Development of Shark Single Domain Antibodies Specific for Human α-Fetoprotein and the Multimerization Strategy in Serum Detection.

Sensitive detection of cancer biomarkers can contribute to the timely diagnosis and treatment of diseases. In this study, the whitespotted bamboo sharks were immunized with human α-fetoprotein (AFP), and a phage-displayed variable new antigen receptor (VNAR) single domain antibody library was constructed. Then four unique VNARs (VNAR1, VNAR11, VNAR21, and VNAR25) against AFP were isolated from the library by biopanning for the first time. All of the sequences belong to type II of VNAR, and the VNAR11 was much different from the rest of the three sequences. Then VNAR1 and VNAR11 were selected to fuse with the C4-binding protein α chain (C4bpα) sequence and efficiently expressed in the Escherichia coli system. Furthermore, a VNAR-C4bpα-mediated sandwich chemiluminescence immunoassay (VSCLIA) was developed for the detection of AFP in human serum samples. After optimization, the VSCLIA showed a limit of detection of 0.74 ng/mL with good selectivity and accuracy. Moreover, the results of clinical serum samples detected by the VSCLIA were confirmed by an automatic immunoanalyzer in the hospital, indicating its practical application in actual samples. In conclusion, the novel antibody element VNAR exhibits great potential for immunodiagnosis, and this study also provides a new direction and experimental basis for AFP detection.

Development of alkaline phosphatase-linked single-chain variable fragment fusion proteins for one-step immunodetection of deoxynivalenol in cereals.

Deoxynivalenol (DON) is a mycotoxin that widely distributes in various foods and seriously threatens food safety. To minimize the consumers' dietary exposure to DON, there is an urgent demand for developing rapid and sensitive detection methods for DON in food. In this study, a bifunctional single-chain variable fragment (scFv) linked alkaline phosphatase (ALP) fusion protein was developed for rapid and sensitive detection of deoxynivalenol (DON). The scFv gene was chemically synthesized and cloned into the expression vector pET25b containing the ALP gene by homologous recombination. The prokaryotic expression, purification, and activity analysis of fusion proteins (scFv-ALP and ALP-scFv) were well characterized and performed. The interactions between scFv and DON were investigated by computer-assisted simulation, which included hydrogen bonds, hydrophobic interactions, and van der Waals forces. The scFv-ALP which showed better bifunctional activity was selected for developing a direct competitive enzyme-linked immunosorbent assay (dc-ELISA) for DON in cereals. The dc-ELISA takes 90 min for one test and exhibits a half inhibitory concentration (IC50) of 11.72 ng/mL, of which the IC50 was 3.08-fold lower than that of the scFv-based dc-ELISA. The developed method showed high selectivity for DON, and good accuracy was obtained from the spike experiments. Furthermore, the detection results of actual cereal samples analyzed by the method correlated well with that determined by high-performance liquid chromatography (R2=0.97165). These results indicated that the scFv-ALP is a promising bifunctional probe for developing the one-step colorimetric immunoassay, providing a new strategy for rapid and sensitive detection of DON in cereals.

Illuminating the path: aggregation-induced emission for food contaminants detection.

Food safety is a global concern that deeply affects human health. To ensure the profitability of the food industry and consumer safety, there is an urgent need to develop rapid, sensitive, accurate, and cost-effective detection methods for food contaminants. Recently, the Aggregation-Induced Emission (AIE) has been successfully used to detect food contaminants. AIEgens, fluorescent dyes that cause AIE, have several valuable properties including high quantum yields, photostability, and large Stokes shifts. This review provides a detailed introduction to the principles and advantages of AIE-triggered detection, followed by a focus on the past five years' applications of AIE in detecting various food contaminants including pesticides, veterinary drugs, mycotoxins, food additives, ions, pathogens, and biogenic amines. Each detection principle and component is comprehensively covered and explained. Moreover, the similarities and differences among different types of food contaminants are summarized, aiming to inspire future researchers. Finally, this review concludes with a discussion of the prospects for incorporating AIEgens more effectively into the detection of food contaminants.

Development of a biotin-streptavidin-amplified nanobody-based ELISA for ochratoxin A in cereal.

A biotin-streptavidin-amplified enzyme-linked immunosorbent assay using a biotinylated nanobody (BA-Nb ELISA) was developed to detect ochratoxin A (OTA) in cereal. The limit of detection (LOD) of the BA-Nb ELISA, which equals to 10% maximal inhibitory concentration, was 0.011 ng/mL for OTA in buffer, and the sensitivity was approximately improved by one order of magnitude compared with the traditional Nb ELISA (LOD = 0.112 ng/mL). Under optimal conditions, the developed assay could be accomplished in 40 min with maximal inhibitory concentration of 0.138 ng/mL and the linear detection range of 0.034-0.460 ng/mL. The average recovery rate of the BA-Nb ELISA ranged from 92.8% to 114%, and the relative standard deviation was in the range of 2.04-9.85%. The developed BA-Nb ELISA was validated by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and the results indicated the reliability of BA-Nb ELISA for the detection of OTA in cereal.

Antimicrobial Susceptibility and Antibacterial Mechanism of Limonene against Listeria monocytogenes.

Limonene is a monoterpenoid compound, which is founded in a lot of plants' essential oils with good antibacterial activity against food-borne pathogens, but it has an ambiguous antimicrobial susceptibility and mechanism against Listeria monocytogenes (L. monocytogenes). In this study, the antimicrobial susceptibility of Limonene to L. monocytogenes was studied, and some new sights regarding its antibacterial mechanism were further explored. Scanning electron microscopy (SEM) verified that limonene caused the destruction of the cell integrity and wall structure of L. monocytogenes. The increase in conductivity and the leakage of intracellular biomacromolecules (nucleic acids and proteins) confirmed that limonene had an obvious effect on cell membrane permeability. The results of Propidium Iodide (PI) fluorescence staining were consistent with the results of the conductivity measurements. This indicated that limonene treatment caused damage to the L. monocytogenes cell membrane. Furthermore, the decrease in ATP content, ATPase (Na+K+-ATPase, Ca2+-ATPase) activity and respiratory chain complex activity indicated that limonene could hinder ATP synthesis by inhibiting the activity of the respiratory complex and ATPase. Finally, differential expression of proteins in the respiratory chain confirmed that limonene affected respiration and energy metabolism by inhibiting the function of the respiratory chain complex.

Development of a Nanobody-AviTag Fusion Protein and Its Application in a Streptavidin-Biotin-Amplified Enzyme-Linked Immunosorbent Assay for Ochratoxin A in Cereal.

Ochratoxin A (OTA) is a common food contaminant that threatens consumers' safety and health. A sensitive and selective biotin-streptavidin-amplified enzyme-linked immunosorbent assay (BA-ELISA) for OTA using a nanobody-AviTag fusion protein (Nb-AviTag) was developed in this study. The prokaryotic expression vector Nb28-AviTag-pAC6 for Nb-AviTag was constructed, followed by transformation to the AVB101 cells for antibody expression and in vivo biotinylation. The purified Nb28-AviTag was used to establish the BA-ELISA and the procedures for this Nb-AviTag-based BA-ELISA were optimized. The Nb-AviTag-based BA-ELISA exhibited the half maximal inhibitory concentration (IC50) of 0.14 ng mL-1 and the limit of detection (LOD = IC10) of 0.028 ng mL-1 for OTA basing on the optimized experiment parameters. The assay sensitivity was improved 4.6 times and 4.3 times compared to Nb-based ELISA, respectively. This method had LODs of 1.4 µg kg-1 in barley, 0.56 µg kg-1 in oats, and 0.84 µg kg-1 in rice for OTA. The average recovery percent was in a range of 84-137%, and the relative standard derivation percent ranged from 0.64% to 7.8%. The content of OTA in contaminated cereal samples was determined by both the developed Nb-AviTag-based method and liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results demonstrated that the Nb-AviTag was a robust and promising bioreceptor in highly sensitive detection of OTA and other low molecular weight compounds using BA system.

Nanobody-Alkaline Phosphatase Fusion Protein-Based Enzyme-Linked Immunosorbent Assay for One-Step Detection of Ochratoxin A in Rice.

Ochratoxin A (OTA) has become one a focus of public concern because of its multiple toxic effects and widespread contamination. To monitor OTA in rice, a sensitive, selective, and one-step enzyme-linked immunosorbent assay (ELISA) using a nanobody-alkaline phosphatase fusion protein (Nb28-AP) was developed. The Nb28-AP was produced by auto-induction expression and retained an intact antigen-binding capacity and enzymatic activity. It exhibited high thermal stability and organic solvent tolerance. Under the optimal conditions, the developed assay for OTA could be finished in 20 min with a half maximal inhibitory concentration of 0.57 ng mL-1 and a limit of detection of 0.059 ng mL-1, which was 1.1 times and 2.7 times lower than that of the unfused Nb28-based ELISA. The Nb28-AP exhibited a low cross-reactivity (CR) with ochratoxin B (0.92%) and ochratoxin C (6.2%), and an ignorable CR (<0.10%) with other mycotoxins. The developed Nb-AP-based one-step ELISA was validated and compared with a liquid chromatography-tandem mass spectrometry method. The results show the reliability of Nb-AP-based one-step ELISA for the detection of OTA in rice.

Studies on the Inhibition Mechanism of Linalyl Alcohol against the Spoilage Microorganism Brochothrix thermosphacta.

The aim of this study was to investigate the bacterial inhibitory ability and mechanism of action of linalyl alcohol against B. thermosphacta. Linalyl alcohol causes the leakage of intracellular material by disrupting the cell wall and exposing the hydrophobic phospholipid bilayer, which binds to bacterial membrane proteins and alters their structure. In addition, linalyl alcohol causes cell membrane damage by affecting fatty acids and proteins in the cell membrane. By inhibiting the synthesis of macromolecular proteins, the normal physiological functions of the bacteria are altered. Linalyl alcohol binds to DNA in both grooved and embedded modes, affecting the normal functioning of B. thermosphacta, as demonstrated through a DNA interaction analysis.

Plasmonic enzyme immunoassay via nanobody-driven controllable aggregation of gold nanoparticles for detection of ochratoxin A in pepper.

Ochratoxin A (OTA) in food poses a serious challenge to public health. Herein, using the nanobody-driven controllable aggregation of gold nanoparticles (AuNPs) in a glucose oxidase-tyramine-horseradish peroxidase (GOx-TYR-HRP) system, we propose a direct competitive plasmonic enzyme immunoassay (dc-PEIA) for OTA detection. The OTA-GOx conjugate catalyzes glucose to produce hydrogen peroxide (H2O2), and then HRP catalyzes H2O2 to generate hydroxyl radical which induces the crosslink of TYR. Crosslinked TYR leads to aggregation of AuNPs through strong electrostatic interactions, which is tunable based on the competition of OTA-GOx and free OTA for binding the immobilized nanobody. The optimized dc-PEIA achieves an instrumental limit of detection (LOD) of 0.275 ng/mL and a visual LOD of 1.56 ng/mL. It exhibits good selectivity for OTA and accuracy in the analysis of pepper samples, with the confirmation of high-performance liquid chromatography. Overall, the dc-PEIA is demonstrated as a useful tool for detecting OTA in food.

Ready-to-use ratiometric bioluminescence immunosensor for detection of ochratoxin a in pepper.

Rapid, portable, and accurate detection tools for monitoring ochratoxin A (OTA) in food are essential for the guarantee of food safety and human health. Herein, as a proof-of-concept, this study proposed a ratiometric bioluminescence immunosensor (RBL-immunosensor) for homogeneous detection of OTA in pepper. The construct of the RBL-immunosensor consists of three components, including the large fragment of the split nanoluciferase (NanoLuc)-tagged nanobody (NLg), the small fragment of the split NanoLuc-tagged mimotope peptide heptamer (MPSm), and the calibrator luciferase (GeNL). The specific nanobody-mimotope peptide interaction between NLg and MPSm induces the reconstitution of the NanoLuc, which catalyzes the Nano-Glo substrate and produces a blue emission peak at 458 nm. Meanwhile, GeNL can produce a green emission peak at 518 nm upon substrate conversion via bioluminescent resonance energy transfer (BRET). Therefore, the concentration of OTA can be linked to the variation of the bioluminescence signal (λ458/λ518) measured by microplate reader and the variation of the blue/green ratio measured by smartphone via the competitive immunoreaction where OTA competes with MPSm to bind NLg. The immunosensor is ready-to-use and works by simply mixing the components in a one-step incubation of 10 min for readout. It has a limit of detection (LOD) of 0.98 ng/mL by a microplate reader and an LOD of 1.89 ng/mL by a smartphone. Good selectivity and accuracy were confirmed for the immunosensor by cross-reaction analysis and recovery experiments. The contents of OTA in 10 commercial pepper powder samples were tested by the RBL-immunosensor and validated by high-performance liquid chromatography. Hence, the ready-to-use RBL-immunosensor was demonstrated as a highly reliable tool for detection of OTA in food.

Colorimetric and surface-enhanced Raman scattering dual-mode lateral flow immunosensor using phage-displayed shark nanobody for the detection of crustacean allergen tropomyosin.

Tropomyosin (TM) is the primary allergenic protein responsible for crustacean food allergies, and thus sensitive and rapid methods are required for the screening of crustacean TM in food. In this study, using the phage-displayed shark nanobody (PSN) as a multifunctional biomaterial, we developed a colorimetric and surface-enhanced Raman scattering dual-mode lateral flow immunosensor (CM/SERS-LFI) for competitive detection of crustacean TM. The SERS tag AuMBA@AgNPs with the Raman signal molecule 4-mercaptobenzoic acid (4-MBA) was prepared and immobilized on the PSN to construct the immunoprobe AuMBA@Ag-PSN. The probe can identify free TM that competes with TM on the T-line, and the optimized CM/SERS-LFI enables quantitative analysis of TM using the probe with a limit of detection (LOD) of 0.0026 µg/mL (SERS mode) and 0.0057 µg/mL (colorimetric mode), respectively. Additionally, it can implement a qualitative analysis by the naked eye with a visual LOD of 0.01 µg/mL. The CM/SERS-LFI exhibited excellent performance in the tests of selectivity, accuracy, precision, and stability. Moreover, the method's effectiveness in the analysis of real samples was confirmed by a commercial ELISA kit. Therefore, the developed CM/SERS-LFI was demonstrated to be a powerful and reliable tool for the rapid and sensitive detection of crustacean TM in food.

Colorimetric and chemiluminescent enzyme immunoassays based on the alkaline phosphatase-tagged single-chain variable fragment fusion tracer for detecting zearalenone in agro-products.

Zearalenone (ZEN) is a non-steroidal estrogenic mycotoxin and seriously threatens food safety, which requires rapid and sensitive detection methods for monitoring ZEN in agro-products. Herein, an alkaline phosphatase-tagged single-chain variable fragment fusion protein (ALP-scFv) was used as a bifunctional tracer to develop a colorimetric enzyme immunoassay (CEIA) and a chemiluminescent enzyme immunoassay (CLEIA) for ZEN. In addition, the interactions between scFv and ZEN were exploited by computer-assisted simulation, and four key amino acid sites were preliminarily identified. After optimization, the CEIA and CLEIA exhibited a limit of detection of 0.02 and 0.006 ng/mL, respectively. Furthermore, both methods showed favorable accuracy in recovery experiments and good selectivity in cross reactions. Moreover, the detection results of the actual samples from both methods correlated well with those from high-performance liquid chromatography. Overall, the ALP-scFv fusion tracer-based CEIA and CLEIA are demonstrated as reliable tools for ZEN detection in food.

Flexible, scalable and simple-fabricated silver nanorod-decorated bacterial nanocellulose SERS substrates cooperated with portable Raman spectrometer for on-site detection of pesticide residues.

Owing to good flexibility, prominent mechanical properties, three-dimensional (3D) nanofibrous structure and low background interference, sustainable bacterial nanocellulose (BNC) is a highly attractive matrix material for surface-enhanced Raman scattering (SERS) sensor. Herein, a highly sensitive, flexible and scalable silver nanorod-decorated BNC (AgNRs@BNC) SERS sensor is developed by a simple vacuum-assisted filtration. The AgNRs were firmly locked in the 3D nanofibrous network of cellulose nanofibers upon vacuum drying process, resulting in the formation of 3D SERS hotspots with a depth of more than 10 µm on the sensor. With 4-aminothiophenol (4-ATP) as a target molecule, a lowest distinguishable level of 10-12 M and a high enhancement factor of 1.1 × 109 were realized by the optimal AgNRs1.5@BNC SERS sensor. Moreover, the AgNRs@BNC SERS sensor exhibits high detectable level of 10-9 M for thiram molecules by integrating with a portable Raman spectrometer. Besides, toxic thiram residues on grape surface could be directly on-site identified by the combination of AgNRs@BNC SERS sensors and a portable Raman spectrometer through a feasible press-and-peel method. The flexible AgNRs@BNC SERS sensor cooperated with portable Raman system demonstrates great potential for on-site detection of pesticide residues on irregular food surfaces.

Lateral Flow Immunochromatographic Assay for Competitive Detection of Crustacean Allergen Tropomyosin Using Phage-Displayed Shark Single-Domain Antibody.

The common food allergy crustacean tropomyosin (TM) poses a significant food safety challenge, which requires rapid and sensitive methods for screening TM in food. Herein, the variable new antigen receptor (VNAR) single-domain antibodies specific for the crustacean TM were isolated from a naïve phage-displayed shark VNAR library. Subsequently, a lateral flow immunochromatographic assay (LFIA) based on the gold nanoparticle-labeled phage-displayed shark VNAR (AuNPs@PSV) probe was developed for the detection of TM in food. The AuNPs@PSV-LFIA took 15 min for one test and had a visual limit of detection (vLOD) of 0.1 µg/mL and an instrumental LOD of 0.02 µg/mL. Good selectivity, accuracy, precision, and stability were confirmed for the AuNPs@PSV-LFIA. Moreover, the test results of 21 commercially available food products consisted of the allergen labels and were validated by a commercial ELISA kit. Therefore, this work demonstrated the great potential of VNAR for detecting TM in food by LFIA.

Combination of nanobody and peptidomimetic to develop novel immunoassay platforms for detecting ochratoxin A in cereals.

Mimotope-based immunoassays for mycotoxins eliminate the requirement for large amounts of mycotoxin standards for the chemosynthesis of artificial antigens. Herein, the nanobody-based magnetic beads were used to screen the mimotope (peptidomimetic) of ochratoxin A (OTA) from the phage-displayed peptide library. The interactions between nanobody and the most sensitive Y4 peptidomimetic were investigated by computer-assisted simulation and compared with those between nanobody and OTA. By combining the nanobody, the phage-displayed Y4 and alkaline phosphatase-tagged Y4 fusion protein as the competing antigens, were used to develop two novel immunoassay platforms (PN-ELISA and APN-ELISA). The two methods are advantageous in the use of nontoxic substitutes of OTA and avoiding the use of monoclonal antibodies. Moreover, good analytical performances of both methods were obtained and confirmed by liquid chromatography tandem mass spectrometry. Therefore, the proposed novel methods based on nanobody and peptidomimetic were demonstrated to be highly reliable for detecting OTA in food.

Nanobody/NanoBiT system-mediated bioluminescence immunosensor for one-step homogeneous detection of trace ochratoxin A in food.

Hazardous small molecules in food and environment seriously threatens human health, which requires sensitive and rapid tools for monitoring. Using a previously identified nanobody against ochratoxin A (OTA), we herein proposed a homogeneous sensing platform "nanobody/NanoLuc Binary Technology (NanoBiT) system" and developed a nanobody/NanoBiT system-mediated bioluminescence immunosensor (NBL-Immunosens) for OTA using LgBiT (Lg) and SmBiT (Sm), two subunits of the split nanoluciferase (NanoLuc). The core elements of NBL-Immunosens include Lg-nanobody fusion (NLg) and Sm-labeled OTA-bovine serum albumin conjugate (OSm). The antigen-antibody interaction between NLg and OSm triggers the reconstitution of NanoLuc for generating luminescence signals. Moreover, free OTA can compete with OSm for binding to NLg, resulting the decrease of dose-dependent signals. NBL-Immunosens can detect OTA in a one-step assay of 5 min without washing and exhibit a limit of detection of 0.01 ng/mL with a linear range of 0.04-2.23 ng/mL. It shows high selectivity for OTA and has good accuracy and precision in the spiking-and-recovery experiments. Furthermore, its effectiveness was evaluated with real cereal samples and confirmed by liquid chromatography tandem mass spectrometry and commercial ELISA kits. Hence, the NBL-Immunosens is a very promising tool for rapid, accurate, and selective detection of trace OTA in food.

Generation of a nanobody-alkaline phosphatase heptamer fusion for ratiometric fluorescence immunodetection of trace alpha fetoprotein in serum.

Alpha fetoprotein (AFP) is an important biomarker for diagnosis of hepatocellular carcinoma (HCC). Whereas, it is always a challenge to detect trace AFP in serum. In this work, a ratiometric fluorescence enzyme immunoassay (RFEIA) was developed using nanobody-alkaline phosphatase (Nb-AP) heptamer and MnFe layered double hydroxides nanoflakes (MnFe LDH) for ultrasensitive detection of AFP. The Nb-AP heptamer (Nb-C4bpα-AP) was constructed by fusion expression of Nb, AP, and α-chain of C4 binding protein (C4bpα), where the C4bpα contributed to multimerization through self-assembly. The dual functional Nb-C4bpα-AP can recognize AFP, dephosphorylate ascorbic acid-2-phosphate (AAP) into ascorbic acid (AA), and thus tune the MnFe LDH-mediated ratiometric fluorescence, which was generated from the oxidization of MnFe LDH on o-phenylenediamine (OPD) and the catalyzation of MnFe LDH on the cyclization reaction between AA and OPD. By integration of Nb-C4bpα-AP, MnFe LDH, AAP, and OPD, the RFEIA showed a limit of detection of 0.013 ng/mL with good selectivity, accuracy and precision. Furthermore, results of clinical serum samples tested by the RFEIA were well confirmed by the automated chemiluminescence immunoassay analyzer. Thus, this work demonstrated that the Nb-C4bpα-AP is a robust immunoreagent and the developed RFEIA could be a very promising tool for diagnosis of HCC.

Fluonanobody-based nanosensor via fluorescence resonance energy transfer for ultrasensitive detection of ochratoxin A.

Ochratoxin A (OTA) contamination in food is a serious threat to public health. There is an urgent need for development of rapid and sensitive methods for OTA detection, to minimize consumer exposure to OTA. In this study, we constructed two OTA-specific fluonanobodies (FluoNbs), with a nanobody fused at the carboxyl-terminal (SGFP-Nb) or the amino-terminal (Nb-SGFP) of superfolder green fluorescence protein. SGFP-Nb, which displayed better fluorescence performance, was selected as the tracer for OTA, to develop a FluoNb-based nanosensor (FN-Nanosens) via the fluorescence resonance energy transfer, where the SGFP-Nb served as the donor and the chemical conjugates of OTA-quantum dots served as the acceptor. After optimization, FN-Nanosens showed a limit of detection of 5 pg/mL, with a linear detection range of 5-5000 pg/mL. FN-Nanosens was found to be highly selective for OTA and showed good accuracy and repeatability in recovery experiments using cereals with various complex matrix environments. Moreover, the contents of OTA in real samples measured using FN-Nanosens correlated well with those from the liquid chromatography with tandem mass spectrometry. Therefore, this work illustrated that the FluoNb is an ideal immunosensing tool and that FN-Nanosens is reliable for rapid detection of OTA in cereals with ultrahigh sensitivity.

Generation of a nanobody-alkaline phosphatase fusion and its application in an enzyme cascade-amplified immunoassay for colorimetric detection of alpha fetoprotein in human serum.

Sensitive detection of liver disease biomarkers can facilitate the diagnosis of primary hepatoma and other benign liver diseases, and the alpha fetoprotein (AFP) was selected as the model macromolecule in this work. Herein an enzyme cascade-amplified immunoassay (ECAIA) based on the nanobody-alkaline phosphatase fusion (Nb-ALP) and MnO2 nanoflakes was developed for detecting AFP. The bifunctional biological macromolecule Nb-ALP serves as the detection antibody and the reporter molecule. The MnO2 nanoflakes mimic the oxidase for catalyzing the 3,3',5,5'-tetramethylbenzidine (TMB) into the blue oxidized TMB, which has a quantitative signal at the wavelength of 650 nm. Moreover, the Nb-ALP could dephosphorylate the ascorbic acid-2-phosphate (AAP) to form the ascorbic acid (AA) that can disintegrate the nanoflakes to reduce their oxidation capacity with the content decrease of the oxidized TMB. Using the constructed TMB-MnO2 colorimetric sensing system for Nb-ALP and the optimized experimental parameters, the ECAIA has a limit of detection (LOD) of 0.148 ng/mL which is 18.7-fold lower than that of the p-nitrophenylphosphate (pNPP)-based method (LOD = 2.776 ng/mL). The ECAIA showed good selectivity for AFP with observed negligible cross-reactions with several common cancer biomarkers. The recovery rate for AFP spiked in human serum ranged from 94.8% to 113% with the relative standard deviation from 0.3% to 6.5%. For analysis of the actual human serum samples, a good linear correlation was found between the results tested by the ECAIA and the automatic chemiluminescence analyzer. Thus, the ECAIA was demonstrated to be a promising tool for highly sensitive and selective detection of AFP, providing a reference for analysis of other macromolecule biomarkers.

Enzyme cascade-amplified immunoassay based on the nanobody-alkaline phosphatase fusion and MnO2 nanosheets for the detection of ochratoxin A in coffee.

Ochratoxin A (OTA) is a common food contaminant with multiple toxicities and thus rapid and accurate detection of OTA is indispensable to minimize the threat of OTA to public health. Herein a novel enzyme cascade-amplified immunoassay (ECAIA) based on the mutated nanobody-alkaline phosphatase fusion (mNb-AP) and MnO2 nanosheets was established for detecting OTA in coffee. The detection principle is that the dual functional mNb-AP could specifically recognize OTA and dephosphorylate the ascorbic acid-2-phosphate (AAP) into ascorbic acid (AA), and the MnO2 nanosheets mimicking the oxidase could be reduced by AA into Mn2+ and catalyze the 3,3',5,5'-tetramethyl benzidine into blue oxidized product for quantification. Using the optimal conditions, the ECAIA could be finished within 132.5 min and shows a limit of detection of 3.38 ng mL-1 (IC10) with an IC50 of 7.65 ng mL-1 and a linear range (IC20-IC80) of 4.55-12.85 ng mL-1. The ECAIA is highly selective for OTA. Good recovery rates (84.3-113%) with a relative standard deviation of 1.3-3% were obtained and confirmed by high performance liquid chromatography with a fluorescence detector. The developed ECAIA was demonstrated to be a useful tool for the detection of OTA in coffee which provides a reference for the analysis of other toxic small molecules.