Label-free Electrochemical DNA Biosensor Research Articles

A label-free electrochemical biosensor for the detection of alpha-thalassemia 1 (SEA deletion) carriers using screen-printed carbon electrodes

A label-free electrochemical DNA biosensor based on electrochemical impedance spectroscopy (EIS) biosensor has been extensively developed for diagnosing human genetic diseases. However, its application has been limited to simulated target DNA. The aim of this study was to develop an EIS biosensor that can identify carriers of alpha (α)-thalassemia 1 with Southeast Asia (SEA) deletion. The biosensor was coupled with screen-printed carbon electrodes (SPCEs) and Hoechst 33,258 dye. To determine the optimal conditions and cutoff criteria, we evaluated forty samples with known DNA genotypes. Our findings suggested that the cutoff value for identifying α-thalassemia 1 (SEA deletion) carriers was more than 26.84 kΩ of negative imaginary impedance (-Z″) and 48.83 kΩ of charge transfer resistance (Rct). The sensitivity of the developed EIS biosensor was comparable to that of conventional gel electrophoresis, with no cross-reactivity observed in α-thalassemia 1 (THAI deletion), α-thalassemia 2 (3.7 deletion), α-thalassemia 2 (4.2 deletion), and beta (β)-thalassemia carriers. The diagnostic potential of the developed EIS biosensor was evaluated using 81 clinical blood samples, demonstrating 100% sensitivity, 88.2% specificity, 83.3% positive predictive value (PPV), 100% negative predictive value (NPV) and 92.6% accuracy for Rct measurement. The developed EIS biosensor presents an attractive screening method for the detecting α-thalassemia 1 (SEA deletion) carriers due to its simplicity, cost-effectiveness, and 45-fold reduction in turnaround time compared to conventional gel electrophoresis. Consequently, the identification of α-thalassemia 1 (SEA deletion) carrier through this approach represents the most effective strategy for the management and prevention of the most severe form of thalassemia.

The Optimization of a Label-Free Electrochemical DNA Biosensor for Detection of Sus scrofa mtDNA as Food Adulterations

Fast, sensitive, and easy-to-use methods for detecting DNA related to food adulteration, health, religious, and commercial purposes are evolving. In this research, a label-free electrochemical DNA biosensor method was developed for the detection of pork in processed meat samples. Gold electrodeposited screen-printed carbon electrodes (SPCEs) were used and characterized using SEM and cyclic voltammetry. A biotinylated probe DNA sequence of the Cyt b S. scrofa gene mtDNA used as a sensing element containing guanine substituted by inosine bases. The detection of probe-target DNA hybridization on the streptavidin-modified gold SPCE surface was carried out by the peak guanine oxidation of the target using differential pulse voltammetry (DPV). The optimum experimental conditions of data processing using the Box–Behnken design were obtained after 90 min of streptavidin incubation time, at the DNA probe concentration of 1.0 µg/mL, and after 5 min of probe-target DNA hybridization. The detection limit was 0.135 µg/mL, with a linearity range of 0.5–1.5 µg/mL. The resulting current response indicated that this detection method was selective against 5% pork DNA in a mixture of meat samples. This electrochemical biosensor method can be developed into a portable point-of-care detection method for the presence of pork or food adulterations.

A label-free electrochemical DNA biosensor used a printed circuit board gold electrode (PCBGE) to detect SARS-CoV-2 without amplification.

The emergence of coronavirus disease 2019 (COVID-19) motivates continuous efforts to develop robust and accurate diagnostic tests to detect severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Detection of viral nucleic acids provides the highest sensitivity and selectivity for diagnosing early and asymptomatic infection because the human immune system may not be active at this stage. Therefore, this work aims to develop a label-free electrochemical DNA biosensor for SARS-CoV-2 detection using a printed circuit board-based gold substrate (PCBGE). The developed sensor used the nucleocapsid phosphoprotein (N) gene as a biomarker. The DNA sensor-based PCBGE was fabricated by self-assembling a thiolated single-stranded DNA (ssDNA) probe onto an Au surface, which performed as the working electrode (WE). The Au surface was then treated with 6-mercapto-1-hexanol (MCH) before detecting the target N gene to produce a well-oriented arrangement of the immobilized ssDNA chains. The successful fabrication of the biosensor was characterized using cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and atomic force microscopy (AFM). The DNA biosensor performances were evaluated using a synthetic SARS-CoV-2 genome and 20 clinical RNA samples from healthy and infected individuals through EIS. The developed DNA biosensor can detect as low as 1 copy per μL of the N gene within 5 minutes with a LOD of 0.50 μM. Interestingly, the proposed DNA sensor could distinguish the expression of SARS-CoV-2 RNA in a patient diagnosed with COVID-19 without any amplification technique. We believe that the proposed DNA sensor platform is a promising point-of-care (POC) device for COVID-19 viral infection since it offers a rapid detection time with a simple design and workflow detection system, as well as an affordable diagnostic assay.

Label free electrochemical DNA biosensor for COVID-19 diagnosis

The COVID-19 pandemic has significantly increased the development of the development of point-of-care (POC) diagnostic tools because they can serve as useful tools for detecting and controlling spread of the disease. Most current methods require sophisticated laboratory instruments and specialists to provide reliable, cost-effective, specific, and sensitive POC testing for COVID-19 diagnosis. Here, a smartphone-assisted Sensit Smart potentiostat (PalmSens) was integrated with a paper-based electrochemical sensor to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A disposable paper-based device was fabricated, and the working electrode directly modified with a pyrrolidinyl peptide nucleic acid (acpcPNA) as the biological recognition element to capture the target complementary DNA (cDNA). In the presence of the target cDNA, hybridization with acpcPNA probe blocks the redox conversion of a redox reporter, leading to a decrease in electrochemical response correlating to SARS-CoV-2 concentration. Under optimal conditions, a linear range from 0.1 to 200 nM and a detection limit of 1.0 pM were obtained. The PNA-based electrochemical paper-based analytical device (PNA-based ePAD) offers high specificity toward SARS-CoV-2 N gene because of the highly selective PNA-DNA binding. The developed sensor was used for amplification-free SARS-CoV-2 detection in 10 nasopharyngeal swab samples (7 SARS-CoV-2 positive and 3 SARS-CoV-2 negative), giving a 100% agreement result with RT-PCR.

Label free flexible electrochemical DNA biosensor for selective detection of Shigella flexneri in real food samples

An effective tool for early-stage selective detection of the foodborne bacterial pathogen Shigella flexneri (S. flexneri) is essential for diagnosing infectious diseases and controlling outbreaks. Here, a label-free electrochemical DNA biosensor for monitoring S. flexneri is developed. To fabricate the biosensor, detection probe (capture probe) is immobilized on the surface of poly melamine (P-Mel) and poly glutamic acid (PGA), and disuccinimidyl suberate (DSS) functionalized flexible indium tin oxide (ITO) electrode. Anthraquinone-2-sulfonic acid monohydrate sodium salt (AQMS) is used as a signal indicator for the detection of S. flexneri. The proposed DNA biosensor exhibits a wide dynamic range with concentration of the targets ranging from 1 × 10−6 to 1 × 10−21 molL−1 with a limit of detection (LOD) of 7.4 × 10−22 molL−1 in the complementary linear target of S. flexneri, and a detection range of 8 × 1010–80 cells/ml with a LOD of 10 cells/ml in real S. flexneri sample. The proposed flexible biosensor provides high specificity for the detection of S. flexneri compared to other target signals such as discrete base mismatches and different bacterial species. The developed biosensor displayed excellent recoveries in detecting S. flexneri in spiked food samples. Therefore, the proposed biosensor can serve as a model methodology for the detection of other pathogens in a broad span of industries.

A label-free electrochemical impedimetric DNA biosensor for genetically modified soybean detection based on gold carbon dots.

A label-free electrochemical impedimetric biosensor was constructed based on gold carbon dots (GCDs) modified screen-printed carbon electrode for the detection of genetic modified (GM) soybean. The structure and property of GCDs were investigated. The GCDs can directly bind to single-stranded DNA probes through Au-thiol interaction and boost electric conductivity for the DNA sensor construction. The quantification of target DNA was monitored by the change of electron-transfer resistance (Ret) upon the DNA hybridization on sensor surface. Under the optimal conditions, the Ret response (vs. Ag reference electrode) increased with the logarithm of target DNA concentrations in a wide linear range of 1.0 × 10-7 - 1.0 × 10-13M with a detection limit of 3.1 × 10-14M (S/N = 3). It was also demonstrated that the proposed DNA sensor possessed high specificity for discriminating target DNA from mismatched sequences. Moreover, the developed biosensor was applied to detect SHZD32-1 in actual samples, and the results showed a good consistency with those obtained from the gel electrophoresis method. Compared with the previous reports for DNA detection, the label-free biosensor showed a comparatively simple platform due to elimination of complicated DNA labeling. Therefore, the proposed method showed great potential to be an alternative device for simple, sensitive, specific, and portable DNA sensor.

A novel and ultrasensitive label-free electrochemical DNA biosensor for Trichomonas vaginalis detection based on a nanostructured film of poly(ortho-aminophenol)

Trichomoniasis, as a major public health concern, is the world’s most common sexually transmissible disease. This infection is caused by a protozoan parasite called Trichomonas vaginalis (TV) that can lead to reproductive and genital area problems. Accordingly, it is of great significance to expand new methods for increasing the sensitivity of TV detection. In this study, a novel label-free electrochemical DNA biosensor for TV quantitation was constructed based on an electropolymerized poly(ortho-aminophenol) thin film simultaneously acting as a transducer as well as a redox indicator. The redox-active polymeric film was utilized to covalent immobilization of a specific thiolated DNA probe. The hybridization process was then monitored by differential pulse voltammetry. The proposed biosensor detected a synthetic TV target sequence with a calibration sensitivity of − 0.0113 µA (log (concentration/mol L−1))−1, a linear concentration range of 1.0 × 10−20 to 1.0 × 10−12 mol L−1, and a detection limit of 3.9 × 10−21 mol L−1. It also showed an ability to differentiate between the complementary sequence and the base-mismatched and non-complementary sequences with a nice selectivity. Moreover, the designed biosensor was able to detect the TV genome with a calibration sensitivity of − 0.0774 µA (log (concentration/ng µL−1))−1, a linear range of 0.55–64 ng mL−1 and a detection limit of 1.0 pg µL−1.

A new strategy to design label-free electrochemical biosensor for ultrasensitive diagnosis of CYFRA 21–1 as a biomarker for detection of non-small cell lung cancer

Lung cancer is one of the most dangerous cancers with high mortality rate among other cancers therefore, early detection of this cancer is very important. Many studies have been reported in ways of diagnostic lung cancer early. According to reports, one of the most important biomarkers to detect lung cancer is Cytokeratin 19 fragment 21–1 (CYFRA21-1), which is significantly related to non-small cell lung cancer, in particular, squamous cell carcinoma. Thus, finding a new method for the early diagnosis of CYFRA 21–1 (DNA target probe) is essential. In the present report, we design a novel label-free electrochemical DNA-biosensor related to the signal of guanine oxidation. The proposed DNA biosensor is fabricated by a modified glassy carbon electrode (GCE) with reduced-graphene oxide (rGO), poly pyrrole (PPy), silver nanoparticles (AgNPs) and single-strand DNA (ssDNA as capture probe) GCE/rGO/PPy/AgNPs/ssDNA. The differential pulse voltammetry (DPV) and cyclic voltammetry (CV) techniques are used to verify the hybridization process between capture and target probes. Electrochemical impedance spectroscopy (EIS), energy diffraction X-ray (EDX) and field-emission scanning microscopy (FE-SEM) techniques are applied to the characterization of different modified GCE surfaces as well as X-ray diffraction (XRD) for graphene oxide synthesis. The XRD pattern of the synthesized GO that its diffraction peak appears at 10.2. The applied CV and DPV for the guanine oxidation are determined under optimal conditions. The label-free DNA biosensor showed a great result for the determination of CYFRA21-1 with a wide linear range from two consecutive linear relationships of peak current and CYFRA21-1 concentration were found (1.0 × 10−14 - 1.0 × 10−10 M, R2 = 0.9936 and 1.0 × 10−9 - 1.0 × 10−6 M, R2 = 0.9955). Proposed electrochemical biosensor displayed low detection limit (2.4 fM).

Electrochemical detection of DNA by formation of efficient electron transfer pathways through adsorbing gold nanoparticles to DNA modified electrodes

The high-performance detection of nucleic acids is increasingly important in the early and accurate disease diagnosis. Herein, we describe a unique label-free electrochemical DNA biosensor, which is based on gold nanoparticles (GNPs) mediated electron transfer through a DNA monolayer modified gold electrode surface. We observed that the different states of DNA monolayers (single-strand DNA or triple-helix DNA) and the different sizes of GNPs have a significant effect on the electron transfer by the DNA modified gold electrodes. This DNA biosensing strategy avoids the complicated fabrication process of the DNA capture probes and minimizes the steps of electrode pre-treatment. Under optimal conditions, this method can sensitively and accurately detect the target DNA ranging from 10 pM to 100 nM with a detection limit of 1.47 pM.

A Highly Sensitive Impedimetric DNA Biosensor Based on Hollow Silica Microspheres for Label-Free Determination of E. coli.

A novel label-free electrochemical DNA biosensor was constructed for the determination of Escherichia coli bacteria in environmental water samples. The aminated DNA probe was immobilized onto hollow silica microspheres (HSMs) functionalized with 3-aminopropyltriethoxysilane and deposited onto a screen-printed electrode (SPE) carbon paste with supported gold nanoparticles (AuNPs). The biosensor was optimized for higher specificity and sensitivity. The label-free E. coli DNA biosensor exhibited a dynamic linear response range of 1 × 10−10 µM to 1 × 10−5 µM (R2 = 0.982), with a limit of detection at 1.95 × 10−15 µM, without a redox mediator. The sensitivity of the developed DNA biosensor was comparable to the non-complementary and single-base mismatched DNA. The DNA biosensor demonstrated a stable response up to 21 days of storage at 4 ℃ and pH 7. The DNA biosensor response was regenerable over three successive regeneration and rehybridization cycles.

Label-Free and Ultrasensitive Electrochemical DNA Biosensor Based on Urchinlike Carbon Nanotube-Gold Nanoparticle Nanoclusters

Nanomaterials have been extensively utilized in biosensing systems for highly sensitive and selective detection of a variety of biotargets. In this work, a facile, label-free, and ultrasensitive electrochemical DNA biosensor has been developed, based on "urchinlike" carbon nanotube-gold nanoparticle (CNT-AuNP) nanoclusters, for signal amplification. Specifically, electrochemical polymerization of dopamine (DA) was employed to modify a gold electrode for immobilization of DNA probes through the Schiff base reaction. Upon sensing the target nucleic acid, the dual-DNA (reporter and linker) functionalized AuNPs were introduced into the sensing system via DNA hybridization. Afterward, the end-modified single-wall carbon nanotubes with DNA (SWCNT-DNA) were attached to the surface of the AuNPs through linker-DNA hybridization that formed 3D radial nanoclusters, which generated a remarkable electrochemical response. Because of the larger contact surface area and super electronic conductivity of CNT-AuNP clusters, this novel designed 3D radial nanostructure exhibits an ultrasensitive detection of DNA, with a detection limit of 5.2 fM (a linear range of from 0.1 pM to 10 nM), as well as a high selectivity that discriminates single-mismatched DNA from fully matched target DNA under optimal conditions. This biosensor, which combines the synergistic properties of both CNTs and AuNPs, represents a promising signal amplification strategy for achieving a sensitive biosensor for DNA detection and diagnostic applications.

A label-free electrochemical DNA biosensor for kanamycin detection based on diblock DNA with poly-cytosine as a high affinity anchor on graphene oxide.

It is urgent to develop a more simple and sensitive method to detect antibiotic residues considering the harm of antibiotic residues in food to the human body. Herein we designed a label-free electrochemical DNA biosensor for the sensitive detection of kanamycin (KAN) based on diblock DNA with a 15-mer of poly-cytosine (poly-C). The diblock DNA can be immobilized on graphene oxide (GO) due to strong physical adsorption between the 15-mer of poly-C and GO. The aptamer of KAN acted as the other block for rapidly binding the target. It can specifically capture the target, which leads to the change of electrochemical signal. Consequently, the DNA biosensor exhibited high sensitivity and specificity towards KAN, the linear range was from 0.05 pM to 100 nM with a detection limit of 0.0476 pM. The developed DNA biosensor was constructed easily and showed promising applications for the detection of antibiotic residues for food safety.

Label-free electrochemical biosensor for the detection of Influenza genes and the solution of guanine-based displaying problem of DNA hybridization

The differentiation of fully matched and unlabelled Influenza A (Inf A) or B (Inf B) target DNA obtained from polymerase chain reaction (PCR)-amplified real samples towards non-complementary sequences have been analyzed with an extremely simple electrochemical methodology by developed label-free electrochemical DNA biosensor without any surface modification. In the meanwhile, this is the first study that contains the solution of the guanine signal-based displaying problem of DNA hybridization which has been solved by designed biosensor. The monitoring of guanine oxidation signal affected by experimental conditions and the response showed significant differences depending on the nature and composition of DNA. In the presented work, the effect of differences in the number of inosine in probe DNA on hybridization imaging was also discussed. The electrochemical oxidation of guanine (approximately +1.00 V) was measured at pencil graphite electrode (PGE) by using differential pulse voltammetry (DPV) technique and evaluated before and after hybridization between probe and target DNAs. Several hybridization solutions and rinsing protocols with different ionic strengths have been utilized to achieve optimum hybridization displaying response. The selectivity of developed genosensor was also tested at the same time with hybridization. The detection limits of sensors were calculated as 35 nM for Inf A and 21 nM for Inf B sequences.

A label-free electrochemical DNA biosensor based on thionine functionalized reduced graphene oxide

In this paper, a transducing interface was designed on a glassy carbon electrode (GCE) to construct a label-free DNA biosensor. The interface was consisted of capture DNA sequence (CP), gold nanoparticles (AuNPs), and thionine functionalized reduced graphene oxide (Thi-rGO). The signal transduction induced directly from redox reaction of thionine because of the excellent electrochemical activity of Thi in the Thi-rGO nanocomposite. However, the redox reaction will be hindered by the biomolecules that immobilized subsequently onto the surface of GCE. Therefore, the determination of target sequence (tDNA) can be realized by monitoring the voltammetric response current of Thi before and after the hybridization between CP and tDNA. Under the optimal conditions, the peak current decreased linearly with the logarithm of concentration of tDNA in the range from 1.0 × 10−17 to 1.0 × 10−12 M with a detection limit of 4.28 × 10−19 M (S/N = 3). The DNA biosensor showed the advantage of acceptable selectivity, good stability and fabrication reproducibility. The proposed DNA biosensor was applied for tDNA detection in a real biological sample, and good recoveries were obtained from 95.61 to 101.8%.

Label-free electrochemical DNA biosensor for guanine and adenine by ds-DNA/poly(L-cysteine)/Fe3O4 nanoparticles-graphene oxide nanocomposite modified electrode

In this study, we aim to design a simple and effective electrochemical DNA biosensor based on a carbon paste electrode modified with ds-DNA/poly(L-cysteine)/Fe3O4 nanoparticles-graphene oxide (ds-DNA/p(L-Cys)/Fe3O4 NPs-GO/CPE) for sensitive detection of adenine (A) and guanine (G). The electrocatalytic oxidation of A and G on the electrode was explored by differential pulse voltammetry (DPV) and cyclic voltammetry (CV). This sensor shows separated and well-defined peaks for A and G, by which one can determine these biological bases individually or simultaneously. The ds-DNA/p(L-Cys)/Fe3O4 NPs-GO/CPE exhibited an increase in peak currents and the electron transfer kinetics and decrease in the overpotential for the oxidation reaction of A and G. Under the optimal conditions a linear relationship is figured out between the peak current and the analytes' concentrations on a range of 0.01–30.0μM and 0.01–25.0μM for simultaneous determination of A and G, with detection limits of 3.48 and 1.59nM, respectively. As well as, individually determination is resulted two linear concentration ranges of 0.01–30.0μM for A and 0.01–25.0μM for G with detection limits of 3.90 and 1.58nM for A and G, respectively. The proposed biosensor exhibited some advantages in terms of simplicity, rapidity, high sensitivity, good reproducibility and long-term stability. Furthermore, the measurements of thermally denatured single-stranded DNA were carried out and the value of (G + C)/(A + T) of DNA was calculated as about 0.77 for various DNA samples. This study also ascertained that the proposed biosensor can be profitable to evaluate DNA bases damage.

Rapid and label-free electrochemical DNA biosensor for detecting hepatitis A virus

Diagnostic systems that can deliver highly specific and sensitive detection of hepatitis A virus (HAV) in food and water are of particular interest in many fields including food safety, biosecurity and control of outbreaks. Our aim was the development of an electrochemical method based on DNA hybridization to detect HAV. A ssDNA probe specific for HAV (capture probe) was designed and tested on DNAs from various viral and bacterial samples using Nested-Reverse Transcription Polymerase Chain Reaction (nRT-PCR). To develop the electrochemical device, a disposable gold electrode was functionalized with the specific capture probe and tested on complementary ssDNA and on HAV cDNA. The DNA hybridization on the electrode was measured through the monitoring of the oxidative peak potential of the indicator tripropylamine by cyclic voltammetry. To prevent non-specific binding the gold surface was treated with 3% BSA before detection. High resolution atomic force microscopy (AFM) confirmed the efficiency of electrode functionalization and on-electrode hybridization. The proposed device showed a limit of detection of 0.65pM for the complementary ssDNA and 6.94fg/µL for viral cDNA. For a comparison, nRT-PCR quantified the target HAV cDNA with a limit of detection of 6.4fg/µL. The DNA-sensor developed can be adapted to a portable format to be adopted as an easy-to- use and low cost method for screening HAV in contaminated food and water. In addition, it can be useful for rapid control of HAV infections as it takes only a few minutes to provide the results.

A label-free Electrochemical DNA Biosensor for the Determination of Low Concentrations of Mitoxantrone in Serum Samples

A label-free Electrochemical DNA Biosensor for the Determination of Low Concentrations of Mitoxantrone in Serum Samples

A label-free electrochemical DNA biosensor for breast cancer marker BRCA1 based on self-assembled antifouling peptide monolayer

Electrochemical assays potentially offer a highly cheap, automated, portable and sensitive method for a great deal of disease biomarker detections, and minimization of nonspecific adsorption is a critical issue for their application in natural complex media. We present herein, the development and application of a highly sensitive and selective electrochemical DNA hybridization biosensor for breast cancer marker BRCA1, based on a zwitterionic peptide self-assembled monolayer (SAM) support serving as the low fouling substrate, the 19-mer BRCA1 related sequence-specific oligonucleotides, and the sensitively label-free sensing technique of electrochemical impedance spectroscopy (EIS). The capability of such peptide SAM in resisting nonspecific protein adsorption was surveyed using EIS, and the biosensor fabrication process and sensing performance were also investigated with X-Ray photoelectron spectroscopy (XPS) and EIS. Significantly, this developed biosensing strategy achieved a linear detection range from 1.0fM to 10.0pM with a limit of detection of 0.3fM, associated with satisfactory sensing properties of facile fabrication, desired sensitivity, high selectivity, and favourable reproducibility and antifouling ability, manifesting its promising potential in practical application.

An electrochemical biosensor for direct detection of DNA using polystyrene-g-soya oil-g-imidazole graft copolymer

A label-free electrochemical DNA biosensor was developed through the attachment of polystyrene-g-soya oil-g-imidazole graft copolymer (PS-PSyIm) onto modified graphene oxide (GO) electrodeposited on glassy carbon electrode (GC). GC/GO electrode was initially functionalised via electrochemical reduction of 4-nitrobenzene diazonium salt, followed by the electrochemical reduction of NO2 to NH2. Subsequent to the electrochemical deposition of gold nanoparticles on modified surface, the attachment of the PS-PSyIm graft copolymer on the resulting electrode was achieved. The interaction of PS-PSyIm with DNA at the bare glassy carbon electrode was studied by cyclic voltammetry technique, and it was found that interaction predominantly takes place through intercalation mode. The selectivity of developed DNA biosensor was also explored by DPV on the basis of considering hybridisation event with non-complementary, one-base mismatched DNA and complementary target DNA sequence. Large decrease in the peak current was found upon the addition of complementary target DNA. The sensitivity of the developed DNA biosensor was also investigated, and detection limit was found to be 1.20 nmol L−1.

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.