to detect and quantify nucleic acid but has not yet been investigated as a diagnostic tool for infectious disease. In this work, we describe a nuclease protection enzyme-linked immunosorbent assay (NP-ELISA) for accurate and sensitive detection of nucleic acid. Briefly, binding of a nucleic acid target to an oligo probe protects it from digestion of un-hybridized nucleic acid by S1 nuclease. Following the workflow of an ELISA, a horseradish peroxidase (HRP)-conjugated antibody binds the probe and oxidizes its substrate to generate signal. The assay was validated with three HRP substrates for absorbance, chemiluminescence, and electrochemical readouts, demonstrating great versatility. Electrochemical detection with 3,3',5,5'-Tetramethylbenzidine (TMB) gave the highest assay sensitivity with a limit of detection of 3.72×103 molecules mL-1. Furthermore, non-complementary targets did not generate a response, indicating a high degree of specificity. This proof of principle serves as a stepping stone towards developing miniaturized, multiplexed nuclease protection assays for point-of-care diagnosis.

Electrochemical Detection of Nucleic Acids Using Graphene-Based Electrodes

We showcase the variation of a popular technique for the detection of nucleic acids that involves differential pulse voltammetry (DPV). We fabricated an electrochemical sensor using reduced graphene oxide (RGO) for the sensitive detection of nucleic acids. As a proof-of-concept, we demonstrated the electrochemical detection of deoxyguanosine triphosphate (dGTP). The dGTP, along with other nucleotides, are detectable with DPV because of their inherent electroactive nature. Each nucleotide has a characteristic redox peak that can be observed with DPV or cyclic voltammetry. The magnitude of each peak, which is measured in current, is proportional to the concentration of the nucleotide in solution. We observed the peaks between 0.7 and 1.0V, which is a characteristic range for dGTP, meaning that the RGO is a viable substrate for future use in rapidly detecting nucleic acids. This work shows the potential capabilities of the RGO as a material useful for a wide range of diagnostic applications, including on-chip real-time polymerase chain reaction for viral load detection and genotyping.

DNA-fueled target recycling-induced two-leg DNA walker for amplified electrochemical detection of nucleic acid

Taking advantage of the homogeneous and heterogeneous electrochemical biosensors, a simple, sensitive, and selective electrochemical biosensor is constructed by combining entropy-driven amplification (EDA) with DNA walker. This electrochemical biosensor realizes the biorecognition and EDA operation in homogeneous solution, which is beneficial to improve the recognition and amplification efficiency. A two-leg DNA walker generated by EDA can walk on the surface of gold electrode for cleaving the immobilized substrate DNA and releasing the electroactive labels, giving rise to a significant decrease of the electrochemical signal. The immobilization of the electroactive labels ensures the reproducibility and reliability of the biosensor. The present cascade amplification assay can be applied to detect target DNA with a detection limit of 0.29 fM, and base mutations can be easily distinguished. Moreover, the proposed electrochemical biosensor shows a satisfactory performance for the detection of target DNA in human serum. Thus, the novel electrochemical biosensor holds promising potential for a future application in disease diagnosis.

Design of DNA nanostructure-based interfacial probes for the electrochemical detection of nucleic acids directly in whole blood.

Here we report a robust and sensitive DNA nanostructure-based electrochemical (E-nanoDNA) sensor that utilizes tetrahedral DNA nanostructures (TDNs) as an interfacial probe to detect biomolecules in a single-step procedure. In this study, we have firstly demonstrated that the use of TDNs can significantly suppress electrochemical background signals compared to traditional linear DNA probes upon introduction of base mismatches in the edges of TDNs. After further optimization of the two functional strands in the TDNs, quantitative, one-step detection of DNA can be achieved in the picomolar range in less than 10 min, and directly in complex media. Moreover, the baseline drift of this biosensor can be greatly decreased even after several hours in flowing whole blood in vitro, which suggests that the sensor holds potential to be employed in live animals. Furthermore, through replacing functional strands with aptamers or other DNA elements, this E-nanoDNA sensor can be easily used to probe various analytes, broadening the application range of the proposed sensor.

Label-free, non-enzymatic and ultrasensitive electrochemical nucleic acid biosensing by tandem DNA-fueled target recycling and hybridization chain reaction

Herein, the DNA-fueled target recycling was effectively married with the hybridization chain reaction (HCR) for the autonomous dual signal amplification, by which an isothermal, non-enzymatic, label-free and ultrasensitive electrochemical DNA biosensor was developed for the first time. The DNA-fueled target recycling was operated onto the three-strand duplex DNA probe immobilized electrode according to the principle of the target DNA-triggered two cascaded toehold-mediated strand displacement reactions, accompanied with the association of plenty of DNA fuel strands on the electrode surface. The introduced DNA fuel strand possesses an emerging DNA fragment, which could be used to further propagate the HCR between two DNA hairpins, inducing the formation of a typical one-dimensional linear DNA concatamer on the electrode surface. The formed DNA concatamers were then bound with Hexaamineruthenium (III) chloride (RuHex) through electrostatic interaction for the significantly enhanced electrochemical response toward target DNA. A very low detection limit of 20aM with an excellent selectivity toward target DNA could be achieved. The developed biosensor creates an opportunity for the simple, flexible and enzyme-free coupling of the target recycling and post-amplification strategies and thus opens a promising avenue for the electrochemical detection of nucleic acid with low abundance in bioanalysis and clinical biomedicine.

Diagnostic Applications of Morpholinos and Label-Free Electrochemical Detection of Nucleic Acids.

Diagnostic applications of morpholinos take advantage of their unique properties including backbone charge neutrality, a weak impact of ionic strength on their hybridization behavior, and their resistance to enzymatic degradation. This chapter overviews how these properties have advanced transduction and other capabilities useful for the analysis of nucleic acids. In many cases, the benefits stem from electrostatic mechanisms; for example, use of low ionic strengths improves sensitivity of detection while decreasing background signals because only the nucleic acid analyte is charged. While most literature reports focus on in vitro assays in buffer, morpholinos have been also used for biodistribution measurements of species such as fungal rRNA and miRNA. After reviewing the diagnostic applications of morpholinos, the chapter describes preparation of morpholino monolayers on metal supports for electrochemical diagnostics and the procedure for performing label-free detection of DNA from changes in surface capacitance.

In Situ Growth of Three-Dimensional Graphene Films for Signal-On Electrochemical Biosensing of Various Analytes.

In this work, an in situ growth protocol is introduced to fabricate three-dimensional graphene films (3D GFs) on gold substrates, which are successfully utilized as working electrode for electrochemical detection of nucleic acid (microRNA) and protein (lysozyme) based on a signal-on sensing mechanism. To realize the bridge between the gold substrate and graphene film, a monolayer of 4-aminothiophenol is self-assembled on the substrate, which is then served as connectors for the growth of 3D GFs on the gold substrate by the hydrothermal reduction (HR) technique. Moreover, given the excellent properties, such as enlarged surface area, strong binding strength between 3D GFs and gold substrate, and improved conductivity, the proposed 3D GF-fabricated gold substrate is readily employed to the construction of electrochemical biosensing platforms through introduction of magnetic nanoparticles (MNPs) as probe carriers. On the basis of the strand displacement reaction and specific binding between aptamer and its target, the developed biosensors achieve signal-on detection of microRNA-155 (miR-155) and lysozyme (Lyz) with high sensitivity and selectivity and further successfully applied to human serum assay. Overall, the proposed strategy for in situ growth of 3D GFs provides a powerful tool for a wide range of applications, which is not limited to electrochemical biosensors and can be extended to other areas, such as electrocatalysis and electronic energy-related systems.