Probe Immobilization Research Articles

Ultrasensitive detection of circulating tumor DNA using a CRISPR/Cas9 nickase-driven 3D DNA walker based on a COF-AuNPs sensing platform.

Aelectrochemical biosensor was designedutilizing a CRISPR Cas9n-driven DNA walker combined with gold-nanosphere-like covalent organic frameworks (COFs-AuNPs) to detect breast cancer markers (PIK3CA E545K ctDNA). The DNA walker probe is activated only in the presence of circulating tumor deoxyribonucleic acid(ctDNA), binding to a support probe to form a double strand that is then specifically cleaved by the Cas9n/sgRNA complex. This cleavage produces numerous DNA fragments for signal amplification. The COF-AuNPs as electrode materials facilitate electronic transfer and provide additional active sites for the immobilization of nucleic acid probes. This setup achieves a detection limit of 1.76 aM, demonstrating high sensitivity. Additionally, Cas9n improves the specificity of the sensor, accurately distinguishing a pair of base-mismatched sequences, and reducing the occurrence of false positives. Overall, the sensor exhibits excellent selectivity, reproducibility, and potential for early diagnosis of breast cancer.

Label-free and sensitive detection of Citrus Bark Cracking Viroid in hop using Ti3C2Tx MXene-modified genosensor

Since the discovery of Citrus bark cracking viroid (CBCVd) in hops in 2007, affected hop-growing countries such as Slovenia and Germany have been actively pursuing efficient and easily accessible diagnostic tools that could contribute to the early detection of CBCVd in the field. In the early stages of CBCVd infection, typical symptoms or subtle signs of spread may not be evident. Detection becomes feasible only when the plant begins to display symptoms. Unfortunately, there is currently no treatment available, which requires the removal of infected plants as the only viable solution. However, this approach leads to significant economic losses on a large scale. This work demonstrates the development and study of a sensitive, selective, and label-free impedimetric genosensor for the detection of CBCVd in total RNA hop samples. The genosensor is based on a supporting glassy carbon electrode modified with streptavidin-agarose beads, which serve as an effective immobilization layer for a biotinylated single-stranded DNA capture probe. The integration of a 2D-layered Ti3C2Tx MXene into the sensing architecture resulted in a significantly improved electroanalytical performance of the genosensor. Several fabrication and operational parameters were optimized, such as the streptavidin-agarose beads deposition time, capture probe immobilization time and concentration, and sample incubation time. The optimized genosensor exhibited a limit of detection of only 0.5 fg μL⁻¹ (5.5 fmol L−1) in combination with a one-hour incubation with the denatured total RNA hop extract, thus eliminating the need for an additional and laborious amplification step.

Immobilization of Protein Probes on Biochips with Brush Polymer Cells

Immobilization of Protein Probes on Biochips with Brush Polymer Cells

Immuno-Rolling Circle Amplification (Immuno-RCA): Biosensing Strategies, Practical Applications, and Future Perspectives.

In the rapidly evolving field of life sciences and biomedicine, detecting low-abundance biomolecules, and ultraweak biosignals presents significant challenges. This has spurred a rapid development of analytical techniques aiming for increased sensitivity and specificity. These advancements, including signal amplification strategies and the integration of biorecognition events, mark a transformative era in bioanalytical precision and accuracy. A prominent method among these innovations is immuno-rolling circle amplification (immuno-RCA) technology, which effectively combines immunoassays with signal amplification via RCA. This process starts when a targeted biomolecule, such as a protein or cell, binds to an immobilized antibody or probe on a substrate. The introduction of a circular DNA template triggers RCA, leading to exponential amplification and significantly enhanced signal intensity, thus the target molecule is detectable and quantifiable even at the single-molecule level. This review provides an overview of the biosensing strategy and extensive practical applications of immuno-RCA in detecting biomarkers. Furthermore, it scrutinizes the limitations inherent to these sensors and sets forth expectations for their future trajectory. This review serves as a valuable reference for advancing immuno-RCA in various domains, such as diagnostics, biomarker discovery, and molecular imaging.

PCR-free and minute-scale electrochemical analysis of porcine DNA adulteration via molecularly amplified DNA sandwich assay

The increasing incidence of meat adulteration and mislabeling poses significant challenges in terms of food safety and consumer trust. This study proposes an electrochemical DNA biosensor for detecting porcine mitochondrial DNA in tainted meat products, offering a novel approach to address the above challenges. Unlike conventional nucleic acid amplification tests that rely on polymerase chain reactions (PCRs), the proposed biosensor employs a molecularly amplified DNA strategy with DNA tracers that bind to two regions of the target DNA, creating an elongated hybridization structure with multiple redox-tagging molecules. This design catalyzes detection signals autonomously, eliminating the need for PCR amplification. One-step DNA probe immobilization using poly-adenine (poly-A) oligonucleotides significantly improves hybridization efficiency and reduces the necessity for extensive sample purification, thereby simplifying the detection process. The proposed biosensor exhibits a linear detection range of 101–106 pM and a limit of detection (LOD) of 2.2 pM in controlled settings. Furthermore, the proposed biosensor distinguishes pork from beef in adulterated samples with a LOD of 1 % w/w. With its stability exceeding 9 weeks and a cost of less than 0.5 USD per test, the proposed biosensor offers a highly sensitive, economically viable solution with significant potential for widespread use in the meat industry and by end-users, effectively combating porcine adulteration.

Label-free, background-free detection of nucleic acid with immobilization-free heterogeneous biosensor and one-pot hybridization chain reaction amplification

Although immobilization-free and label-free electrochemical DNA (E-DNA) biosensors have engaged tremendous interest due to their superior properties, such as easy operation, time-saving and cost-saving, most of them are fabricated in homogeneous modes and usually produce high background current. In the present work, we proposed a new immobilization-free and label-free heterogeneous E-DNA assay based on a dual-blocker-aided multibranched hybridization chain reaction (HCR) for one-pot nucleic acid detection with zero background. The target nucleic acid triggers the HCR involving cascaded hybridization between two metastable hairpins, resulting in the generation of HCR products with multibranched arms, which can be captured onto the electrode via π-π stacking interactions between multibranched arms and reduced graphene oxide (rGO). Prior to the incubation process with an electrode, two blockers are designed to prohibit the nonspecific absorption of unreacted hairpin probes. Thus, an immobilization-free and label-free heterogeneous electrochemical assay for one-pot nucleic acid detection with zero background is readily realized. This strategy also presents additional merits of simplicity and cheap cost, since probe immobilization, signal tag labeling, and multiple incubation processes are avoided. Therefore, the as-proposed effective and versatile biosensor has great potential to be applied in nucleic acid-related practical biosensing.

Carbon nanotubes integrated photonic barcodes in Herringbone Microfluidics for Multiplex Biomarker Quantification

Early monitoring of cardiovascular disease (CVD) is crucial for its treatment and prognosis. Hence, highly specific and sensitive detection method is urgently needed. In this study, we propose a novel herringbone microfluid chip with aptamer functionalized core-shell photonic crystal (PhC) barcode integration for high throughput multiplex CVD detection. Based on the PhC derived from co-assembled carboxylated single-wall carbon nanotubes and silicon dioxide nanoparticles, we obtain core-shell PhC barcodes by hydrogel replicating and partially etching. These core-shell PhC barcodes not only retain the original structural colors coding element, but also fully expose a large number of carboxyl elements in the ore for the probe immobilization. We further combine the functionalized barcodes with herringbone groove microfluidic chip to elucidate its acceptability in testing clinical sample. It is demonstrated that the special design of microfluidic chip can significantly enhance fluid vortex resistance and contact frequency, improving the sample capture efficiency and detection sensitivity. These features indicate that our core-shell PhC barcodes-integrated herringbone microfluidic system possesses great potential for multiplex biomarker detection in clinical application.

AptaShield: A Universal Signal-Transduction System for Fast and High-Throughput Optical Molecular Biosensing.

Biosensing technologies are often described to provide facile, sensitive, and minimally to noninvasive detection of molecular analytes across diverse scientific, environmental, and clinical diagnostic disciplines. However, commercialization has been very limited mostly due to the difficulty of biosensor reconfiguration for different analyte(s) and limited high-throughput capabilities. The immobilization of different biomolecular probes (e.g., antibodies, peptides, and aptamers) requires the sensor surface chemistry to be tailored to provide optimal probe coupling, orientation, and passivation and prevent nonspecific interactions. To overcome these challenges, here we report the development of a solution-phase biosensor consisting of an engineered aptamer, the AptaShield, capable of universally binding to any antigen recognition site (Fab') of fluorescently labeled immunoglobulins (IgG) produced in rabbits. The resulting AptaShield biosensor relies on a low affinity dynamic equilibrium between the fluorescently tagged aptamer and IgG to generate a specific Förster resonance energy transfer (FRET) signal. As the analyte binds to the IgG, the AptaShield DNA aptamer-IgG complex dissociates, leading to an analyte concentration-dependent decrease of the FRET signal. The biosensor demonstrates high selectivity, specificity, and reproducibility for analyte quantification in different biological fluids (e.g., urine and blood serum) in a one-step and low sample volume (0.5-6.25 μL) format. The AptaShield provides a universal signal transduction mechanism as it can be coupled to different rabbit antibodies without the need for aptamer modification, therefore representing a robust high-throughput solution-phase technology suitable for point-of-care applications, overcoming the current limitations of gold standard enzyme-linked immunosorbent assays (ELISA) for molecular profiling.

Outer-Surface Functionalized Solid-State Nanochannels for Enhanced Sensing Properties: Progress and Perspective.

Solid-state nanochannel-based sensing systems have been established as vigorous tools for sensing plentiful biomarkers due to their label-free, highly sensitive, and high-throughput screening. However, research on solid-state nanochannels has predominantly centered on the functional groups modified on the inner wall, neglecting investigations into the outer surface. Actually, the outer surface, as a part of the nanochannels, also plays a key role in regulating ionic current. When the target nears the entrance of the nanochannel and prepares to pass through, it would also interact with functional groups located on the nanochannel's outer surface, leading to subsequent alterations in the ionic current. Recently, the probes on the outer surface have experimentally demonstrated their ability to independently regulate ionic current, unveiling advantages in in situ target detection, especially for targets larger than the diameter of the nanochannels that cannot pass through them. Here, we review the progress over the past decade in nanochannels featuring diverse outer-surface functionalization aimed at enhanced sensing performance, including charge modification, wettability adjustment, and probe immobilization. In addition, we present the promises and challenges posed by outer-surface functionalized nanochannels and discuss possible directions for their future deployments.

Graphene nanoribbon derived from multi-walled carbon nanotube: An efficient viral gene hosting and biosensing molecular platform for the electroanalysis of HIV-1 gene

Graphene nanoribbon (GNR) is a carbon nanomaterial with strikingly distinct characteristics compared to graphene. GNRs possess high aspect ratio, variable band gap, high surface area and high density of reactive edge states which make it an emerging flat molecular platform alternative to graphene. GNR is not much explored in sensing applications compared to graphene. Here, for the first time, we are reporting the application of GNRs, derived from multiwalled carbon nanotube (MWCNT), as an efficient flat molecular platform for hosting and biosensing of Human immune deficiency virus-1 (HIV-1) gene. For the construction of HIV-1 sensor, GNRs were synthesized by unzipping of MWCNTs and deposited onto glassy carbon electrode (GCE) followed by the immobilization of HIV-1 probe (ssDNA) onto the resulting GCE/GNR platform to obtain GCE/GNR/ssDNA sensor. Subsequently, the sensor was employed in the analysis of target gene (cDNA) by measuring the voltammetric response generated from hybridization between ssDNA and cDNA. Differential pulse voltammetry (DPV) responses recorded at different target gene concentrations displayed perfect linear correlation (R2 = 0.991). The sensor exhibited low detection limit (2.3 aM), low quantification limit (7.67 aM) and high sensitivity (5.5 μA aM−1 cm−2) which is possibly due to synergic effects of GNRs. Sensor exhibited acceptable stability, repeatability and displayed good selectivity when tested with mismatched target DNA sequences. Further, sensor’s diagnostic applicability was verified by spiking target gene in human blood sample which showed high recovery (4 %) indicating its practical utility in the electroanalysis of HIV-1 gene in human blood samples.

Microfluidic Device for Simple Diagnosis of Plant Growth Condition by Detecting miRNAs from Filtered Plant Extracts

Plants are exposed to a variety of environmental stress, and starvation of inorganic phosphorus can be a major constraint in crop production. In plants, in response to phosphate deficiency in soil, miR399, a type of microRNA (miRNA), is up-regulated. By detecting miR399, the early diagnosis of phosphorus deficiency stress in plants can be accomplished. However, general miRNA detection methods require complicated experimental manipulations. Therefore, simple and rapid miRNA detection methods are required for early plant nutritional diagnosis. For the simple detection of miR399, microfluidic technology is suitable for point-of-care applications because of its ability to detect target molecules in small amounts in a short time and with simple manipulation. In this study, we developed a microfluidic device to detect miRNAs from filtered plant extracts for the easy diagnosis of plant growth conditions. To fabricate the microfluidic device, verification of the amine-terminated glass as the basis of the device and the DNA probe immobilization method on the glass was conducted. In this device, the target miRNAs were detected by fluorescence of sandwich hybridization in a microfluidic channel. For plant stress diagnostics using a microfluidic device, we developed a protocol for miRNA detection by validating the sample preparation buffer, filtering, and signal amplification. Using this system, endogenous sly-miR399 in tomatoes, which is expressed in response to phosphorus deficiency, was detected before the appearance of stress symptoms. This early diagnosis system of plant growth conditions has a potential to improve food production and sustainability through cultivation management.

Upconversion-based intelligent dual-mode hydrogel nanosensor for visual quantitative detection of formaldehyde

Formaldehyde (FA) is a highly toxic carbonyl compound to both living organisms and the environment, which requires a rapid and sensitive detection method. The cost-effective paper-based fluorescent sensors are robust tools for FA detection while the immobilization of fluorescent probes onto paper substrates results in reduced sensitivity. To overcome these challenges, an intelligent hydrogel nanosensor was prepared by embedding an upconversion optical probe in the 3D porous polyacrylamide hydrogel for the multiplex chroma detection of FA. The hydrogel nanosensor provided a liquid-phase detection environment for the upconversion probe, ensuring the detection sensitivity compared to paper-based sensors. Moreover, the near-infrared excited UCNPs effectively avoided the background fluorescence generated by the hydrogel matrix under UV light, thus enhancing the precision of FA detection. The prepared hydrogel nanosensor exhibited fluorescent and colorimetric dual-mode response for FA, with a limit of detection (LOD) of 10 nM in fluorescent mode and 25 nM in colorimetric mode. Moreover, a portable sensing platform has been further developed to address practical requirements, enabling cost-effective and instant detection. This novel UCNPs-based hydrogel nanosensor provides a convenient and precise sensing strategy for the detection of hazardous substances in food, with the potential to promote the practical application of portable fluorescent sensing devices in food safety monitoring.

Electrochemical aptasensor fabricated by anchoring recognition aptamers and immobilizing redox probes on bipolar silica nanochannel array for reagentless detection of carbohydrate antigen 15-3.

Timely, convenient, and efficient detection of carbohydrate antigen 15-3 (CA15-3) levels in serum holds significant importance in early screening, diagnostic assistance and prognosis prediction of breast cancer. The development of efficient and convenient electrochemical aptasensors with immobilized redox probes for label-free detection of CA15-3 is highly desirable. In this work, a bipolar silica nanochannel array film (bp-SNA) with two distinct functional domains including nanochannels and an outer surface was employed for the immobilization of recognition ligands and electrochemical redox probes, enabling the construction of a probe-integrated aptasensor for reagentless electrochemical detection of CA15-3. Cost-effective and readily available indium tin oxide (ITO) was used as the supporting electrode for sequential growth of a negatively charged inner layer (n-SNA) followed by a positively charged outer layer (p-SNA). The preparation process of bp-SNA is convenient. Functionalization of amino groups on the outer surface of bp-SNA was modified by aldehyde groups for covalent immobilization of recognition aptamers, further establishing the recognition interface. Within the nanochannels of bp-SNA, the electrochemical redox probe, tri (2,2'-dipyridyl) cobalt (II) (Co(bpy)3 2+) was immobilized, which experienced a dual effect of electrostatic attraction from n-SNA and electrostatic repulsion from p-SNA, resulting in high stability of the immobilized probes. The constructed aptasensor allowed for reagentless electrochemical detection of CA15-3 ranged from 0.001U/mL to 500 U/mL with a low detection limit (DL), 0.13mU/mL). The application of the constructed aptasensor for CA15-3 detection in fetal bovine serum was also validated. This sensor offers advantages of a simple and readily obtainable supporting electrode, easy bp-SNA fabrication, high probe stability and good stability.

An electrochemical metallic nanowire aptasensor for rapid and ultrasensitive detection of cardiac troponin I

Cardiac troponin I (cTnI) is regarded as a significant protein marker of cardiovascular diseases. Slight increase of blood cTnI level could reflect cardiomyocyte injury, presenting great clinical value in early diagnosis and prognostic monitoring of heart failure (HF). Here, we report for the first time, a metallic nanowire aptasensor with high sensitivity and short detection time as a promising strategy for POC detection of cTnI. We designed and fabricated a novel single Au nanowire electrode with stable anchor structure, and successfully attained immobilization of cTnI-specific aptamer probes. The metallic nanowire aptasensor exhibited high-rate mass transfer and low background current, as well as high affinity and selectivity of the aptamers that facilitated excellent detection performance such as high sensitivity (limit of detection: 13 fg/mL, S/N = 3), short detection time (≤ 15 min), high specificity and good reproducibility in serum samples. These unique properties enabled the nanowire aptasensor for POC detection of cTnI in early diagnosis and prognostic monitoring of HF. Moreover, this nanowire aptasensor was also featured with high stability and regeneration capability, which was envisioned to be further integrated into portable devices, providing potential applications in continuous monitoring of cardiomyocyte injury.

Biochip with Cells of Brush Polymers Carrying Carboxyl Groups for DNA Analysis

A method for manufacturing biochips by photolithography with hydrogel cells made of brush copolymers based on acrylic acid and acrylamide, fixed at one end on the surface of a polymer substrate has been developed. Hydrogel cells with reactive carboxyl groups were used for covalent immobilization of oligonucleotide probes. The effectiveness of the method was demonstrated in the hybridization analysis of DNA by targets of different lengths of the sequence site 7 of the exon of the human ABO gene.

Immunosensor for bovine anaplasmosis diagnosis on graphite platform functionalized with poly (3-hydroxybenzoic acid)

This study describe the development of an electrochemical immunosensor with a transducer platform functionalized with the poly-(3- hydroxybenzoic acid), to detection of antibodies against the surface protein (Am1) of Anaplasma marginale. This biological probe was immobilized on the polymer under graphite and characterized electrochemically. The detections of antigen-antibody interactions were conducted using the signal obtained from the oxidation of 4-aminoantipyrine (4 -AAP), interleaved with Am1, to enable the use of voltammetry technique of differential pulse. The detections of antibodies of A. marginale, using the graphite electrode functionalized with the probe Am1 was 66% higher than the graphite electrode not functionalized. The time of storage of the immunosensor was satisfactory, with reduced peak current by 35%, after 90 days. The results demonstrate the excellent applicability of this new functionalized platform for probe immobilization and diagnosis of anaplasmosis without the interference of the most similar bovine diseases

Integrating multiple probes for simplifying signal-on photoelectrochemical biosensing of microRNA with ultrasensitivity and wide detection range based on biofunctionalized porous ferroferric oxide and hypotoxic quaternary semiconductor

A facile and signal-on photoelectrochemical (PEC) biosensing strategy was designed based on hypotoxic Cu2ZnSnS4 NPs nanoparticles (NPs) and biofunctionalized Fe3O4 NPs that integrated recognition units with signal elements, without the need for immobilization of probes on the electrode. Cu2ZnSnS4 NPs were used as the PEC substrate to produce intensive and stable photocurrent. The porous magnetic Fe3O4 NPs displayed favorable loading capacity for CdS QDs and easy biofunctionalization by negatively charged capture DNA (cDNA). cDNA sealed the pore of Fe3O4 NPs, avoiding the escape of CdS QDs as a PEC sensitizer. After hybridizing with target microRNA (miRNA), cDNA split away off Fe3O4 NPs whose porous channel might open and release sealed CdS QDs (signal element), resulting in a dramatical enhancement of PEC response. Herein, miRNA hardly contacted with CdS QDs, effectively avoiding harm to the target miRNA. This proposed strategy simplified procedures of assembly and made the biorecognition process sufficient for promoting a stationary quantity of probes, which was expected to obtain satisfactory performance for bioassay. Using miRNA-155 as a model analyte and combining with duplex-specific nuclease (DSN)-assisted amplification, a simplified and signal-on PEC biosensing platform for miRNA-155 with wonderful performance was proposed. DSN-assisted amplification further promoted PEC signal increment, leading to ulteriorly improving sensitivity (detection limit of 0.17 fM) and linear range (6.5 orders of magnitude) for miRNA-155 assay. Moreover, the developed PEC biosensing platform exhibited satisfactory stability, excellent specificity, and favorable accuracy for miRNA-155, which would have a promising prospect for monitoring miRNA expression in tumor cells.

Point‐of‐Care Testing (POCT) Devices for DNA Detection: A Comprehensive Review

DNA chips play a crucial role in point‐of‐care diagnostics by enabling rapid and accurate detection of genetic information. These chips offer high sensitivity and selectivity, allowing for the identification of specific DNA sequences associated with diseases and pathogens. Integration into lab‐on‐chip platforms streamlines and miniaturizes diagnostic workflows, paving the way for cost‐effective, portable, and user‐friendly testing devices that can revolutionize healthcare delivery. In this review, a comprehensive description of the platforms utilized in DNA analysis, including microfluidic devices and integrated DNA chips, is provided. It explores the selection and immobilization of DNA probes for improved selectivity. Additionally, it covers diverse detection techniques such as optical detection (colorimetry and fluorescence) and electrochemical techniques. In these discussions, it is aimed to provide a thorough understanding of the current state of the art in DNA biosensor‐integrated lab‐on‐chip technology for point‐of‐care testing. The continued advancements in DNA chip technology hold immense promise for the development of next‐generation point‐of‐care diagnostics, where integrated sample preparation and rapid results generation can further enhance patient outcomes and contribute to the effective management of diseases.

Monitoring the Effects Brought By a Drug on Myocardial Cells Using Field Effect Transistor (FET) Based Sensing Platform

: Ion channels are the membrane proteins that modulate the active flow of ions between cellular environment. These therapeutic targets are expressed in every active cell and can be used as important targets for cellular changes. The ion channels play an important role in different physiological processes including blood regulation, cell proliferation, nerve relaxation, etc. Various cardiac and neurological diseases have been reported to be related to ion channel regulation. They are brought through by the ion channel mutations. Several techniques are currently deployed for monitoring drug effectiveness of ion channels. Among these techniques, patch clamp technology is considered as a golden standard for monitoring the ion channel function. Unfortunately, the conventional patch clamp technologies are not enough to be employed in the early stages of drug discovery. Similarly, serval fluorescence detection methods and combination technologies have been developed for calcium ion channel detection. These assays are not suitable for large scale detection due to less selectivity and sensitivity.At present, field-effect transistor-based biosensors (BioFETs) have been widely employed in various applications due to their non-invasive detection, and high sensitivity. In this work, we adopted an extended gated EDL-FET platform for rapid drug screening on myocardial cell lines (H9c2). The drug effectiveness was studied by tested nifedipine on myocardial cells which were mounted over an EDL-FET platform. Two different surface conditions were studied using Ca2+ treatment and the capability the of EDL-FET sensor as a drug screening platform was discussed.Surface functionalization and Measurements An extended gate chip was used as the sensor array. Before probe immobilization, the sensor electrode was treated with oxygen plasma. Cell attachment over the sensor platform was facilitated using a glycoprotein- Fibronectin. Traut’s reagent was used to modify fibronectin to initiate the covalent attachment to the sensor surface. Optical images and electrical readings were taken to ensure proper functionalization of the fibronectin-attached surface before the cells were seeded over it. Myocardial cells (H9c2 cells), removed from their culture media, were incubated over the functionalized electrodes for ~2 hours following which, electrical measurements were taken to establish a baseline reading using the BioFET device. Different concentrations of nifedipine [0 to 50 μM] and calcium ions [18 μM to 1.8 mM] were used to make testing solutions, which were used for monitoring various changes occurring on the ion channels of the myocardial cells. Figure 1

Ultrasensitive rapid detection of antibiotic resistance genes by electrochemical ratiometric genosensor based on 2D monolayer Ti3C2@AuNPs

As an important emerging pollutant, antibiotic resistance genes (ARGs) monitoring is crucial to protect the ecological environment and public health, but its rapid and accurate detection is still a major challenge. In this study, a new single-labeled dual-signal output ratiometric electrochemical genosensor (E-DNA) was developed for the rapid and highly sensitive detection of ARGs using a synergistic signal amplification strategy of T3C2@Au nanoparticles (T3C2@AuNPs) and isothermal strand displacement polymerase reaction (ISDPR). Specially, two-dimensional monolayer T3C2 nanosheets loaded with uniformly gold nanoparticles were prepared and used as the sensing platform of the E-DNA sensor. Benefiting from excellent conductivity and large specific surface area of Ti3C2@AuNPs, the probe immobilization capacity of the E-DNA sensor is doubled, and electrochemical response signals of the E-DNA sensor were significantly improved. The proposed single-labeled dual-signal output ratiometric sensing strategy exhibits three to six times higher sensitivity for the sul2 gene than the single-signal sensing strategy, which significantly reduces cost meanwhile retaining the advantages of high sensitivity and reliability offered by conventional dual-labeled ratiometric sensors. Coupled with ISDPR amplification technology, the E-DNA sensor has a wider linear range from 10 fM to 10 nM and a limit of detection as low as 2.04 fM (S/N=3). More importantly, the E-DNA sensor demonstrates excellent specificity, good stability and reproducibility for target ARGs detection in real water samples. The proposed new sensing strategy provides a highly sensitive and versatile tool for the rapid and accurate quantitative analysis of various ARGs in environmental water samples.