Reaction Signal Amplification Research Articles

Ratiometric electrochemical biosensor based on hybridization chain reaction signal amplification for sensitive microRNA-155 detection.

In this work, a sensitive ratiometric electrochemical biosensor for microRNA-155 (miRNA-155) detection is reported based on a hybridization chain reaction amplifying the electrochemical signal. The biosensor was fabricated using Au NPs as a modified material to assemble capture DNA labeled with ferrocene (Fc) molecules, and a DNA probe labeled with methylene blue (MB) was employed for the signal probe. In the presence of target miRNA-155, it can be dual hybridized with capture and signal probe, especially with signal probe to continuously produce long concatemers containing lots of MB molecules. The electrochemical signal of Fc was used for the internal signal, and the signal from MB was used as an indicator signal. As the concentration of miRNA-155 was altered, the internal reference signal of Fc remained constant, and only the indicator signal changed in a sensitive way. The change in the ratio (IMB/IFc) between the indicator signal of MB and internal reference signal of Fc can be used to monitor the concentration of miRNA-155. Under optimal conditions, the prepared ratiometric biosensor could detect miRNA-155 within a wide linear range from 100 fM to 100 nM with low detection limit of 33 fM (at S/N = 3). Moreover, the biosensor was evaluated with human serum samples, and satisfactory recoveries were obtained, indicating that the ratiometric biosensor can be applied to clinical sample analysis.

Coreactant-Free Zirconium Metal-Organic Framework with Dual Emission for Ratiometric Electrochemiluminescence Detection of HIV DNA.

Owing to the limitations of dual-signal luminescent materials and coreactants, constructing a ratiometric electrochemiluminescence (ECL) biosensor based on a single luminophore is a huge challenge. This work developed an excellent zirconium metal-organic framework (MOF) Zr-TBAPY as a single ECL luminophore, which simultaneously exhibited cathodic and anodic ECL without any additional coreactants. First, Zr-TBAPY was successfully prepared by a solvothermal method with 1,3,6,8-tetra(4-carboxyphenyl)pyrene (TBAPY) as the organic ligand and Zr4+ cluster as the metal node. The exploration of ECL mechanisms confirmed that the cathodic ECL of Zr-TBAPY originated from the pathway of reactive oxygen species (ROS) as the cathodic coreactant, which is generated by dissolved oxygen (O2), while the anodic ECL stemmed from the pathway of generated Zr-TBAPY radical itself as the anodic coreactant. Besides, N,N-diethylethylenediamine (DEDA) was developed as a regulator to ECL signals, which quenched the cathodic ECL and enhanced the anodic ECL, and the specific mechanisms of its dual action were also investigated. DEDA can act as the anodic coreactant while consuming the cathodic coreactant ROS. Therefore, the coreactant-free ratiometric ECL biosensor was skillfully constructed by combining the regulatory role of DEDA with the signal amplification reaction of catalytic hairpin assembly (CHA). The ECL biosensor realized the ultrasensitive ratio detection of HIV DNA. The linear range was 1 fM to 100 pM, and the limit of detection (LOD) was as low as 550 aM. The outstanding characteristic of Zr-TBAPY provided new thoughts for the development of ECL materials and developed a new way of fabricating the coreactant-free and single-luminophore ratiometric ECL platform.

Enzyme-free and label-free detection of HER2 in human serum based on Ag+-released hybridization chain reaction signal amplification

The overexpression of human epidermal growth factor receptor 2 (HER2) has been linked to aggressive breast cancer and worse clinical outcomes. However, accurately measuring HER2 remains challenging, due to its low serum content. Herein, we developed a novel method for HER2 detection in serum by the combination of aptamer with C-Ag+-C structure, Ag+-released hybridization chain reaction (Ag+-HCR) and coprecipitation of Ag+ and gold nanoparticles (AuNPs) in the solution including Eosin Y (EY) and AgI colloids. Following the aptamer bound to HER2, signal amplification occurred through the release of numerous Ag+ by Ag+-HCR. This process subsequently induced AuNPs aggregation on the surface of AgI colloids, resulting in reduced fluorescence intensity of EY in the supernatant. The reduced value of EY fluorescence intensity in the supernatant was proportional to the level of HER2. Under optimized reaction conditions, a wide detection range was obtained between 0.09 and 9000 pg/mL with a limit of detection of 11.7 fg/mL in diluted human serum, and 96 serum samples in a 96-well plate could be measured simultaneously by a microtiter plate reader within 2 h. Moreover, the quantitative results of HER2 were consistent with those obtained using the enzyme-linked immunosorbent assay kit (r = 0.9995). Therefore, leveraging the delicate design of Ag+-HCR signal amplification, this enzyme-free, label-free, and effective method has demonstrated to be capable of detecting HER2 in serum due to its high sensitivity. Remarkably, this method may be extended to detect other cancer-related proteins and genes by replacing the corresponding target sequences.

Ultrasensitive upconverting nanoprobes for in situ imaging of drug-induced liver injury using miR-122 as the biomarker

Drug-induced liver injury (DILI) is a frequent adverse drug reaction. The current clinical diagnostic methods are inadequate for accurate and early detection of DILI due to the lack of effective diagnostic biomarkers. Hepatocyte-specific miR-122 is released from injured hepatocytes promptly and its efflux is significantly correlated with the progression of DILI. Therefore, achieving precise in situ detection of miR-122 with high sensitivity is vital for early visualization of DILI. Herein, a new nanoprobe, consisting of miR-122 aptamer, upconversion nanoparticles (UCNPs) and Prussian blue nanoparticles (PBNPs) was introduced for the early and sensitive detection of DILI in situ. As the nanoprobes reached in the liver, miR-122 aptamer-based entropy-driven strand displacement (ESDR) signal amplification reaction was triggered and luminescence resonance energy transfer (LRET) between UCNPs and PBNPs was responded to achieve the high-fidelity detection of DILI. A negative correlation was observed between the intensity of upconversion luminescence (UCL) and the concentration of miR-122. UCL imaging conducted both in vivo and ex vivo indicated that a reduction in miR-122 concentration led to an increase in UCL intensity, revealing a precise state of DILI. The detection technique demonstrated a positive correlation between signal intensity and severity, offering a more straightforward and intuitive method of visualizing DILI.

Repair-driven DNA tetrahedral nanomachine combined with DNAzyme for 8-oxo guanine DNA glycosylase activity assay, drug screening and intracellular imaging.

8-oxo guanine DNA glycosylase (8-oxoG DNA glycosylase), a crucial DNA repair enzyme, is essential for maintaining genome integrity and preventing diseases caused by DNA oxidative damage. Imaging 8-oxoG DNA glycosylase in living cells requires a dependable technique. In this study, we designed a DNAzyme-modified DNA tetrahedral nanomachine (DTDN) powered by 8-oxoG restoration. Incorporating a molecular beacon probe (MB), the constructed platform was used for amplified in situ monitoring of 8-oxoG DNA glycosylase. Under normal conditions, duplexing with a complementary strand modified with two 8-oxoG sites inhibited the activity of DNAzyme. The restoration of DNAzyme activity by the repair of intracellular 8-oxoG DNA glycosylase on 8-oxoG bases can initiate a signal amplification reaction. This detection system can detect 8-oxoG DNA glycosylase activity linearly between 0 and 20 U mL-1, with a detection limit as low as 0.52 U mL-1. Using this method, we were able to screen 14 natural compounds and identify 6 of them as 8-oxoG DNA glycosylase inhibitors. In addition, a novel approach was utilized to assess the activity of 8-oxoG DNA glycosylase in living cells. In conclusion, this method provides a universal tool for monitoring the activity of 8-oxoG DNA glycosylase in vitro and in living cells, which holds great promise for elucidating the enzyme's functionality and facilitating drug screening endeavors.

FEN1-assisted DNA logic amplifier circuit for fast and compact DNA computing.

This work developed DNA amplifier logic gates (AND-OR, OR-AND, FAN-IN, FAN-OUT, and 4-bit square-root circuits) using a flap endonuclease 1 (FEN1)-catalyzed signal amplification reaction, for the fastest and compact DNA computing. Moreover, the logic circuit can use input strands with concentrations of less than 1 nM, which is more than 100 times lower than the input concentration of other DNA logic circuits, providing a promising methodology for constructing fast and compact DNA computations.

Tailoring high-energy self-powered sensing system by Walker-mediated CRISPR/Cas12a cascade signal amplification and hybridization chain reaction for ultrasensitive microRNA detection

MicroRNAs (miRNAs) are important cancer-associated biomarkers for early detection. However, accurately measuring miRNAs is challenging due to their low abundance and high homogeneity in human serum. CRISPR technology can greatly improve the specificity and sensitivity of detection in biosensor construction. In this study, we develop a remarkably responsive self-powered biosensing platform for detecting microRNA-21 (miRNA-21) using hybridization chain reaction (HCR) and CRISPR/Cas12a amplification cascade mediated by 3D DNA walkers. Carbon-coated molybdenum disulfide nanotubes with a large surface area and excellent conductivity are fabricated on flexible carbon paper as the anode and cathode for biological fuel cell construction. Glucose oxidase is loaded onto the anode surface, while serving as the biological cathode, it initiated HCR, generating abundant double-stranded DNA. The negatively charged DNA backbone captures electron acceptors [Ru(NH3)6]3+ through electrostatic adsorption, resulting in a significant open circuit voltage (EOCV) signal. Through CRISPR/Cas12a amplification cascade mediated by 3D DNA walkers, a smaller EOCV is produced, enabling sensitive target detection. The method exhibits a detection range spanning from 0.0001 to 10,000 pM and capable of detecting as low as 19.0 aM (S/N = 3). This highly selective and sensitive method provides an effective approach for accurately detecting tumor markers in clinical applications.

DNAzyme-driven bipedal DNA walker and catalytic hairpin assembly multistage signal amplified electrochemical biosensor based on porous AuNPs@Zr-MOF for detection of Pb2+

As a highly toxic and refractory heavy metal contaminant, Pb2+ seriously endangers human health. The problems of low sensitivity and high cost of signal labeling widely exist in common electrochemical biosensors. Herein, a Pb2+ electrochemical biosensor was constructed using a DNAzyme-driven bipedal DNA Walker and catalytic hairpin assembly as the multistage signal amplification strategy. Compared with Zr-MOF, AuNPs@Zr-MOF has a larger porosity and specific surface area, which can effectively load MB to amplify the current signal. Pb2+ can trigger a dual signal amplification reaction to gradually accumulate the signal of methylene blue/gold nanoparticle @ zirconium-based metal organic frameworks (MB/AuNPs@Zr-MOF) on the electrode. The ingeniously designed sensing strategy realized the analysis of Pb2+ with a wide linear range from 0.05 to 1000 nmol/L and a lower limit of detection (LOD) of 4.65 pmol/L. In addition, the sensor has strong anti-interference ability and can accurately detect Pb2+ in various food samples.

Copper nanoclusters electrochemiluminescence with tunable near-infrared emission wavelength for ultrasensitive detection of matrix metalloproteinase-2

Herein, the methionine (Met)/N-acetyl-L-cysteine (NAC) templated copper nanoclusters (Met/NAC-Cu NCs) with tunable near-infrared region (NIR) electrochemiluminescence (ECL) emission wavelength was firstly synthesized as emitter for the ultrasensitive detection of matrix metalloproteinase-2 (MMP-2). Significantly, the NAC played the role of template and reductant of cupric to acquire Cu NCs, and the surface defect regulator Met was used to connect NAC through -S-S- bond, which could heighten the surface defect of Cu NCs to continuously regulate the maximum ECL emission by successively controlling the molar ratio of Met and NAC, leading to the ECL emission wavelength of Cu NCs ranged from 680 nm to 750 nm. In addition, a rapid target triggered catalyst hairpin assembly (CHA) recycling amplification strategy was constructed through orderly and equidistantly arranging hairpin to increase its local concentration, resulting in greatly accelerated signal amplification efficiency and reaction rate. As a proof of concept, based on Met/NAC-Cu NCs as NIR ECL emitter and effective signal amplification tactic, a super-sensitive ECL biosensor was fabricated to detect target MMP-2 with the detection limit (LOD) as low as 1.65 fg/mL and successfully utilized for detecting of MMP-2 that from Hela and MCF-7 cancer cells. This research provided a wonderful avenue for regulating the optical performance of metal nanoclusters-based ECL emitters, and the developed neoteric NIR ECL emitter with the merits of less photochemical damage and deeper tissue penetration exhibited great potential in ultrasensitive biosensing and high-definition ECL imaging.

Dual-Signal Cascaded Nucleic Acid Amplification Circuit-Loaded Metal-Organic Frameworks for Accurate and Robust Imaging of Intracellular MicroRNA.

Cascaded signal amplification technologies play an important role in the sensitive detection of lowly expressed biomarkers of interests yet are constrained by severe background interference and low cellular accessibility. Herein, we constructed a metal-organic framework-encapsulating dual-signal cascaded nucleic acid sensor for precise intracellular miRNA imaging. ZIF-8 nanoparticles load and deliver FAM-labeled upstream catalytic hairpin assembly (CHA) and Cy5-modified downstream hybridization chain reaction (HCR) hairpin reactants to tumor cells, enabling visualization of the target-initiated signal amplification process for double-insurance detection of analytes. The pH-responsive ZIF-8 nanoparticles effectively protect DNA hairpins from degradation and allow the release of them in the acid tumor microenvironment. Then, intracellular target miRNAs orderly trigger cascaded nucleic acid signal amplification reaction, of which the exact progress is investigated through the analysis of the fluorescence recovering process of FAM and Cy5. In addition, DNA@ZIF-8 nanoparticles improve measurement accuracy by dual-signal colocalization imaging, effectively avoiding nonspecific false-positive signals and enabling in situ imaging of miRNAs in living cells. A dual-signal colocalization strategy allows accurate target detection in living cells, and DNA@ZIF-8 provides a promising intracellular sensing platform for signal amplification and visual monitoring.

Spherical Nucleic Acid‐Amplified Digital Flow Cytometric Bead Assay for the Ultrasensitive Detection of Protein and Exosome†

Comprehensive SummaryMost conventional digital bioassays rely on the use of fully‐sealed microchambers as independent units to compartmentalize the target molecules and the signal generation reaction, which require specialized equipment or proprietary reagents/consumables. Herein, we report a microchamber‐free and spherical nucleic acid (SNA)‐amplified digital flow cytometric bead assay (dFBA) for ultrasensitive protein and exosome analysis with simple workflows, easily accessible instruments/reagents, and high discriminating ability towards the fluorescence‐positive and fluorescence‐negative beads. In this dFBA, microbeads are employed as independent carriers to anchor the single target molecule‐initiated signal amplification reaction, avoiding the use of sealed droplets or microwell microchambers. Meanwhile, antibody‐functionalized SNAs (FSNAs) with a high density of DNA probes act as a bridge for efficiently amplified target‐to‐DNA signal conversion, which allows the use of DNA‐based rolling circle amplification (RCA) as the fluorescence signal amplification technique to quantify non‐nucleic acid targets. Even a single target‐induced on‐bead RCA and fluorescence enriching are sufficient to make the target‐loaded bead bright enough to be clearly discriminated from the negative ones just by use of a most common flow cytometer (FCM). This dFBA has successfully realized the digital analysis of ultralow levels of protein and exosome biomarkers, enlarging the toolbox of digital bioassays for clinical applications.

Self-powered DNA nanomachines for fluorescence detection of lead.

A versatile DNA nanomachine detection system has been developedvia the combination ofDNAzyme with catalytic hairpin assembly (CHA) technology for achieving accurate and sensitive detection of lead ions (Pb2+). In the presence of target Pb2+, capture DNA nanomachine formed by AuNP and DNAzyme recognized and reacted with Pb2+, which yielded an "active" DNAzyme, that induced the cleavage of substrate strand, and then released the initiator DNA (TT) for CHA. With the help of the initiator DNA TT, self-powered CHA was activated to achieve the signal amplification reaction in the detection of DNA nanomachine. Meanwhile, the initiator DNA TT was released and hybridized with the other H1 strand to initiate another CHA, replacement, and turnovers, producing enhanced fluorescence signal of fluorophore FAM (excitation 490nm/emission 520nm) for sensitive determination of Pb2+. Under the optimized conditions, the DNA nanomachine detection system revealed high selectivity toward Pb2+ in the concentration range 50-600pM, with the limit of detection (LOD) of 31pM. Recovery tests demonstrated that the DNA nanomachine detection system has excellent detection capability in real samples. Therefore, the proposed strategy can be extended and act as a basic platform for highly accurate and sensitive detection of various heavy metal ions.

Self-Stacking Autocatalytic Molecular Circuit with Minimal Catalytic DNA Assembly.

Isothermal autocatalytic DNA circuits have been proven to be versatile and powerful biocomputing platforms by virtue of their self-sustainable and self-accelerating reaction profiles, yet they are currently constrained by their complicated designs, severe signal leakages, and unclear reaction mechanisms. Herein, we developed a simpler-yet-efficient autocatalytic assembly circuit (AAC) for highly robust bioimaging in live cells and mice. The scalable and sustainable AAC system was composed of a mere catalytic DNA assembly reaction with minimal strand complexity and, upon specific stimulation, could reproduce numerous new triggers to expedite the whole reaction. Through in-depth theoretical simulations and systematic experimental demonstrations, the catalytic efficiency of these reproduced triggers was found to play a vital role in the autocatalytic profile and thus could be facilely improved to achieve more efficient and characteristic autocatalytic signal amplification. Due to its exponentially high signal amplification and minimal reaction components, our self-stacking AAC facilitated the efficient detection of trace biomolecules with low signal leakage, thus providing great clinical diagnosis and therapeutic assessment potential.

A homogeneous fluorescence assay for rapid and sensitive quantification of the global level of abasic sites in genomic DNA

The apurinic/apyrimidinic (AP) sites are frequent DNA lesions in genomic DNA (gDNA). Here we report a facile approach for rapid quantification of the AP sites in gDNA with high selectivity and sensitivity. With the assistance of T4 pyrimidine dimer glycosylase, we covalently labeled the AP sites with 5′-hydroxylamine-modified oligonucleotide strand with high chemical selectivity against to naturally occurring formylated-bases, such as 5-formylcytosine and 5-formyluracil. Next, we sequentially removed the excessive labeling strands and triggered a signal amplification reaction with the labeled strands in a homogeneous system by flexible variation of the 3′ or 5′ terminal bases of an assistant strand and a fluorescent probe in the presence of a versatile exonuclease (lambda exonuclease). The detection of AP sites in gDNA was realized with an input of gDNA less than 500 ng and a limit of detection down to 0.2 fmol. The method enabled quantification of AP sites in gDNA from both normal cells and cells exposed to external damaging agents, showing the variation of AP sites level along with damaging and repair processes. The work has also provided a useful strategy for the rapid detection of other targeted sites in gDNA in a homogeneous system.

A facile enzyme-assisted multiple recycling amplification strategy for ultrasensitive fluorescence detection of HIV-1 DNA

We developed an enzyme-assisted multiple signal amplification reaction for target nucleic acid detection. In this strategy, the target nucleic acid as triggers, and in the presence of two specific primers, DNA polymerase and DNA nicking endonuclease act in concert to promote the repetitive cycle DNA replication process, leading to an exponential increase in nucleic acid sequence. Both ends of each sequence of the amplification products and the specific primer could from a unit of Mg2+-DNAzyme which in the presence of cofactor Mg2+ could recognize and cyclically cleave the hairpin probes in the solution and thus realize real-time fluorescence detection of HIV-1 DNA. Under optimal conditions, the detection limit of this method is 10−17 M. The simple in design, isothermal conditions and easy fluorescence measurement of this method exhibits superior sensitivity and specificity, and thus holds great potential to be a promising assay for quantitative detection of DNA or RNA in theoretical and clinic studies.

Microneedle Array Encapsulated with Programmed DNA Hydrogels for Rapidly Sampling and Sensitively Sensing of Specific MicroRNA in Dermal Interstitial Fluid.

Author: Please verify that the changes made to improve the English still retain your original meaning.Detection of microRNA (miRNA) in dermal interstitial fluid (ISF) has emerged as clinically useful in health status monitoring. However, it remains a great challenge owing to the difficult sampling and low abundance. Here, we report a DNA hydrogel microneedles (MNs) array to realize rapid enrichment and sensitive detection of miRNA in ISF. The MNs' patch consists of methacrylate hyaluronic acid (MeHA) equipped with a smart DNA circuit hydrogels' system (MeHA/DNA), in which an appropriate miRNA input enables triggering a cascading toehold-mediated DNA displacement reaction to catalytically cleave cross-linking points to generate amplified fluorescence (FL) for miRNA detection. The MeHA/DNA-MNs patch with high mechanical strength can extract adequate ISF in a short time (0.97 ± 0.2 mg in 5 min) in vivo because of its supreme water affinity. Additionally, the cascading toehold-mediated DNA displacement signal amplification reaction allows for sensitive detection of the low-abundant miRNAs down to 241.56 pM. The DNA hydrogels' MNs present potential for minimally invasive personalized diagnosis and real-time health monitoring in clinical applications.

A phage-based magnetic relaxation switching biosensor using bioorthogonal reaction signal amplification for Salmonella detection in foods

Phages are uniquely suited for bacterial detection due to their low cost and ability to recognize live bacteria. Herein, our work establishes the proof-of-concept detection of Salmonella in orange juice based on a phage-mediated portable magnetic relaxation switching (MRS) biosensor. The limit of quantification (LOQ) could reach 5 CFU/mL (95 % confidence interval [CI]: 4–7, N = 4) with a linear range of 102-108 CFU/mL, which has improved 10-fold than that without bioorthogonal signal amplification. The recovery rate of the phage-based MRS biosensor was 95.0 % (95 % confidence interval [CI]: 89.0 %-100.9 %, N = 6). The specificity of the phage-based MRS biosensor was 100 % without false-positive results. In addition, this sensor was able to detect <10 CFU per 25 mL of Salmonella in orange juice with 4-h pre-enrichment. The result from the phage-based MRS biosensor is consistent with that from the standard plate count method. This sensor provides a reliable and ultrasensitive detection platform for pathogens.

Wavelength-resolved photoelectrochemical biosensor triggered by cascade signal amplification reactions for RNA methylation analysis on a single interface

Among ribonucleic acid (RNA) methylation modifications, those of m6A and m5C are the two most abundant forms, and the bias of m6A and m5C modification levels plays an essential role in many biological processes. Therefore, constructing a mechanism that can simultaneously analyze the two modifications would be potentially very significant. Herein, we propose a wavelength-resolved photoelectrochemical (PEC) biosensor based on cascade signal amplification reactions for m6A-RNA and m5C-RNA analysis on a single interface. First, a target sequence was transformed with antibody-functionalized magnetic beads to obtain the initial sequences, triggering the 3D DNA nanomachine to transform and amplify targets by generating many output sequences. Subsequently, the output sequences were combined with rolling-circle-amplification (RCA) primer scaffolds, and then a DNA origami structure was formed based on RCA. A DNA origami could immobilize many photoelectrochemical mediators to amplify photocurrent responses. As expected, the proposed PEC biosensor exhibited excellent analytical performances for both m6A-RNA and m5C-RNA. The detection limits were as low as 1.96 fM and 7.37 pM, respectively. The successful fabrication of the PEC biosensor opened new possibilities for epigenetic research and the diagnosis and treatment of methylation-related diseases.

A Sample and Detection Microneedle Patch for Psoriasis MicroRNA Biomarker Analysis in Interstitial Fluid

Skin interstitial fluid (ISF) containing a great variety of molecular biomarkers derived from cells and subcutaneous blood capillaries has recently emerged as a clinically potential component for early diagnosis of a wide range of diseases; however, the minimally invasive sampling and detection of cell-free biomarkers in ISF is still a key challenge. Herein, we developed microneedles (MNs) that consist of gelatin methacryloyl (GelMA) and graphene oxide (GO) for the enrichment and sensitive detection of multiple microRNA (miRNA) biomarkers from skin ISF. The GO-GelMA MNs exhibited robust mechanical properties, fast sampling kinetics, and large swelling capacity, which enabled collecting ISF volume high to 21.34 μL in 30 min, facilitating effective miRNA analysis. It preliminarily realized the sensitive detection of three types of psoriasis-related miRNAs biomarkers either on the patch itself or in solution after release from the hydrogel by combining catalytic hairpin assembly signal amplification reaction. The automated and minimally invasive ISF miRNA detection technology of GO-GelMA MNs has great potential to monitor cell-free clinically informative biomarkers for personalized diagnosis and prognosis.

A novel electrochemical immunosensor for dibutyl phthalate based on Au@Pt/PEI-rGO and DNA hybridization chain reaction signal amplification strategy

Herein, a sensitive immunosensor for the rapid detection of Dibutyl phthalate (DBP) small molecules was developed. Its principle is based on Au@Pt/Polyethyleneimine (PEI)-rGO and hybridization chain reaction signal amplification system. First, Au@Pt/PEI-rGO modified electrode is used as a signal amplification platform and the antibody is fixed. In the presence of target DBP, it competes with DBP-bovine serum albumin-S0 for binding antibody. Finally, the DNA concatemer carrying a large number of signal molecules methylene blue combines with its S0 to produce a reduced electrochemical signal. Under the most reasonable conditions, this immunosensor showed a good linear range from1 pg mL−1 to 0.1 μg mL−1with a low detection limit of 0.276 pg mL−1. It has been verified that this immunosensor has good selectivity, reproducibility and stability. The designed biosensing solution will open up a new path for DBP monitoring and other small molecule targets in the food field.