A Highly Sensitive and Group-Specific Enzyme-Linked Immunosorbent Assay (ELISA) for the Detection of AFB1 in Agriculture and Aquiculture Products.

Aflatoxins (AFs) including AFB1, AFB2, AFG1 and AFG2 are widely found in agriculture products, and AFB1 is considered one of the most toxic and harmful mycotoxins. Herein, a highly sensitive (at the pg mL-1 level) and group-specific enzyme-linked immunosorbent assay (ELISA) for the detection of AFB1 in agricultural and aquiculture products was developed. The AFB1 derivative containing a carboxylic group was synthesized and covalently linked to bovine serum albumin (BSA). The AFB1-BSA conjugate was used as an immunogen to immunize mice. A high-quality monoclonal antibody (mAb) against AFB1 was produced by hybridoma technology, and the mAb-based ELISA for AFB1 was established. IC50 and limit of detection (LOD) of the ELISA for AFB1 were 90 pg mL-1 and 18 pg mL-1, respectively. The cross-reactivities (CRs) of the assay with AFB2, AFG1, and AFG2 were 23.6%, 42.5%, and 1.9%, respectively, revealing some degree of group specificity. Corn flour, wheat flour, and crab roe samples spiked with different contents of AFB1 were subjected to ELISA procedures. The recoveries and relative standard deviation (RSD) of the ELISA for AFB1 in spiked samples were 78.3-116.6% and 1.49-13.21% (n = 3), respectively. Wheat flour samples spiked with the mixed AF (AFB1, AFB2, AFG1, AFG2) standard solution were measured by ELISA and LC-MS/MS simultaneously. It was demonstrated that the proposed ELISA can be used as a screening method for evaluation of AFs (AFB1, AFB2, AFG1, AFG2) in wheat flour samples.

Ultrasensitive detection of zearalenone based on electrochemiluminescent immunoassay with Zr-MOF nanoplates and Au@MoS2 nanoflowers.

In this work, an effective competitive-type electrochemiluminescence (ECL) immunosensor was constructed for zearalenone determination by using Zr-MOF nanoplates as the ECL luminophore and Au@MoS2 nanoflowers as the substrate material. Zr-MOF have an ultra-thin sheet-like structure that accelerates the transfer of electrons, ions and co-reactant intermediates, which exhibited strong and stable anodic luminescence. The three-dimensional Au@MoS2 nanoflowers would form a thin film modification layer on the glassy carbon electrode (GCE). And its good electrical conductivity and higher specific surface area utilization further improving the sensitivity of the ECL immunosensor. Under the optimized conditions, the proposed immunosensor exhibited satisfactory stability, sensitivity and accuracy, and its ECL signal was proportional to the logarithm of ZEN concentration (0.0001-100 ng/mL) and the limit of detection (LOD) was 0.034 pg/mL. In addition, the results of recovery experiment acquired for wheat flour and pig urine samples further proved the feasibility of the immunosensor for the detection of real samples, indicating its potential for ultrasensitive detection of ZEN.

Development of a flow injection chemiluminescence immunoassay based on DES-mediated CuCo2O4 nanoenzyme for ultrasensitive detection of zearalenone in foods.

Nanoenzymes have been widely used to construct biosensors because of their cost-effectiveness, high stability, and easy modification. At the same time, the discovery of deep eutectic solvents (DES) was a great breakthrough in green chemistry, and their combination with different materials can improve the sensing performance of biosensors. In this work, we report an immunosensor using CuCo2O4 nanoenzyme combined with flow injection chemiluminescence immunoassay for the automated detection of zearalenone (ZEN). The immunosensor exhibited excellent sensing performance. Under the optimal conditions, the detection range of ZEN was 0.0001-100 ng mL-1, and the limit of detection (LOD) was 0.076 pg mL-1 (S/N = 3). In addition, the immunosensor showed excellent stability with a relative standard deviation (RSD) of 2.65% for  15 repetitive  injections. The method has been successfully applied to the analysis of real samples with satisfactory recovery results, and can hence provide a reference for the detection of small molecules in food and feed.

Electrochemiluminescence immunosensor based on tin dioxide quantum dots and palladium-modified graphene oxide for the detection of zearalenone.

Developing low-cost and efficient methods to enhance the electrochemiluminescence (ECL) intensity of luminophores is highly desirable and challenging. Herein, we developed an efficient ECL system based on palladium-modified graphene oxide as a substrate and tin dioxide quantum dot-modified spike-like gold-silver alloy as an immunoprobe. Specifically, palladium-modified graphene oxide was rationally selected as the sensor substrate for the attachment of zearalenone antigens while facilitating the amplification of the ECL signal through enhanced electron transfer efficiency. A spike-like gold-silver alloy modified with tin dioxide quantum dots was attached to the zearalenone antibody as an immunoprobe, and the sensor exhibited remarkable sensitivity due to the exceptional ECL performance of the quantum dots. To demonstrate the practical feasibility of the principle, zearalenone levels were detected in actual samples of maize and pig urine, and the sensor showed a broad linear range (0.0005-500 ng mL-1) and low detection limit (0.16 pg mL-1) in the high-sensitivity detection of Zearalenone. Overall, this work first reports the construction of a highly sensitive ECL immunosensor for the detection of zearalenone using a protruding gold-silver alloy modified with tin dioxide as an immunoprobe and a palladium modified graphene oxide as a substrate. It provides a novel approach for the detection of small molecule toxin-like substances.

A sensitive monoclonal antibody-based ELISA integrated with immunoaffinity column extraction for the detection of zearalenone in food and feed samples.

Zearalenone (ZEN) is one of the most toxic mycotoxins widely found in agricultural products. In this study, a sensitive enzyme-linked immunosorbent assay (ELISA) integrated with immunoaffinity column extraction for the detection of ZEN in food and feed samples was developed. A ZEN derivative containing a carboxylic group was first synthesized and then linked to bovine serum albumin (BSA). The formed ZEN-BSA conjugate was used as the immunogen for the production of the monoclonal antibody (mAb) against ZEN. The hybridoma clones (1G5) capable of secreting antibodies against ZEN were successfully selected. Based on this mAb, the IC50 and LOD of the ELISA for ZEN were 0.37 ng mL-1 and 0.04 ng mL-1, respectively, which were 1.6-308.1 times lower than those in the published ELISAs, indicating the high sensitivity of our assay. There was no cross-reactivity of the mAb with other four mycotoxins (patulin, AFB1, DON, and OTA). Due to the high similarity in molecular structures among ZEN and its homologs (α-zearalanol, ß-zearalanol, zearalanone, α-zearalenol, ß-zearalenol), the CR values of the mAb with the homologs were within 3.59%-105.71%. Taking advantage of plenty of mAb, the immunoaffinity column was prepared by immobilizing the mAb on Sepharose-4B gel and filling it into an SPE column. ZEN spiked samples (corn, wheat, feed) were extracted using an immunoaffinity column and measured by ELISA and HPLC-FLD simultaneously. The recoveries of the ELISA for ZEN in the spiked samples were 92.46-105.48% with RSDs of 4.87-10.11%. A good correlation between ELISA (x) and HPLC-FLD (y) with the linear regression equation y = 1.0589x + 1.43815 (R2 = 0.998, n = 6) was obtained. To verify the applicability, the proposed ELISA was also applied to some real samples randomly collected from a local market. It was proven that the newly produced mAb-based ELISA was a feasible and sensitive method for the detection of ZEN in food and feed samples.

Potential-Resolved Electrochemiluminescence Multiplex Immunoassay for Florfenicol and Chloramphenicol in a Single Sample.

The simultaneous detection of multiple antibiotic residues in food is of great significance for food safety. In this work, a novel dual-potential electrochemiluminescence (ECL) immunoassay was designed for the simultaneous detection of chloramphenicol and fluorfenicol residues in food. Ru@MOF was used as an anodic probe, and SnS2 QDs-PEI-Au-MoS2 was used as a cathodic probe. Notably, the coreactant for both luminophores was K2S2O8, avoiding interactions caused by different kinds of coreactants. Au nanoparticles functionalized with a nitrogen- and sulfur-doped graphene oxide-modified glassy carbon electrode to improve the electron transfer efficiency and provide a larger surface area for immobilization of antigen. The linear range for the detection of florfenicol was determined to be 0.1-1000 ng mL-1 with a detection limit of 0.03 ng mL-1, and the linear range for the detection of chloramphenicol was 0.01-1000 ng mL-1 with a detection limit of 3.2 pg mL-1 by recording the ECL responses at two different excitation potentials. The proposed immunoassay achieved a more stable recovery in the detection of actual samples and provided a new analytical method for the simultaneous detection of florfenicol and chloramphenicol residues with high sensitivity and specificity.

Electrochemiluminescence resonance energy transfer immunoassay based on a porphyrin metal-organic framework and AuNPs/NSG for the sensitive detection of zearalenone.

In this study, a novel electrochemiluminescence resonance energy transfer (ECL-RET) immunoassay was developed for the first time for the detection of zearalenone (ZEN). A porphyrin metal-organic framework (PCN-222), an emerging porphyrin-based ECL luminophore, was prepared by a simple hydrothermal method using tetrakis(4-carboxyphenyl) porphyrin, which has excellent ECL emission as well as good ECL efficiency. Because the ECL emission spectrum of PCN-222 is highly matched to the absorption spectrum of gold nanoparticle-modified graphene oxide (AuNPs/NSG) nanocomposites, they were used as donor-acceptor counterparts in this work for the ECL-RET strategy. Under optimal conditions, the ECL immunosensor showed a sensitive response to ZEN in a wide detection range, with a linearity of 0.0005-1000 ng mL-1 and a detection limit of 0.15 pg mL-1. In addition, the sensor showed good potential for application in the detection of wheat and corn samples, providing a new approach for the detection of mycotoxin-like contaminants such as ZEN in food grains.

An Extremely Highly Sensitive ELISA in pg mL-1 Level Based on a Newly Produced Monoclonal Antibody for the Detection of Ochratoxin A in Food Samples.

In this study, an extremely highly sensitive enzyme-linked immunosorbent assay (ELISA) based on a newly produced monoclonal antibody (mAb) for the detection of ochratoxin A (OTA) in food samples was developed. OTA-Bovine serum albumin (BSA) conjugate was prepared and used as the immunogen for the production of the mAb. Among four hybridoma clones (8B10, 5C2, 9B7, and 5E11), the antibody from 8B10 displayed the highest affinity recognition for OTA. Based on the mAb (8B10), the IC50 and LOD of the ELISA for OTA were 34.8 pg mL-1 and 1.5 pg mL-1, respectively, which was 1.53~147 times lower than those in published ELISAs, indicating the ultra-sensitivity of our assay. There was no cross-reactivity of the mAb with the other four mycotoxins (AFB1, ZEN, DON, and T-2). Due to the high similarity in molecular structures among OTA, ochratoxin B (OTB), and ochratoxin C (OTC), the CR values of the mAb with OTB and OTC were 96.67% and 22.02%, respectively. Taking this advantage, the ELISA may be able to evaluate total ochratoxin levels in food samples. The recoveries of the ELISA for OTA in spiked samples (corn, wheat, and feed) were 96.5-110.8%, 89.5-94.4%, and 91.8-113.3%; and the RSDs were 5.2-13.6%, 8.2-13.0%, and 7.7-13.7% (n = 3), respectively. The spiked food samples (corn) were measured by ELISA and HPLC-FLD simultaneously. A good correlation between ELISA (x) and HPLC-FLD (y) with the linear regression equation of y = 0.918x - 0.034 (R2 = 0.985, n = 5) was obtained. These results demonstrated that the newly produced mAb-based ELISA was a feasible and ultra-sensitive analytical method for the detection of OTA in food samples.

Ultra-sensitive detection of 5-fluorouracil by flow injection chemiluminescence immunoassay based on Fenton-like effect of single atom Co nanozyme.

Single atom nanozymes (SAzymes) are considered as the most hopeful candidates for replacing natural enzymes. In this work, a flow-injection chemiluminescent immunoassay (FI-CLIA) based on a Fenton-like activity single atom cobalt nanozyme (Co SAzyme) was developed for the rapid and sensitive detection of 5-fluorouracil (5-Fu) in serum for the first time. Co SAzyme was prepared by an in-situ etching method at room temperature using ZIF-8 metal-organic frameworks (ZIF-8 MOFs). With excellent chemical stability and ultra-high porosity of ZIF-8 MOFs as the core, Co SAzyme presents high Fenton-like activity which can catalyze the decomposition of H2O2 to produce large amounts of superoxide radical anions, thus effectively amplifying the chemiluminescence of the Luminol-H2O2 system. In addition, carboxyl-modified resin beads were used as the substrate to load more antigens due to its advantages of good biocompatibility and large specific surface area. Under optimal conditions, the detection range of 5-Fu was 0.001-1000 ng mL-1 with a limit of detection of 0.29 pg mL-1 (S/N = 3). Furthermore, the immunosensor was successfully applied for the detection of 5-Fu in human serum samples with satisfactory results, displaying the potential application of this strategy for bioanalysis and clinical diagnosis.

A resonant energy transfer electrochemiluminescence immunosensor based on low trigger potential of Zn-metal organic framework and CoOOH nanosheets for 5-fluorouracil detection.

The organic luminophores have inspired widespread interest in electrochemiluminescence (ECL). Herein, a novel rod-like metal-organic framework was formed by chelating Zn ion with 9,10-di(p-carboxyphenyl)-anthracene (DPA), defined as Zn-MOF for simplicity. In this proposal, the prepared Zn-MOF was first used as a powerful organic luminophore with low trigger potential, thus developing a competitive ECL immunoassay for ultrasensitive detection of 5-fluorouracil (5-FU) with 1,4-diazabicyclo[2.2.2]octane (D-H2) as the coreactant. The absorption spectrum of cobalt oxyhydroxide (CoOOH) nanosheets and the ECL emission spectrum of Zn-MOF could be highly matched, which ensured the occurrence of resonance energy transfer (RET). For that, ECL-RET was applied in the assembly strategy of the ECL biosensor, and Zn-MOF was used as the energy donor and CoOOH nanosheets as the acceptor. Taking advantage of the luminophore and ECL-RET, the immunoassay can be used for ultra-sensitive quantitative detection of 5-fluorouracil. The proposed ECL-RET immunosensor showed satisfactory sensitivity and accuracy with a wider linear range from 0.001 to 1000 ng/mL, and a lower detection limit (0.52 pg/mL). Hence, it is worth believing that this strategy can pave a bright research direction for the detection of 5-FU or other biological small molecules.

A SERS-based lateral flow immunochromatographic assay using Raman reporter mediated-gap AuNR@Au nanoparticles as the substrate for the detection of enrofloxacin in food samples.

A lateral flow immunochromatographic assay (LFIA) based on surface-enhanced Raman scattering (SERS) for sensitive and specific detection of antibiotic enrofloxacin (ENR) in food samples was developed. 1,4-benzenedithiol (BDT) was selected as the Raman reporter, and the BDT mediated-gap AuNR@Au nanoparticles (NPs) were synthesized, characterized and used as the substrate in SERS-LFIA due to the existence of the anisotropic gold nanorods (AuNRs) and the nano-gap with the high SERS enhancement. AuNRs were prepared, then covered by monolayer BDT. Under reduction condition and in presence of HAuCl4, the reduced gold was deposited and grown on AuNRs to form AuNRBDT@Au NPs. As the two thiol groups on para-positions in BDT were respectively linked to AuNR (core) and Au (shell), the gap size inside the NPs was uniform. The immunoprobe (e.g. AuNRBDT@Au-Ab) was obtained by immobilizing Ab against ENR on the surface of AuNRBDT@Au NPs. The performance of SERS-LFIA was similar to that in colloidal gold based-LFIA, and the entire assay time was within 15 min. After LFIA procedures, the specific SERS intensity of BDT at 1560 cm-1 on the test line was measured for the quantitative detection of ENR. The IC50 and limit of detection (LOD) of the LFIA for ENR were 59 pg mL-1 and 0.12 pg mL-1 (e.g. 71 pg g-1 and 0.14 pg g-1 in real sample), respectively. There was no cross-reactivity (CR) of the LFIA with other five antibiotics. The recoveries of ENR from spiked food samples were in range of 89.2%-102.4% with the relative standard deviation (RSD) of 1.70%-6.38%. It was proven that the proposed method was able to simply and rapidly detect ENR in food samples with high sensitivity, specificity, accuracy and precision. The platform can be also an alternative platform for the detection of other target analytes using corresponding Abs.

Electrochemiluminescence immunoassay strategies based on a hexagonal Ru-MOF and MoS2@GO nanosheets: detection of 5-fluorouracil in serum samples.

Herein, a competitive-type electrochemiluminescence immunosensor for ultrasensitive detection of 5-fluorouracil (5-FU) was fabricated. Ruthenium(II)-metal-organic framework (Ru-MOF) nanosheets were selected to act a promising ECL luminophore using tris(4,4'-dicarboxylic acid-2,2'-bipyridyl) ruthenium(II) dichloride (Ru(dcbpy)32+) as the organic ligand. The two-dimensional (2D) Ru-MOF nanosheets achieved an increased loading of Ru(dcbpy)32+ and effectively prevented leakage of the ECL emitter during application, which exhibited satisfactory ECL performance. Thin two-dimensional MoS2@GO was used to modify the electrode as the sensing platform for improving the electron transfer rate and loading more 5-FU coating antigens due to its large specific surface area and piezoelectric catalytic efficiency. Under the optimized conditions, the proposed immunosensor presented high sensitivity, a wide detection range (0.0001 ng-100 ng mL-1), a low limit of detection (0.031 pg mL-1, S/N = 3), good specificity and stability. Furthermore, the immunosensor was successfully applied for the detection of 5-FU in human serum samples with satisfactory results, proving this strategy has potential applications in bioanalysis and clinical diagnosis.

A SERS/electrochemical dual-signal readout immunosensor using highly-ordered Au/Ag bimetallic cavity array as the substrate for simultaneous detection of three ß-adrenergic agonists.

A surface-enhanced Raman scattering (SERS)/electrochemical dual-signal readout immunosensor was developed for simultaneous detection of ß-adrenergic agonists salbutamol (SAL), ractopamine (RAC) and phenylethanolamine A (PA). The highly-ordered gold/silver bimetallic cavity array (BMCA) was prepared by electrodepositing Au/Ag nanoparticles to the interstice of highly ordered close-packed polystyrene templates. After electrochemical and SERS characterization, the BMCA was used as the substrate for constructing SERS/electrochemical dual-signal readout immunosensor. 3,3',5,5'-tetramethylbenzidine (TMB), methylene blue (MB) and Nile blue (NB) were selected as the dual-signal reporters, and hybridization chain reaction (HCR) was used as the signal amplifier. The immunoprobe was prepared by absorption of the antibody (Ab) and constructing HCR system embedded with electro/SERS reporter on Au nanoparticles (NPs). After competitive immuno-reaction between coating antigen and analyte for limited Ab on immunoprobe, the SERS/electrochemical dual-signals on BMCA were measured for quantitatively detecting SAL, RAC and PA simultaneously. SAL, RAC and PA were detected in concentration range of 1 pg mL-1 to 100 ng mL-1 with LOD of 0.8, 0.4, and 1.3 pg mL-1, respectively. The applicability of the proposed immunosensor in spiked pork liver samples was verified by the recovery of 95.0%-108.5% with RSD of 6.9%-10.7%. It was proven that the immunosensor was able to detect multiple ß-adrenergic agonists with high sensitivity, specificity, accuracy and precision. The immunosensor can be used as a platform for the determination of other small molecular compounds in biological, food and environmental analytical fields.

Recent progress in assembly strategies of nanomaterials-based ultrasensitive electrochemiluminescence biosensors for food safety and disease diagnosis.

The Electrochemiluminescence (ECL)-based biosensors have received considerable attention in food contaminants and disease diagnosis, due to their fascinating advantages such as low cost, fast analysis speed, wide linear range, high sensitivity, and excellent anti-interference ability. Meanwhile, with the vigorous development and improvement of nanotechnology, biosensor assembly strategies tend to diversify and be multifunctional. This review focuses on the representative ECL biosensors in food safety and disease diagnosis reported by our research group and other research groups based on nanomaterials assembly strategies in recent years. According to the different roles of nanomaterials played in the constitution of ECL biosensors, nanomaterials would be divided into the following two categories to be summarized: (1) Nanomaterials for signal amplification. (2) Nanomaterials as ECL emitters. Finally, this review prospects the perspectives on the future development direction of ECL biosensor in food safety and disease diagnosis.

Ultrasensitive and Specific Detection of Anticancer Drug 5-Fluorouracil in Blood Samples by a Surface-Enhanced Raman Scattering (SERS)-Based Lateral Flow Immunochromatographic Assay.

5-Fluorouracil (5-FU) is an effective anticancer drug widely used in the world. To improve therapy efficiency and reduce side effects, it is very important to frequently detect the concentration of 5-FU in blood samples of patients. In this work, a new type of lateral flow immunochromatographic assay (LFIA) based on surface-enhanced Raman scattering (SERS) for ultrasensitive and specific detection of 5-FU in blood samples was developed. Au@Ag/Au nanoparticles (NPs) employing Au particles as the core and Ag/Au alloy as the shell were synthesized, characterized and used as the substrate in SERS-LFIA due to their high SERS enhancement and biocompatibility. The immunoprobe was made in the form of AuMBA@Ag/Au-Ab in which mercaptobenzoic acid (MBA, a common Raman active reporter) was embedded in the core-shell layer and the monoclonal antibody (mAb) against 5-FU was immobilized on the surface. The performance of SERS-LFIA was similar to that in colloidal gold based-LFIA, and the entire assay time was within 20 min. According to the color intensity on the testing (T) lines of LFIA strips visualized by eyes, the contents of 5-FU in the samples could be qualitatively or semi-quantitatively identified. Furthermore, by measuring the characteristic Raman intensities of MBA on T lines, quantitative detection of 5-FU in the samples were achieved. The IC50 and limit of detection (LOD) of the LFIA for 5-FU were found to be 20.9 pg mL-1 and 4.4 pg mL-1, respectively. There was no cross-reactivity (CR) of the LFIA with nine relative compounds, and the CR with cytosine, tegafur and carmofur were less than 4.5%. The recoveries of 5-FU from spiked blood samples were in the range of 78.6~86.4% with the relative standard deviation (RSD) of 2.69~4.42%. Five blood samples containing 5-FU collected from the Cancer Hospital were measured by SERS-LFIA, and the results were confirmed by LC-MS/MS. It was proven that the proposed method was able to simply and rapidly detect 5-FU in blood samples with high sensitivity, specificity, accuracy and precision.

Development of a highly sensitive and specific monoclonal antibody-based ELISA coupled with immuno-affinity extraction for the detection of anticancer drug 5-fluorouracil in blood samples.

5-Fluorouracil (5-FU) is an effective anticancer drug widely used in cancer treatment. In this study, two 5-FU derivatives containing a spacer arm with the carboxylic group at the end were synthesized, which were linked to the carrier proteins to form 5-FU-protein conjugates used as the immunogens for the production of monoclonal antibody (mAb). Based on the produced mAb, the highly sensitive and specific enzyme-linked immunosorbent assay (ELISA) for 5-FU detection was established. The IC50 and LOD values of the assay were found to be 19.5 ng mL-1 and 0.5 ng mL-1, respectively. There was no cross-reactivity (CR) of the ELISA with cytosine, thymine and uracil, which avoided the interference from inherent pyrimidines. The CR values of the assay with three substitutes of 5-FU (tegafur, 5-fluoro-2'-deoxyuridine, carmofur) were within 9.7%-17.6%. The produced mAb was also applied in sample extraction. The immuno-affinity column capable specific capturing 5-FU was prepared by immobilizing the mAb on Sepharose-4B gel and filling into a SPE column. The recoveries of 5-FU in spiked samples measured by ELISA were 72.4%-90.7% with RSD of 3.6%-8.3%. Five blood samples collected from patients were extracted by immuno-affinity column, then measured by ELISA and confirmed by HPLC-MS/MS. There was a good correlation between HPLC-MS/MS and ELISA. It is demonstrated that the developed ELISA combined with immuno-affinity extraction can be a powerful alternative method for the detection of 5-FU in blood samples.

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

A competitive 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 the bimetal. 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 102 ng mL-1, and a detection limit of 3.1 × 10-5 ng mL-1, where the scan potential was varied from 0 to 0.6 V vs 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.

Ultra-sensitive detection of florfenicol by flow injection chemiluminescence immunoassay based on Nickel/Cobalt bimetallic metal-organic framework nanozymes.

The emergence and progress of metal-organic frameworks (MOFs) with high stability, large surface area, and abundant unsaturated active sites, once again promote the development of nanozymes, making nanozymes more advantageous to replace natural enzymes and will increase the applications of chemiluminescence immunoassay. In this study, a flow injection chemiluminescence immunoassay based on Ni/Co metal-organic framework (Ni/Co-MOF) nanozymes was developed, which can quickly and highly sensitively detect florfenicol (FF) in animal-derived food residues. Ni/Co-MOF0.75 nanospheres can not only form stable immune probes with antibodies but also act as nanozymes to efficiently catalyze H2O2 for amplifying the chemiluminescence signal of the luminol-H2O2 system. In addition, due to good biocompatibility and large specific surface area, carboxyl-modified resin beads are used as a suitable material for loading more coating antigens. Based on the principle of competitive immunity, FF standard solution will compete with coating antigen loaded on the carboxyl resin beads for the limited binding sites on the FF antibody. Under the best experimental conditions, the detection range of FF is 0.0001-1000 ng mL-1, and the detection limit (LOD) is 0.033 pg mL-1 (S/N = 3). Furthermore, this method has been successfully applied to the analysis of actual samples with satisfactory results, which will provide a certain reference for the detection of small molecules in food and environmental analysis.

A novel electrochemiluminescence immunoassay based on highly efficient resonance energy transfer for florfenicol detection.

A novel competitive mechanism electrochemiluminescence (ECL) immunoassay based on resonance energy transfer was used to detect florfenicol for the first time. In this work, CeO2@TiO2 nanocomposite, which was used as a donor, was prepared in sol-gel method and the effective band gap of TiO2 could be reduced by CeO2, which promoted the ECL emission of TiO2 and made the ECL performance of the donor more outstanding. The absorption spectrum of Cu2S and the ECL emission spectrum of the donor could be highly matched, which ensured the occurrence of electrochemiluminescence resonance energy transfer (ECL-RET). In addition, the snowflake-like structure of cuprous sulfide could load more antibodies. It is worth mentioning that as far as we know, there have been no reports of this material as an ECL receptor before. Furthermore, the ECL-RET system based on this has shown excellent performance in the detection of florfenicol. The proposed immunoassay showed satisfactory sensitivity with a wide linear range from 0.001 to 1000 ng mL-1 and a low detection limit (0.3 pg mL-1). Due to the remarkable quenching effect and simple assembly process, the immunoassay is of great practical significance and has reference value for the detection of florfenicol or other biological small molecules.

A novel electrochemiluminescence immunosensing strategy fabricated by Co(OH)2 two-dimensional nanosheets and Ru@SiO2-Au NPs for the highly sensitive detection of enrofloxacin.

In this work, a novel sensitive electrochemiluminescence immunosensor based on Ru@SiO2-Au NPs and Co(OH)2 two-dimensional nanosheets (2D Co(OH)2) is constructed for the detection of enrofloxacin (ENR). Ruthenium bipyridine silica spheres and modified gold nanoparticles were synthesized as immune probe materials, which were combined with ENR antibodies (Abs) to form the immune probe part. 2D Co(OH)2 with a large specific surface area and good catalytic effect was firstly used as an immune substrate material, and at the same time, it was conjugated with the coating antigen (Ae) of ENR to form an immune substrate. Based on the principle of competitive immunity, ENR and ENR coated antigen could jointly compete for the specific binding sites on the ENR antibody, so as to achieve efficient detection of ENR. Under optimal conditions, the prepared immunosensor exhibited high sensitivity with a wide linear range from 0.0001 to 1000 ng mL-1 and a low detection limit (LOD) of 0.063 pg mL-1. The proposed immunosensor has been successfully applied to the detection of ENR residues in poultry, aquatic products and lake water.