Catalytic Signal Amplification Research Articles

Simple and Ultrasensitive Detection of Glioma-Related ctDNAs in Mice Serum by SERS-Based Catalytic Hairpin Assembly Signal Amplification Coupled with Magnetic Aggregation.

Circulating tumor DNA (ctDNA) is more representative and accurate than biopsy and is also conducive to dynamic monitoring, facilitating accurate diagnosis and prognosis of glioma. Therefore, the present study aimed to establish and validate a novel amplified method for the detection of IDH1 R132H and BRAF V600E, which were associated with the genetic diagnosis of glioma. A dual-signal amplification method based on magnetic aggregation and catalytic hairpin assembly (CHA) was constructed for the simultaneous detection of ctDNAs. When target ctDNAs are present, the CHA reaction is initiated and leads to the assembly of Au-Ag nanoshuttles (Au-Ag NSs) onto magnetic beads (MBs). Further enrichment of MBs under an external magnetic field facilitated the dual-signal amplification of SERS. The limit of detection (LOD) for IDH1 R132H and BRAF V600E in serum was as low as 6.01 aM and 5.48 aM. The reproducibility and selectivity of the proposed SERS analysis platform was satisfactory. Finally, the platform was applied to quantify IDH1 R132H and BRAF V600E in the serum of subcutaneous-tumor‑bearing nude mice, and the results obtained by SERS were consistent with those from quantitative real-time polymerase chain reaction (qRT-PCR). The present study showed that the dual-signal amplification method is a simple and ultrasensitive strategy for gliomas-associated ctDNAs detection, which is crucial for early diagnosis and dynamic monitoring.

Ultrasensitive lateral flow biosensor based on PtAu@CNTs nanocomposite catalytic chromogenic signal amplification strategy for the detection of nucleic acid

A rapid and ultrasensitive lateral flow biosensor was developed, which based on gold and platinum nanoparticles-decorated carbon nanotubes (PtAu@CNTs) nanocomposite catalytic chromogenic signal amplification strategy for the detection of nucleic acid. Independent platinum and gold nanoparticles modified functional carbon nanotubes (PtAu@CNTs) were prepared by in-situ reduction. Sandwich-type hybridization reaction occurred between PtAu@CNTs-labeled DNA probe, target DNA and Biotin-modified DNA probes, which was captured on test zone of the strip. Accumulation of PtAu@CNTs nano-labels formed a characteristic colored band. After systematic optimization and catalytic chromogen, the naked eye detection limit of PtAu@CNTs-LFA was about 2 pM, and the theoretical detection limit of target DNA is calculated to be 0.43 pM according to the standard curve. The results indicates a rapid, sensitive and specific methods for DNA detection in biological samples, showing great promise for biomedical diagnosis in some malignant diseases in clinical application.

Coupling Bifunctional Nanozyme-Mediated Catalytic Signal Amplification and Label-Free SERS with Immunoassays for Ultrasensitive Detection of Pathogens in Milk Samples.

Rapid and sensitive detection of foodborne bacteria is of great significance in guaranteeing food safety and preventing foodborne diseases. A bifunctional Au@Pt core-shell nanozyme with excellent catalytic properties and high surface-enhanced Raman scattering (SERS) activity was developed for the highly sensitive detection of Salmonella typhimurium based on a label-free SERS strategy. The ultrathin Pt shell (about 1 nm) can catalyze Raman-inactive molecules into Raman-active reporters, greatly amplifying the amount of signal molecules. Moreover, the Au core serves as an active SERS substrate to enhance the signal of reporter molecules, further significantly improving the detection sensitivity. Benefiting from the excellent properties, such a bifunctional Au@Pt nanozyme was integrated with a magnetic immunoassay to construct a label-free SERS platform for the highly sensitive detection of S. typhi with a low detection limit of 10 CFU mL-1. Also, the Au@Pt-based SERS platform exhibited excellent selectivity and was successfully utilized for the detection of S. typhi in milk samples by a portable Raman spectrometer. Our demonstration of the bifunctional nanozyme-based SERS strategy provides an efficient pathway to improve the sensitivity of label-free SERS detection of pathogens and holds great promise in food safety, environmental analysis, and other biosensing fields.

Sensitive Electrochemical Sensor for Glycoprotein Detection Using a Self-Serviced-Track 3D DNA Walker and Catalytic Hairpin Assembly Enzyme-Free Signal Amplification.

Approaches for the detection of targets in the cellular microenvironment have been extensively developed. However, developing a method with sensitive and accurate analysis for noninvasive cancer diagnosis has remained challenging until now. Here, we reported a sensitive and universal electrochemical platform that integrates a self-serviced-track 3D DNA walker and catalytic hairpin assembly (CHA) triggering G-Quadruplex/Hemin DNAzyme assembly signal amplification. In the presence of a target, the aptamer recognition initiated the 3D DNA walker on the cell surface autonomous running and releasing DNA (C) from the triple helix. The released DNA C as the target-triggered CHA moiety, and then G-quadruplex/hemin, was formed on the surface of electrode. Eventually, a large amount of G-quadruplex/hemin was formed on the sensor surface to generate an amplified electrochemical signal. Using N-acetylgalactosamine as a model, benefiting from the high selectivity and sensitivity of the self-serviced-track 3D DNA walker and the CHA, this designed method showed a detection limit of 39 cell/mL and 2.16 nM N-acetylgalactosamine. Furthermore, this detection strategy was enzyme free and exhibited highly sensitive, accurate, and universal detection of a variety of targets by using the corresponding DNA aptamer in clinical sample analysis, showing potential for early and prognostic diagnostic application.

Delayed delivery of chromogenic substrate to nanozyme amplified aptamer lateral flow assay for acetamiprid

Nanozyme amplified lateral flow assay (LFA) demonstrated many attractive performances than the traditional gold nanoparticles (AuNPs) based LFA, however, they required several additional operations to implement the catalytic reaction of substrate. In this work, a chromogenic substrate integrated and nanozyme amplified aptamer lateral flow assay (CSNLFA) was constructed through automatic delivery of chromogenic substrate on strip. By introducing a sucrose-fixed chromogenic substrate loaded fiber (SCF) onto the sample pad of LFA strip, the CSNLFA platform achieved automated sequential catalytic reaction of substrate oxidation deposition after Au@Pt nanozyme was fully captured at T line of strip. This design was applied in an acetamiprid aptamer LFA and the developed aptamer CSNLFA (Apt-CSNLFA) achieved a linear range of 1–150 ng/mL for acetamiprid with the limit of detection (LOD) of 0.17 ng/mL. The Apt-CSNLFA demonstrated similar analytical performance to the conventional aptamer NLFAs but eliminated the additional operations for catalytic signal amplification. In addition, the new method was applied to the detection of tomato samples spiked with acetamiprid. The recovery ranged from 95% to 106.4% was obtained, which demonstrated the high application potential in rapid detection of acetamiprid.

Target/aptamer binding-induced inhibition of enzyme activity for amplified electrochemical detection of Sonic Hedgehog protein

The high expression of Sonic Hedgehog (SHh) is responsible for the known biological activity of Hedgehog, which closely correlates with cancer metastasis, drug resistance and poor prognosis of hepatocellular carcinoma. In this study, with a new target/aptamer binding-induced inhibition of enzyme activity strategy, we report the construction of an amplified and sensitive electrochemical biosensor for detecting SHh. The analytes bind hairpin aptamer probes to form SHh/aptamer complexes, which inhibit cleavage of aptamers by exonuclease III via the steric hindrance effect not to yield the displacement strands that can displace the biotin-modified signal probes from the sensor electrode. The streptavidin-horseradish peroxidase (STV-HRP) subsequently bind the biotin-probes and are confined on electrode surface through biotin/streptavidin interactions to show highly enhanced catalytic currents with the presence of 3, 3′, 5, 5′-tetramethylbenzidine (TMB)/H2O2 for realizing SHh detection down to 3.34 pM with high selectivity. The interrogation of SHh in diluted serums by this sensor has also been demonstrated. With the verification for SHh assay and the advantages in terms of aptamer recognition, simplicity by electrochemical transduction and sensitivity by catalytic current signal amplification, our strategy can be a convenient aptamer-based approach for detecting different proteins for various biological studies and disease diagnosis purposes.

An Artificial Miniaturized Peroxidase for Signal Amplification in Lateral Flow Immunoassays.

Signal amplification strategies are widely used for improving the sensitivity of lateral flow immunoassays (LFiAs). Herein, the artificial miniaturized peroxidase Fe(III)-MimochromeVI*a (FeMC6*a), immobilized on gold nanoparticles (AuNPs), is used as a strategy to obtain catalytic signal amplification in sandwich immunoassays on lateral flow strips. The assay scheme uses AuNPs decorated with the mini-peroxidase FeMC6*a and anti-human-IgG as a detection antibody (dAb), for the detection of human-IgG, as a model analyte. Recognition of the analyte by the capture and detection antibodies is first evidenced by the appearance of a red color in the test line (TL), due to the accumulation of AuNPs. Subsequent addition of 3,3',5,5'-tetramethylbenzidine (TMB) induces an increase of the test line color, due to the TMB being converted into an insoluble colored product, catalyzed by FeMC6*a. This work shows that FeMC6*a acts as an efficient catalyst in paper, increasing the sensitivity of an LFiA up to four times with respect to a conventional LFiA. Furthermore, FeMC6*a achieves lower limits of detection that are found in control experiments where it is replaced with horseradish peroxidase (HRP), its natural counterpart. This study represents a significant proof-of-concept for the development of more sensitive LFiAs, for different analytes, based on properly designed artificial metalloenzymes.

A Diode-Like Dye-Cu Anisotropic Junction in a Coordination Polymer as a Logic State Ratchet for Intratumor Redox Photomodulation.

Redox logic materials offer new avenues to modulate intracellular pathologic redox environment area-specifically, but the unambiguity of redox logic states and their unidirectional and repetitive switchability are challenging to realize. By merging a bistable diisophthalic phenazine dye ligand with CuII salt, a multistable coordination polymer (CP) was constructed, of which the dye-Cu anisotropic junction achieved the diode-like unidirectional electron transfer and logic state ratchet for the first time. Radical cationic CP maintained OFF status with low toxicity in healthy tissues, but was reduced to the neutral SERVO state by the overexpressed glutathione (GSH) in hypoxic tumors. After photoirradiation, the stabilized charge-separated ON status promoted photo-Fenton reaction for reactive oxygen species (ROS) signal transduction, and simultaneously recovered the initial state for catalytic signal amplification of ROS, furnishing intratumor redox photomodulation for therapy.

A Diode‐Like Dye‐Cu Anisotropic Junction in a Coordination Polymer as a Logic State Ratchet for Intratumor Redox Photomodulation

AbstractRedox logic materials offer new avenues to modulate intracellular pathologic redox environment area‐specifically, but the unambiguity of redox logic states and their unidirectional and repetitive switchability are challenging to realize. By merging a bistable diisophthalic phenazine dye ligand with CuII salt, a multistable coordination polymer (CP) was constructed, of which the dye‐Cu anisotropic junction achieved the diode‐like unidirectional electron transfer and logic state ratchet for the first time. Radical cationic CP maintained OFF status with low toxicity in healthy tissues, but was reduced to the neutral SERVO state by the overexpressed glutathione (GSH) in hypoxic tumors. After photoirradiation, the stabilized charge‐separated ON status promoted photo‐Fenton reaction for reactive oxygen species (ROS) signal transduction, and simultaneously recovered the initial state for catalytic signal amplification of ROS, furnishing intratumor redox photomodulation for therapy.

Polyhedral MnSe microparticles with specific Hg2+-suppressed oxidase-like activity: Toward a green and low-cost turn-off method for Hg2+ detection

With the attractive advantage of catalytic signal amplification, noble metal-based peroxidase-mimicking nanomaterials have been intensively used to establish optical methods for hypertoxic Hg2+ detection. However, in these methods the requirement of high-concentration H2O2 can lead to secondary pollution, and the noble metals used result in these assays’ high cost. Therefore, exploring new categories of enzyme mimics to develop environmentally friendly and low-cost approaches for Hg2+ sensing is highly desired. Herein we proposed a novel noble metal-free material, MnSe microparticles, featuring specific Hg2+-suppressed oxidase-like catalytic activity, to fabricate a green, cheap, and high-performance turn-off approach for Hg2+ detection. The MnSe microparticles with a polyhedral structure were obtained via a one-pot solvothermal process, showing excellent oxidase-like activity to catalyze the oxidation of colorless 3,3′,5,5′-tetramethylbenzidine (TMB) to its blue product. The introduction of Hg2+ can significantly mask their active sites via forming an inert layer of HgSe outside the MnSe microparticles, thus suppressing the TMB catalytic color reaction. Based on the turn-off principle, colorimetric quantification of Hg2+ was realized with high sensitivity and specificity. By integrating smartphone sensing with 3D-printed accessories and paper strips, a portable platform for the convenient detection of Hg2+ was further established, demonstrating its great practicability in real analysis.

MMP-responsive nanomaterials.

Matrix metalloproteinases (MMP) are enzymes that degrade the extracellular matrix and regulate essential normal cell behaviors. Inhibition of these enzymes has been a strategy for anti-cancer therapy since the 1990s, but with limited success. A new type of MMP-targeting strategy exploits the innate selective hydrolytic activity and consequent catalytic signal amplification of the proteinases, rather than inhibiting it. Using nanomaterials, the enzymatic chemical reaction can trigger the temporal and spatial activation of the anti-cancer effects, amplify the associated response, and cause mechanical damage or report on cancer cells. We analyzed nearly 60 literature studies that incorporate chemical design strategies that lead to spatial, temporal, and mechanical control of the anti-cancer effect through four modes of action: nanomaterial shrinkage, induced aggregation, formation of cytotoxic nanofibers, and activation by de-PEGylation. From the literature analysis, we derived chemical design guidelines to control and enhance MMP activation of nanomaterials of various chemical compositions (peptide, lipid, polymer, inorganic). Finally, the review includes a guide on how multiple characteristics of the nanomaterial, such as substrate modification, supramolecular structure, and electrostatic charge should be collectively considered for the targeted MMP to result in optimal kinetics of enzyme action on the nanomaterial, which allow access to amplification and additional levels of spatial, temporal, and mechanical control of the response. Although this review focuses on the design strategies of MMP-responsive nanomaterials in cancer applications, these guidelines are expected to be generalizable to systems that target MMP for treatment or detection of cancer and other diseases, as well as other enzyme-responsive nanomaterials.

Dual II-scheme nanosheet-like Bi2S3/Bi2O3/Ag2S heterostructures for ultrasensitive PEC aptasensing of aflatoxin B1 coupled with catalytic signal amplification by dendritic nanorod-like Au@Pd@Pt nanozyme

As one of the most toxic chemical substances, aflatoxin B1 (AFB1) has a strong carcinogenic effect even at a trace level in human and animal, which severely threatens human health and even causes cancers. Therefore, ultrasensitive detection of AFB1 is of significant importance. For such analysis, dual II-scheme sheet-like Bi2S3/Bi2O3/Ag2S heterostructures were prepared by the in-situ growth method, which exhibited high separation efficiency for the electron-hole (e−-h+) pairs, prominent stability, and high photoactivity. Moreover, the dendritic nanorod-like Au@Pd@Pt (Au@Pd@Pt DNRs) nanozyme was homely synthesized, whose peroxidase-like activity was scrupulously investigated by catalytical oxidation of diaminobenzidine (DAB) in the presence of H2O2. Integration by the aptasensing strategy, a photoelectrochemical (PEC) “signal-on” aptasensor was prepared, which exhibited a broader linear range of 0.5 pg mL−1–100 ng mL−1 with a lower limit of detection (LOD = 0.09 pg mL−1, S/N = 3). This work provides a feasible strategy to develop advanced PEC biosensors for actual analysis of environmental pollutants.

High efficient electrochemical biosensor based on exonuclease-Ⅲ-assisted dual-recycling amplification for ultrasensitive detection of kanamycin

A target-triggered and exonuclease-Ⅲ-assisted strand displacement, dual-recycling amplification reaction-based biosensor was developed for the rapid, ultrasensitive and accurate detection of kanamycin. The robust profiling platform was constructed using high conductive MXene/VS2 for the electrode surface modification and high active CeCu2O4 bimetallic nanoparticles as nanozyme to improve the sensitivity as well as the catalytic signal amplification of the biosensor. Using the dual supplementary recycling of primer DNA and hairpin DNA, the electrochemical platform could accurately detect kanamycin to as low as 0.6 pM from the range of 5 pM to 5 μM. By profiling five other antibiotics, this platform exhibited high specificity, enhanced repeatability and reproducibility. Based on these intrinsic characteristics and by utilizing milk and water samples, the as-designed biosensor offers a remarkable strategy for antibiotic detection due to its favorable analytical accuracy and reliability, thereby demonstrating potential application prospect for various antibiotic biosensing in food quality control, water contamination detection and biological safety analysis.

Convenient colorimetric-fluorescent dual-mode recognition of I− in agricultural products and visual determination of Hg2+ in drinking beverages using Ag-Pt bimetal quantum dot nanozyme

Conveniently and efficiently monitoring I− and Hg2+ in agricultural products or drinking beverages for the protection of human health is currently a great challenge. With this aim, a Ag-Pt bimetal quantum-dot nanozyme boosted by bioactive folic acid (FA@Ag-Pt QDs) was first developed for multichannel monitoring of I− and Hg2+ in this work using a two-step liquid-phase reduction method. Not only did the present FA@Ag-Pt QDs possess superior peroxidase-like activity with Michaelis constant (Km) and maximal reaction rate (Vmax) of 0.01 mM/2.95 × 10−8 M·s−1 and 1.15 mM/3.88 × 10−8 M·s−1, respectively, trace Hg2+ or I− could exclusively alter their enzyme-mimic performance with obvious color changes from blue to colorless or dark blue. I− could also strengthen the inherent fluorescence property of FA@Ag-Pt QDs. When applied for visual monitoring of I− and Hg2+ in real beverages or iodine-containing agricultural products, the detection recoveries were 93.9 %–105.3 % and 96.8–104.3 % with low detection limits of 6.56 × 10−8 mol/L and 4.00 × 10−10 mol/L (S/N = 3), respectively. The recovery and detection limit for fluorescent detection of I− were 95.8 %–104.1 % and 1.75 × 10−8 mol/L (S/N = 3), respectively. The mechanisms driving the improved peroxidase-like activity of FA@Ag-Pt QDs and their selective monitoring of Hg2+ and I− were illustrated in detail. The proposed FA@Ag-Pt QDs will act as an efficient sensor for the practical multichannel monitoring of Hg2+ and I−, with superior catalytic signal amplification.

Atomically thin bismuthene nanosheets for sensitive electrochemical determination of heavy metal ions

Developing effective electrocatalysts to achieve highly sensitive and selective detection of heavy metal ions is one of the challenges in the field of environmental monitoring. Herein, bismuth (Bi) metallene (Bi-ene) in atomic thickness is successfully synthesized and applied as a conceptual application in electrochemical sensors for the detection of lead ion (Pb2+) and cadmium ion (Cd2+) both individually and simultaneously, exhibiting superior sensitivity and anti-interference performance. Density functional theory (DFT) calculations reveal that the Bi-ene has a stronger adsorption capability for Pb and Cd than that of Bi nanosheets (Bi-NSs). This work not only achieves Bi-ene-based catalytic signal amplification for sensitive detection of heavy metal ions but also holds promising application of atomic scale materials in environmental monitoring.

Small molecule photocatalysis enables drug target identification via energy transfer

Over half of new therapeutic approaches fail in clinical trials due to a lack of target validation. As such, the development of new methods to improve and accelerate the identification of cellular targets, broadly known as target ID, remains a fundamental goal in drug discovery. While advances in sequencing and mass spectrometry technologies have revolutionized drug target ID in recent decades, the corresponding chemical-based approaches have not changed in over 50 y. Consigned to outdated stoichiometric activation modes, modern target ID campaigns are regularly confounded by poor signal-to-noise resulting from limited receptor occupancy and low crosslinking yields, especially when targeting low abundance membrane proteins or multiple protein target engagement. Here, we describe a broadly general platform for photocatalytic small molecule target ID, which is founded upon the catalytic amplification of target-tag crosslinking through the continuous generation of high-energy carbene intermediates via visible light-mediated Dexter energy transfer. By decoupling the reactive warhead tag from the small molecule ligand, catalytic signal amplification results in unprecedented levels of target enrichment, enabling the quantitative target and off target ID of several drugs including (+)-JQ1, paclitaxel (Taxol), dasatinib (Sprycel), as well as two G-protein-coupled receptors-ADORA2A and GPR40.

Versatile Electrochemiluminescence Biosensing Platform Based on DNA Nanostructures and Catalytic Hairpin Assembly Signal Amplification.

Achieving rapid and highly sensitive detection of biomarkers is crucial for disease diagnosis and treatment. Here, a highly sensitive and versatile dual-amplification electrochemiluminescence (ECL) biosensing platform was constructed for target detection based on DNA nanostructures and catalyzed hairpin assembly (CHA). Specifically, when the target DNA was present, it would hybridize with the auxiliary strands (D1 and D2) to form an I-shaped nanostructure, which in turn triggered the subsequent catalytic hairpin assembly reaction to generate plenty of double-stranded DNA complexes (H1-H2). The resulting double-stranded complex could be trapped on the electrode surface and adsorbed the ECL signal probe Ru(phen)32+.We found that the I-shaped nanostructure-triggered CHA reaction had higher amplification efficiency compared with traditional CHA amplification. Thus, a sensitive "signal-on" ECL biosensor was constructed for target DNA detection with a detection limit of 1.09 fM. Additionally, by combining the binding properties of C-Ag+-C with an elaborately designed "Ag+-helper" probe, the proposed strategy could be immediately utilized for the highly sensitive and selective detection of silver ions, demonstrating the versatility of the developed biosensing platform. This strategy provided a new approach with potential applications in disease diagnosis and environmental monitoring.

Self-Assembly of DNA Tiles with G-Quadruplex DNAzyme Catalytic Activity for Sensing Applications.

DNA tiles form through self-assembly of a small number of DNA strands that interact through basic repeated interactions, allowing the growth of nanoscale structures seeded by molecular inputs. If an approach for catalytic signal amplification can be integrated into the resultant nanostructure, then one can anticipate biosensing or diagnostic applications mediated by DNA tile self-assembly. Here, two-dimensional DNA tiles with split quadruplexes were designed as diagnostic tools for nucleic acid sensing without the use of protein enzymes. The presence of a target sequence leads to formation of extended microscale structures with arrayed multiple G-quadruplexes across the tile plane, with catalytic activity coupled to a colorimetric reporter. Such a mechanism has potential for low-cost signal amplification using unmodified DNA without the use of protein enzymes for biosensing.

An ultrasensitive label-free photoelectrochemical aptasensor based on terminal deoxynucleotidyl transferase amplification and catalytic reaction of G-quadruplex/hemin

An ultrasensitive photoelectrochemical (PEC) analysis method based on terminal deoxynucleotidyl transferase (TdT) amplification effect and G-quadruplex/hemin (G4/hemin) catalytic reaction assisted signal amplification was prepared for the determination of ampicillin. A novel Au NPs@SnIn4S8-graphene sensitized structure was used as photoactive material to attain an intense basic photocurrent. In the presence of the target, P1 obtained by strand displacement reaction was introduced into the electrode surface, and abundant G4/hemin was formed in the presence of TdT and hemin. Subsequently, G4/hemin with horseradish peroxidase-mimicking activity catalyzed the oxidation of 4-chloro-1-naphthol by H2O2 to form benzo-4-chlorohexadienone precipitation on the modified electrode surface, which severely hindered the electron transfer and effectively inhibited the photocurrent output. The detection range of the PEC aptasensor for ampicillin was 10 fM-30 nM, and the limit of detection was 4.97 fM. The aptasensor had good stability and high sensitivity, and possessed a promising application in biological analysis and environmental monitoring.

Highly sensitive competitive electrochemiluminescence immunosensor based on ABEI-H2O2 system with cobalt hydroxide nanosheets and bimetal PdAg as co-enhancer for detection of florfenicol.

Acompetitive electrochemiluminescence immunoassay was established based on the isoluminol-H2O2 (ABEI-H2O2) system catalyzed by cobalt hydroxide (Co(OH)2) to detect florfenicol residues in food. First , ultra-thin two-dimensional Co(OH)2 nanosheets were used as the catalyst of ABEI-H2O2 system, and excellent catalytic effects were acquired by catalytic decomposition of hydrogen peroxide with cobalt ions. Then, bimetal PdAg (Pd/Ag) alloy nanoparticles were used as a bridge to connect ABEI and antibody due to their good biocompatibility; Pd/Ag alloy nanoparticles also had a catalytic effect to further amplify the ECL signal in the system due to the synergistic catalytic effect of thebimetal. A competitive immunoassay strategy was used to detect florfenicol, where the florfenicol in the sample will compete with the antibody for the limited binding sites on the coating antigen. The ECL immunosensor for florfenicol detection shows high sensitivity, with a linear range from 10-4 to 102ngmL-1, and a detection limit of 3.1 × 10-5ngmL-1, where the scan potential was varied from 0 to 0.6Vvs Ag/AgCl. This work was the first to use Co(OH)2 nanosheets and bimetal PdAg catalytic signal amplification methods to design the sensor, which provides a novel, convenient and reliable strategy for ultra-sensitive detection of florfenicol, and other biological small molecules. A novel ECL immunosensor based on ABEI-H2O22.