DNA Supersandwich Research Articles

A Single Set of Well‐Designed Aptamer Probes for Reliable On‐site Qualitative and Ultra‐Sensitive Quantitative Detection

AbstractAptamer‐based probes are pivotal components in various sensing strategies, owing to their exceptional specificity and versatile programmable structure. Nevertheless, numerous aptamer‐based probes usually offer only a single function, limiting their capacity to meet the diverse requirements of multi‐faceted sensing systems. Here, we introduced supersandwich DNA probes (SSW‐DNA), designed and modified on the outer surface of nanochannels with hydrophobic inner walls, enabling dual functionality: qualitative detection for on‐site analysis and quantitative detection for precise analysis. The fragmented DNAs resulting from the target recognition, are subsequently identified through lateral flow assays, enabling robust on‐site qualitative detection of microcystin‐LR with an impressively low limit of detection (LOD) at 0.01 μg/L. Meanwhile, the nanochannels enable highly sensitive quantification of microcystin‐LR through the current analysis, achieving an exceptionally low LOD at 2.5×10−7 μg/L, with a broad dynamic range spanning from 1×10−6 to 1×102 μg/L. Furthermore, the process of target recognition introduces just a single potential error propagation, which reduces the overall risk of errors during the entire qualitative and quantitative detection process. This sensing strategy broadens the scope of applications for aptamer‐based composite probes, holding promising implications across diverse fields, such as medical diagnosis, food safety, and environmental protection.

A Single Set of Well-Designed Aptamer Probes for Reliable On-site Qualitative and Ultra-Sensitive Quantitative Detection.

Aptamer-based probes are pivotal components in various sensing strategies, owing to their exceptional specificity and versatile programmable structure. Nevertheless, numerous aptamer-based probes usually offer only a single function, limiting their capacity to meet the diverse requirements of multi-faceted sensing systems. Here, we introduced supersandwich DNA probes (SSW-DNA), designed and modified on the outer surface of nanochannels with hydrophobic inner walls, enabling dual functionality: qualitative detection for on-site analysis and quantitative detection for precise analysis. The fragmented DNAs resulting from the target recognition, are subsequently identified through lateral flow assays, enabling robust on-site qualitative detection of microcystin-LR with an impressively low limit of detection (LOD) at 0.01 μg/L. Meanwhile, the nanochannels enable highly sensitive quantification of microcystin-LR through the current analysis, achieving an exceptionally low LOD at 2.5×10-7 μg/L, with a broad dynamic range spanning from 1×10-6 to 1×102 μg/L. Furthermore, the process of target recognition introduces just a single potential error propagation, which reduces the overall risk of errors during the entire qualitative and quantitative detection process. This sensing strategy broadens the scope of applications for aptamer-based composite probes, holding promising implications across diverse fields, such as medical diagnosis, food safety, and environmental protection.

Conductive Ink-Coated Paper-Based Supersandwich DNA Biosensor for Ultrasensitive Detection of Neisseria gonorrhoeae

Herein, we report results of the studies relating to the development of an impedimetric, magnetic bead-assisted supersandwich DNA hybridization assay for ultrasensitive detection of Neisseria gonorrhoeae, the causative agent of a sexually transmitted infection (STI), gonorrhea. First, a conductive ink was formulated by homogenously dispersing carboxylated multiwalled carbon nanotubes (cMWCNTs) in a stable emulsion of terpineol and an aqueous suspension of carboxymethyl cellulose (CMC). The ink, labeled C5, was coated onto paper substrates to fabricate C5@paper conductive electrodes. Thereafter, a magnetic bead (MB)-assisted supersandwich DNA hybridization assay was optimized against the porA pseudogene of N. gonorrhoeae. For this purpose, a pair of specific 5' aminated capture probes (SCP) and supersandwich detector probes (SDP) was designed, which allowed the enrichment of target gonorrheal DNA sequence from a milieu of substances. The SD probe was designed such that instead of 1:1 binding, it allowed the binding of more than one T strand, leading to a 'ladder-like' DNA supersandwich structure. The MB-assisted supersandwich assay was integrated into the C5@paper electrodes for electrochemical analysis. The C5@paper electrodes were found to be highly conductive by a four-probe conductivity method (maximum conductivity of 10.1 S·cm-1). Further, the biosensing assay displayed a wide linear range of 100 aM-100 nM (109 orders of magnitude) with an excellent sensitivity of 22.6 kΩ·(log[concentration])-1. The clinical applicability of the biosensing assay was assessed by detecting genomic DNA extracted from N. gonorrhoeae in the presence of DNA from different non-gonorrheal bacterial species. In conclusion, this study demonstrates a highly sensitive, cost-effective, and label-free paper-based device for STI diagnostics. The ink formulation prepared for the study was found to be highly thixotropic, which indicates that the paper electrodes can be screen-printed in a reproducible and scalable manner.

Target-promoted specific activation of m6A-DNAzyme for SPEXPAR-amplified and highly sensitive non-label electrochemical assay of FTO demethylase.

The demethylase of fat mass and obesity related protein (FTO) is critical to regulate the dynamic N6-methyladenosine (m6A) modification of eukaryotic mRNAs, and its overexpression has found to be closely related to the initiation of several cancers. On the basis of a target-promoted specific activation of DNAzyme strategy coupled with self-primer exponential amplification reaction (SPEXPAR) cycles and DNA supersandwich assemblies, the highly sensitive and label-free electrochemical FTO assay approach is established. The modification of the catalytic core nucleobase of the DNAzyme probe by m6A can inhibit its cleavage activity. The presence of target FTO catalyzes the elimination of the methyl group to restore the DNAzyme activity, which cleaves the hairpin substrates to trigger the SPEXPAR for yielding many ssDNAs. The capture of these DNAs on the sensor electrode leads to the initiation of supersandwich assembly formation of long dsDNAs. Tremendous electrochemical signal probe of [Ru(NH3)6]Cl3 are then absorbed on these dsDNAs to produce highly amplified catalytic currents with the assistance of K3[Fe(CN)6] for detecting trace FTO with 63.1fM detection limit. Furthermore, the sensor can be employed for selective assay of FTO in cell lysates, revealing the great potential of this sensing strategy for biomedical and biological study applications.

A novel cloth-based supersandwich electrochemical aptasensor for direct, sensitive detection of pathogens

Traditional detection methods for food-borne pathogens are usually expensive and laborious, so there is an urgent need for an economical, facile and sensitive method. In this work, a novel cloth-based supersandwich electrochemical aptasensor (CSEA) is firstly developed for direct detection of pathogens. Carbon ink- and wax-based screen-printing is used to make cloth-based electrodes and hydrophilic/hydrophobic regions respectively to fabricate the sensing devices. Two well-designed, specific single-stranded DNA sequences arise a cascade hybridization reaction to form the DNA supersandwich (DSS) whose grooves can be inserted by methylene blue (MB), which effectively amplifies the current signal to greatly improve the detection sensitivity. Taking the detection of Salmonella typhimurium (S. typhimurium) as an example, the aptamers bind to S. typhimurium to form the target-aptamers complex, which can simultaneously bind to the capture probe and DSS, resulting in detection of S. typhimurium. Moreover, the addition of tail sequences of aptamer makes the proposed CSEA versatile. Under optimized conditions, the electrochemical signal increases linearly with the logarithm of S. typhimurium concentration over the range from 102 to 108 CFU mL−1, with a limit of detection of 16 CFU mL−1. Additionally, the CSEA efficiently determined the levels of S. typhimurium in milk samples. Experimental results illustrate that the fabricated CSEA is sensitive, specific, reproducible and stable. Moreover, when Ru(bpy)32+ replaces MB, the electrochemiluminescence (ECL) can be performed. Thus, for the proposed sensing strategy, the dual-mode detection of electrochemistry and ECL is easily realized.

High-Sensitive Assay of Nucleic Acid Using Tetrahedral DNA Probes and DNA Concatamers with a Surface-Enhanced Raman Scattering/Surface Plasmon Resonance Dual-Mode Biosensor Based on a Silver Nanorod-Covered Silver Nanohole Array.

A novel surface-enhanced Raman scattering/surface plasmon resonance (SERS/SPR) dual-mode biosensor prepared on a silver nanorod-covered silver nanohole (Ag NR-NH) array by surface modification of tetrahedral DNA probes is proposed for highly sensitive detecting nucleic acids by a special signal amplification strategy of DNA supersandwich. The Ag NR-NH with a large area and uniformly arrayed nanostructure possesses excellent anisotropic extraordinary optical transmission and strong localized surface plasmon resonance, which lead to sensitive SPR response to the change of a local refractive index and strong localized electric fields for excellent SERS activity. To obtain high sensitivity and specificity, smart tetrahedral DNA probes are immobilized onto the Ag NR-NH array and the DNA supersandwich sensing strategy, including the signal amplification of DNA concatamers, is used. About 10 times signal enhancement for SPR and 4 times for SERS are achieved by this sensing strategy. In the detection of the target DNA in the human serum, the two sensing modes have complementary performances, i.e., the limit of detection for the SPR array is high (0.51 pM), while for SERS, it is low (0.77 fM), but the specificity for SPR is much higher than that of SERS. This improves the robustness of the DNA sensors, and subsequent recovery tests also confirm good reliability of the biosensor. The proposed SERS/SPR dual-mode biosensor has a great potential for high performance and reliable detection of trace disease-related nucleic acid biomarkers in the serum and is a powerful sensing platform for early-stage disease diagnosis.

Dual-Signal Readout of DNA Methylation Status Based on the Assembly of a Supersandwich Electrochemical Biosensor without Enzymatic Reaction.

A highly sensitive and selective biosensing system was designed to analyze DNA methylation using a dual-signal readout technique in combination with the signal amplification of supersandwich DNA structure. Through the ingenious design of target-triggered cascade of hybridization chain reaction, one target DNA could initiate the formation of supersandwich structure with multiple signal probes. As a result, one-to-multiple amplification effect was achieved, which conferred high sensitivity to target molecular recognition. Based on probe 1 labeled with ferrocene and probe 2 modified with methylene blue, the target DNA was clearly recognized by two electrochemical signals at independent potentials, which was helpful for the acquisition of more accurate detection results. Taking advantage of bisulfite conversion, the methylation status of cytosine (C) was changed to nucleic acid sequence status, which facilitated the hybridization-based detection without enzymatic reaction. Consequently, the methylated DNA was detected at the femtomolar level with satisfactory analytical parameters. The proposed system was effectively used to assess methylated DNA in human blood serum samples, illuminating the possibility of the sensing platform for applications in disease diagnosis and biochemistry research.

Nanopore-based DNA Supersandwich Structure for Detection of Streptavidin

Natural and synthetic nanopores are increasingly popular tools in biosensors. In this work, the DNA su-persandwich structure, which was made from two specially designed probes has been used to be fabricated in solid nanopores. Integrating the idea of affinity between streptavidin and biotin, the DNA supersandwich structure with biotins was successfully constructed for streptavidin detection, and the limitation of detection was found to be 10 fmol/L. This nanodevice allows specific, sensitive and versatile detection of diverse analytes with easy operations, thus we believe that it could be developed to detect some disease-related molecular targets and play a considerable role in biotechnology.

CdTe QD-CeO2 Complex as a Strong Photoelectrochemical Signal Indicator for the Ultrasensitive microRNA Assay.

The photoelectrochemical (PEC) signal can be enhanced by constructing sensitization structures containing photoactive materials and appropriate sensitizers. However, usually, the photoactive materials and sensitizers were separated in independent nanostructures, thereby producing long electron-transfer path and large energy loss, which could further result in limited photoelectric conversion efficiency and PEC signals. Herein, we designed a novel sensitization nanostructure simultaneously containing the photoactive material cerium dioxide (CeO2) and its sensitizer CdTe quantum dots (QDs) as the strong PEC signal indicator (CdTe QD-CeO2 complex), which prominently enhanced photoelectric conversion efficiency because of the shortened electron-transfer path and reduced energy loss. The proposed CdTe QD-CeO2 complex was used to construct a PEC biosensor for achieving ultrasensitive determination of microRNA-141 (miRNA-141) coupling with target converting amplification and DNA supersandwich structure amplification. The designed PEC biosensor demonstrated a wide linear range from 0.5 fM to 5 nM with a detection limit of 0.17 fM for miRNA-141. Impressively, this work provided a new and strong PEC signal indicator for the construction of PEC sensing platform and would extend the application of PEC sensors in bioanalysis and early disease diagnosis.

A novel ECL method for histone acetyltransferases (HATs) activity analysis by integrating HCR signal amplification and ECL silver clusters

A novel electrogenerated chemiluminescence (ECL) method combining ECL property of silver clusters and hybridization chain reaction (HCR) signal amplification strategy has been designed for the analysis of histone acetyltransferases (HATs) activity and inhibitor evaluation. In this strategy, the substrate peptide of HAT released from the electrode surface due to the charge change based on the acetylated reaction in the presence of HATs, and then the exposed DNA on the electrode initiated the HCR to form the supersandwich DNA sequence, which can adsorb Ag+, and the silver clusters (AgNCs) generated by the electrochemical reduction. The ECL signal generated by AgNCs can be utilized for HATs activities detection. The detection limit of the as-prepared ECL method was 0.49 nM (S/N = 3). The novel ECL method can be used for HATs activity analysis and inhibition in MCF-7 cell lysates which shows high promise in HATs-related clinical diagnostics.

Optical fiber amplifier for quantitative and sensitive point-of-care testing of myoglobin and miRNA-141

A simple, sensitive, quantitative point-of-care testing (POCT) was developed integrating enzyme-linked DNA supersandwich amplification with optical fiber amplifier. The point-of-care (POC) assay can work in both “Turn-off” mode and “Turn-on” mode. Using myoglobin (Myo) and miRNA-141 as the model biomarker, as low as 0.5 nM Myo and 10 pM miRNA-141 could be detected. More importantly, the optical fiber amplifier, which was used as signal readout, exhibited low-cost, small size, rapid response, and easy operation. Although it did not need large equipment and professional technical personnel, the assay was successfully applied to detect Myo and miRNA-141 in 40% human serum. Since the entire process of the strategy could be done directly in the centrifugal tube, requiring a few easy steps, it showed great potential application for early diagnosis in the world, especially in developing countries or remote regions. This work provided a new avenue for developing the portable and sensitive biosensor.

DNA supersandwich assemblies as artificial receptors to mediate intracellular delivery of catalase for efficient ROS scavenging.

Aptamer-tailored DNA supersandwich assemblies are proposed as artificial receptors anchored onto a cell membrane, enabling catalase delivery with an 8-fold enhancement in efficiency. The catalase transfection led to inhibition of intracellular ROS, while the cell viability was also remarkably improved even when exposed to H2O2 and lipopolysaccharide environments.

Ultrasensitive Photoelectrochemical Assay with PTB7-Th/CdTe Quantum Dots Sensitized Structure as Signal Tag and Benzo-4-chlorohexadienone Precipitate as Efficient Quencher.

Herein, an ultrasensitive photoelectrochemical (PEC) assay was developed for the monitoring of microRNA-141 (miRNA-141) based on poly{4,8-bis[5-(2-ethylhexyl)thiophen-2-yl]benzo[1,2- b:4,5- b']dithiophene-2,6-diyl- alt-3-fluoro-2-[(2-ethylhexyl)carbonyl]thieno[3,4- b]thiophene-4,6-diyl}/CdTe quantum dots ( PTB7-Th/CdTe QDs) sensitized structure as signal tag and the benzo-4-chlorohexadienone (4-CD) precipitate as efficient quencher. PTB7-Th and CdTe QDs were successively modified on the electrode surface to form a novel sensitized structure with eminent photovoltaic performances and surpassing film-forming ability. The PTB7-Th/CdTe QDs sensitized structure served as signal tag for achieving a strong initial PEC signal. Besides, a tiny amount of target (miRNA-141) could be transformed into numerous DNA products via enzyme-assisted target cycling procedure, which further triggered the formation of a DNA supersandwich structure on the electrode surface for loading abundant manganese porphyrin (MnPP). Thereafter, MnPP as mimetic enzyme could catalyze 4-chloro-1-naphthol (4-CN) to generate 4-CD precipitate on the sensing interface in the presence of H2O2, which could efficiently block electron transfer, leading to a significantly quenched PEC signal for determination of miRNA-141. The designed PEC biosensor performed a wide detection range from 0.1 fM to 1 nM with a low detection limit of 33 aM for miRNA-141, which paved a new avenue for highly accurate and ultrasensitive monitoring of multifarious analytes in bioanalysis and clinical diagnosis.

Ultrasensitive electrochemical biosensor for silver ion based on magnetic nanoparticles labeling with hybridization chain reaction amplification strategy

Silver ion (Ag+) is a highly toxic heavy metal ion to aquatic organisms and accumulates in the human body via the food chain. Therefore, fast and accurate detection of Ag+ in water and food resources has become a critical issue within the scope of human health. Herein, we developed an ultrasensitive electrochemical biosensor for detection of Ag+ based on magnetic Fe3O4@gold core-shell nanoparticles (Fe3O4@Au NPs) labeling with hybridization chain reaction (HCR) amplification strategy. In this sensing strategy, the magnetic Fe3O4@Au NPs were selected for labeling with HCR product and enrichment on the surface of magnetic gold electrode. Thiolated-oligonucleotide (S1) was firstly immobilized on the surface of Fe3O4@Au NPs through Au–S chemical bond. In the presence of Ag+, cytosine-rich DNA oligonucleotide S2 hybridized with S1 to form an intramolecular duplex, in which Ag+ can selectively bind to cytosine–cytosine mismatches forming C–Ag+–C complex. The exposed stem of the C–Ag+–C complex opened two alternating ferrocene-labeled DNA hairpins (H1 and H2) in turn and triggered HCR to form a supersandwich DNA structure on the surface of Fe3O4@Au NPs. The HCR products modified Fe3O4@Au NPs were brought to the surface of magnetic gold electrode for direct electrochemical measurements. The proposed strategy led to a low detection limit of 0.5fM and a wide dynamic range of 1fM–100pM for target Ag+. The developed biosensor was highly selective and its practical applicability in tap water and lake water samples was also investigated with a satisfactory result.

Electrogenerated chemiluminescence biosensing method for the detection of DNA demethylase activity: Combining MoS2 nanocomposite with DNA supersandwich

We report an ultrasensitive electrogenerated chemiluminescence (ECL) biosening method for the detection of DNA demethylase activity based on the site-specific cleavage of the HpaII endonuclease in conjunction with signal amplification by a Thionin-MoS2 (Th-MoS2) nanocomposite and supersandwich DNA. The Thionin (Th) served as an effective mediator for the immobilization of the capture probe onto the surface of MoS2, after which the long concatamers containing the methylated site 5′-CmCGG-3′ were hybridized with the capture probe, creating a supersandwich DNA structure. Ru(phen)32+ ECL reagents can be intercalated into the grooves of the formed supersandwich DNA structure with high affinity leading to enhanced signaling. At the biosensing electrode (Ru(phen)32+/supersandwich DNA/Th-MoS2/glassy carbon electrode) after demethylation with demethylase, the demethylated supersandwich DNA could be specifically cleaved using HpaII, resulting in a decreased ECL signal from Ru(phen)32+. An extremely low detection limit of 0.16pgmL−1 was achieved for the detection of DNA demethylase activity. This work demonstrates that the combination of dual signal amplification using a MoS2-thionin nanocomposite and supersandwich with an inherently sensitive ECL method is a promising tactic for the detection of DNA demethylation and assay of the activity of demethylase.

Surface plasmon resonance biosensor for sensitive detection of microRNA and cancer cell using multiple signal amplification strategy

A sensitive and versatile surface plasmon resonance (SPR) biosensor was proposed for the detection of microRNA (miRNA) and cancer cell based on multiple signal amplification strategy. Thiol-modified hairpin probe, including a sequence complementary to the target miRNA, was first immobilized on the Au film. In the presence of target miRNA, the stem-loop structure of hairpin probe was unfolded, and then DNA-linked Au nanoparticles (AuNPs) were hybridized with the terminus of the unfolded hairpin probe. Subsequently, DNA-linked AuNPs initiated the formation of DNA supersandwich structure through the addition of two report DNA sequences. Owing to the electronic coupling between localized plasmon of the AuNPs and the surface plasmon wave, as well as the enhancement of the refractive index of the medium over the Au film induced by DNA supersandwich structure, the SPR response was significantly enhanced. Next, numerous positively charged silver nanoparticles (AgNPs) were absorbed onto the long-range DNA surpersandwich equably, resulting in a further increase of SPR response. Due to the enzyme-free multiple signal amplification strategy, as low as ca. 0.6 fM miRNA-21 could be detected. In addition, this biosensor showed high selectivity toward single-base mismatch. More importantly, this SPR biosensor was also used for cancer cell detection coupled with the cell-specific aptamer modified magnetic nanoparticles. Given that the biosensor avoided enzyme introduction, the limitation of the enzyme was overcome. The versatile biosensor has great potential for the broad applications in the field of clinical analysis.

Sensitive Zn(2+) sensor based on biofunctionalized nanopores via combination of DNAzyme and DNA supersandwich structures.

The sensitivity of detection based on biofunctionalized nanopores is limited since the target-to-signal ratio is 1 : 1. Isothermal amplification is a promising amplification strategy at constant temperature due to its easy operation, quick results, PCR-like sensitivity, low cost and energy efficiency. In the present work, the isothermally amplified detection of Zn(2+) is achieved by using a DNA supersandwich structure and Zn(2+)-requiring DNAzymes. The DNA supersandwich structures, due to the multiple amplification of nucleic acids, heavily plug the nanopore. Simultaneously, the DNA supersandwich structures bond with the sessile probe (SP) of the substrate in the nanopore which partially hybridizes with DNAzymes. In the presence of Zn(2+), the Zn(2+)-requiring DNAzyme cleaves the SP into two fragments, while the DNA supersandwich structures are peeled off and the ionic pathway is unimpeded. A steep drop and a sequential complete recovery of the current occur in the I-V plot when the DNA supersandwich structures are decorated and peeled off. In the present system, the reliable detection limit of Zn(2+) is as low as 1 nM. Discrimination between different types of ions (Cu(2+), Hg(2+), Pb(2+)) is achieved.

Surface plasmon resonance biosensor for enzyme-free amplified microRNA detection based on gold nanoparticles and DNA supersandwich

A novel enzyme-free amplified surface plasmon resonance (SPR) biosensor for microRNA (miRNA) detection was developed based on gold nanoparticles (AuNPs) coupled with DNA supersandwich. In the detection strategy, the DNA-linked AuNPs as the primary amplification element, not only hybridized with the capture DNA on the Au film to amplify SPR signal but also initiated the subsequent secondary amplification, i.e. DNA supersandwich formation of two report probes. In the presence of target, stem–loop structure of capture DNA on the Au film surface was unfolded, and DNA-linked AuNPs were bound to Au film by hybridization with terminus of capture DNA. Then, the carried assistant DNA on the AuNPs could trigger an alternative hybridization reaction of two report probes, resulting in the formation of DNA supersandwich. Due to the electronic coupling between localized plasmon of AuNPs and the surface plasmon wave associated with Au film, as well as the enhancement of the refractive index of the medium next to the metal film caused by DNA supersandwich structure, the shift of resonance angle was enhanced obviously. By employing the enzyme-free dual signal amplification strategies, as low as ca. 8fM miRNA-21 could be detected. Moreover, this assay also showed high selectivity toward single-base mismatch, and demonstrated its applicability for the target detection in human serum. This work may provide great potential applications in future clinical analysis.

An amplified electrochemical aptasensor based on hybridization chain reactions and catalysis of silver nanoclusters.

In the present study, based on the mimic oxidase catalytic character of nucleic-acid-stabilized silver nanoclusters (DNA/AgNCs) and hybridization chain reactions for signal amplification, the fabrication of a label-free sensitive "turn-on" electrochemical aptasensor for the amplified determination of lysozyme was demonstrated. First, the designed DNA duplex was modified on the electrode. With the specific binding of the target, lysozyme and its aptamer, the lysozyme-binding DNA sequence was liberated, exposing the induced DNA sequence, which in turn triggered the formation of the supersandwich DNA structure. Because the cytosine-rich sequence was designed ingeniously on the DNA sequence, DNA/AgNCs were formed on the supersandwich DNA structure. The peroxidase-like character of DNA/AgNCs produced detectable electrochemical signals for the lysozyme aptasensor, which showed a satisfying sensitive detection of lysozyme with a low detection limit of 42 pM and a wide linear range of 10(-10) M to 10(-5) M.

An ultrasensitive supersandwich electrochemical DNA biosensor based on gold nanoparticles decorated reduced graphene oxide

In this article, a supersandwich-type electrochemical biosensor for sequence-specific DNA detection is described. In design, single-strand DNA labeled with methylene blue (MB) was used as signal probe, and auxiliary probe was designed to hybridize with two different regions of signal probe. The biosensor construction contained three steps: (i) capture DNA labeled with thiol was immobilized on the surface of gold nanoparticles decorated reduced graphene oxide (Au NPs/rGO); (ii) the sandwich structure formation contained “capture–target–signal probe”; and (iii) auxiliary probe was introduced to produce long concatamers containing signal molecule MB. Differential pulse voltammetry (DPV) was used to monitor the DNA hybridization event using peak current changes of MB in phosphate-buffered saline (PBS) containing 1.0M NaClO4. Under optimal conditions, the peak currents of MB were linear with the logarithm of the concentration of target DNA in the range of 0.1μM to 0.1fM with a detection limit of 35aM (signal/noise=3). In addition, this biosensor exhibited good selectivity even for single-base mismatched target DNA detection.