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Luminogens with aggregation-induced emission (AIEgens) properties have numerous broad applications in fields of chemical and biological analyses due to their exceptional photostability, excellent signal reliability, high quantum yield, and large Stokes' shift. In particular, AIEgens also bring new blood for immunoassay. Since publication of the first 2004 paper, AIEgens-based immunoassays have received significant attention because of their high sensitivity, specificity, accuracy, and reliability. However, until now, there have been no comprehensive literature reviews focused on the evolving field of AIEgens-based immunoassays. Thus, we have extensively reviewed AIEgens-based immunoassays from their basic working principles to specific applications. We focus on several fundamental elements of AIEgens-based immunoassays, including the typical structures of AIEgens, emission mechanism of AIEgens probes, function of AIEgens in immunoassays, and platform of AIEgens-based immunoassays. Then, the representative applications of AIEgens-based immunoassays in food safety, medical diagnostics, and environmental monitoring are explored. Thus, proposals on how to further improve the AIEgens-based immunoassay performance are also discussed, as well as future challenges and perspectives, aiming to provide brief and valid guidelines for choosing suitable AIEgens-based immunoassays according to specific application requirements.

Recently, urinary tract infection (UTI) triggered by bacteria carrying pan-drug-resistant genes, including carbapenem resistance gene blaNDM and blaKPC, colistin resistance gene mcr-1, and tet(X) for tigecycline resistance, have been reported, posing a serious challenge to the treatment of clinical UTI. Therefore, point-of-care (POC) detection of these genes in UTI samples without the need for pre-culturing is urgently needed. Based on PEG 200-enhanced recombinase polymerase amplification (RPA) and a refined Chelex-100 lysis method with HRP-catalyzed lateral flow immunoassay (LFIA), we developed an MCL-PRPA-HLFIA cascade assay system for detecting these genes in UTI samples. The refined Chelex-100 lysis method extracts target DNA from UTI samples in 20 min without high-speed centrifugation or pre-incubation of urine samples. Following optimization, the cascade detection system achieved an LOD of 102 CFU/mL with satisfactory specificity and could detect these genes in both simulated and actual UTI samples. It takes less than an hour to complete the process without the use of high-speed centrifuges or other specialized equipment, such as PCR amplifiers. The MCL-PRPA-HLFIA cascade assay system provides new ideas for the construction of rapid detection methods for pan-drug-resistant genes in clinical UTI samples and provides the necessary medication guidance for UTI treatment.

Phallotoxins, toxic cyclopeptides found in wild poisonous mushrooms, are predominant causes of fatal food poisoning. For the early and rapid diagnosis mushroom toxin poisoning, a highly sensitive and robust monoclonal antibody (mAb) against phallotoxins was produced for the first time. The half-maximum inhibition concentration (IC50) values of the mAb-based indirect competitive ELISAs for phallacidin (PCD) and phalloidin (PHD) detection were 0.31 ng mL−1 and 0.35 ng mL−1, respectively. In response to the demand for rapid screening of the type of poisoning and accurate determination of the severity of poisoning, colloidal gold nanoparticle (GNP) and time-resolved fluorescent nanosphere (TRFN) based lateral flow assays (LFA) were developed. The GNP-LFA has a visual cut-off value of 3.0 ng mL−1 for phallotoxins in human urine sample. The TRFN-LFA provides a quantitative readout signal with detection limit of 0.1 ng mL−1 in human urine sample. In this study, urine samples without pretreatment were used directly for the LFA strip tests, and both two LFAs were able to accomplish analysis within 10 min. The results demonstrated that LFAs based on the newly produced, highly sensitive, and robust mAb were able to be used for both rapid qualitative screening of the type of poisoning and accurate quantitative determination of the severity of poisoning after accidental ingestion by patients of toxic mushrooms.

Apart from the obvious benefit of "trash-to-treasure", the acquisition of natural nanomaterials from cheap and renewable waste has been intensively researched because of various bioactivities and physical-chemical features. Herein, for the first time, we employed natural cuttlefish ink nanoparticles (CINPs) as a multifunctional label and designed colorimetric-photothermal dual-mode lateral flow immunoassays (CINPs-mediated CPLFIA) for sensitive detection of clenbuterol (CL). The accessibility and renewability of CINPs overcome barriers that artificial nanomaterials face, such as complex manufacturing and relatively high costs. Additionally, inspired by the mussel adhesion, the bio-affinity of CINPs, such as antibody coupling and preservation, was investigated and showed to be considerably superior to Au NPs, leading to significantly increased immunosensor sensitivity. Meanwhile, CINPs exhibit excellent photothermal conversion efficiency for dual-signal production, avoiding the effect of environmental elements (particularly light) for colorimetric mode. Besides, the biosensor was integrated with a smartphone and a thermal imager for portable sensing. After optimization, the detection limit of CINPs-mediated CPLFIA was 0.179 ng mL

Stimulated surface-enhanced Raman scattering (SERS) in combination with engineered nano-tracer offers extraordinary potential in lateral flow immunoassays (LFIAs). Nonetheless, the investigation execution of SERS-LFIA is often compromised by the intricacy and overlap of the Raman fingerprint spectrum as well as the affinity-interference of nano-tracer to antibody. To circumvent these critical issues, an engineered core-shell multifunctional nano-tracer (named APNPs) with precise control of the size of nano-core (AuNPs) and coating of the nano-shell (Prussian blue nanomaterials) is prepared for SERS-LFIA via a modified enlarging particle size and coating modification strategy. Importantly, this nano-tracer exhibits enhanced coupling efficiency, highly retained affinity, reinforced colloid stability, and unique SERS signal (2156 cm

Long-term dietary exposure of aristolochic acids (AAs)-contaminated food proved to be one of the main culprits of Endemic Nephropathy, renal failure; and urothelial cancer. The antibodies utilized in immunoassays for AAs suffer from low affinity and failure of recognition to the family of AAs. This study, we prepared a broad-specificity monoclonal antibody (mAb) 5H5 with highly and uniform affinity for AAs by help of computational chemistry fully exposing the AAs common structures of methoxy and hydroxyl groups. The mAb 5H5 exhibited half inhibitory concentrations of AAA, AAB, AAC, AAD were 0.03, 0.06, 0.05, 0.03 ng/mL. To explain the broad-specificity profile of mAb 5H5, molecular docking was performed. Results shown that multiple conformations of AAs can be flexibly oriented in the spacious cavity of single-chain variable fragment antibody (scFv) 5H5 and the specific hydron bonds were formed by ASN62 and GLY64 of scFV 5H5 to the nitro group of AAs which gave an explanation of the high cross-reactivity of mAb 5H5. The ELISA based on the broad-specificity mAb 5H5with detection limits of 0.04-0.11 μg/kg and 0.02-0.06 μg/kg for four AAs in flour and soil samples, respectively. The study provided a promising method for the family of AAs in environmental and food samples.

The pursuit of the limit between dimensionalities is a scientific goal with high applicability. Sandwich immunoassay, usually based on two antibodies binding two epitopes, is one of the most popular mainstay tools in both academic and industrial fields. Herein, we determined and evaluated the minimum distance of two epitopes in sandwich immunoassays for small molecules. Briefly, nine model analytes comprising two hapten epitopes, i.e., melamine (MEL) and p-nitroaniline (NIA), were designed by increasing the linear chain linkers brick by brick. Two groups of monoclonal antibodies (mAbs) were produced with different recognition properties toward MEL and NIA using 12 new haptens with different spacer arms. The results indicated that two epitopes of the analyte with a distance of only 2.4 Å could be simultaneously bound by two mAbs, which is the known limit of epitope distance in sandwich immunoassays thus far. We further found that an epitope distance of below 8.8 Å for the analyte generally induces noticeable steric hindrance of antibodies, preventing a sandwich immunoassay with high probability. These observations were investigated and evaluated by molecular docking, molecular dynamics, and surface plasmon resonance and using model and real analytes. Altogether, we determined the minimum distance of two epitopes and explored the molecular mechanism of the antibody–analyte–antibody ternary complex in sandwich immunoassays, providing a theoretical basis for hapten design, antibody discovery and development, and sandwich immunoassay establishment for small molecules.

Multiplex lateral flow immunoassay (mLFIA) has attracted great attention due to the increasing need for rapid detection of multiple analytes. However, it has a number of disadvantages with regard to accuracy and interference because of difficulties in simplifying the process of preparing nanomaterial-based probes. In this work, inspired by protein self-assembly, for the first time, a facile natural antibody network (NAN)-based mLFIA for multiple chloramphenicol (CAP) and streptomycin (STR) determination was designed. The NAN structure was constructed by introducing a second antibody (Ab

As antibodies are the main biological binder for hazards in food samples, their performance directly determines the sensitivity, specificity, and reproducibility of the developed immunoassay. The overwhelmingly used mammalian-derived antibodies usually suffer from complicated preparation, high cost, frequent bleeding of animals, and sometimes low titer and affinity. Chicken yolk antibody (IgY) has recently attracted considerable attention in the bioanalytical field owing to its advantages in productivity, animal welfare, comparable affinity, and high specificity. However, a broad understanding of the application of IgY-based immunoassay for the detection of chemical and biological hazards in food samples remains limited. Here, we briefly summarized the diversity, structure, and production of IgY including polyclonal and monoclonal formats. Then, a comprehensive overview of the principles, designs, and applications of IgY-based immunoassays for these hazards was reviewed and discussed, including food-borne pathogens, food allergens, veterinary drugs, pesticides, toxins, endocrine disrupting chemicals, etc. Thus, the trend of IgY-based immunoassays is expected, and more IgY types, higher sensitivity, and diversification of recognition-to-signal manners are necessary in the future.

Sandwich lateral flow immunoassays (LFIAs) based on paired antibodies are the most frequently used platform for food-borne pathogen detection. Although label-free strategies are used in LFIAs to avoid the utilization of paired antibodies, challenges of probe design and detection reliability still remain. Here, we report a new label-free and dual-readout LFIA (LD-LFIA) mediated by a 'Three-To-One' multi-functional nanocomposite with a unique combination of magnetic-adhesion-color-nanozyme properties. The strengths of the new designed nanocomposite are: (i) the Fe

We previously found that the immune response to haptens is positively correlated with molecular hydrophobicity. The antibodies used in immunoassays for capsaicinoids (CPCs) in waste oil suffer from low affinity and loose recognition to structural analogues. To address this issue, four new haptens (hapten1-4), maximally exposing the hydrophobic alkane chain (noncommon moiety of CPCs), were designed and expected to produce antibodies with high affinity and accurate recognition to CPCs based upon our findings. The assumption was first evidenced by computational chemistry and animal immunization successively. Compared with four reported haptens (hapten5-8) that expose the hydrophilic vanillyl amide moiety (common structure of CPCs and other vanillin alkaloids), antisera from hapten1-4 showed an approximately 1000-fold increase in affinity and significantly improved recognition profiles for CPCs. The molecular recognition study showed that the high affinity of the antibody from new haptens mainly originated from hydrophobic forces. An indirect competitive enzyme-linked immunosorbent assay based on a monoclonal antibody from hapten1 was developed and exhibited limits of detection as low as 0.73-3.29 μg/kg for four CPCs in oils and with insignificant cross-reactivities for other eight vanillin alkaloids, which have been never achieved in previous reports.

Ultrasensitive and fast screening of small molecule contaminants by immunochromatography assay (ICA) is still challenging in field conditions. Here, a novel indirect probe based ICA (ipICA) for highly sensitive detection of 17β-estradiol (E2) was introduced. The strategy was achieved using carboxyl modified Fe3O4 magnetic nanoparticles (MNPs) to label goat anti-mouse antibody (GAMA). The free anti-E2 monoclonal antibody (mAb) rather than labeled mAb was adopted to capture E2 molecules. For comparison, MNPs-based traditional and dual-probe ICAs were also prepared. This strip sensor demonstrated higher sensitivity, with a lowest visible detection limit of 0.2 ng mL−1, which was 5 and 2 times better than that of the MNPs-based traditional strip and dual-probe strip, respectively. The outstanding performance of this novel sensor mainly benefited from the decreased amount of free mAb and the signal amplification of this strategy. Additionally, this method was successfully applied in the detection of E2 in milk, prawn, fish and chicken samples. The proposed sensor exhibited acceptable accuracy for on-site detection of target analyte in food safety monitoring, also has potential applications for the other small molecule contaminants.

Many food safety hazard factors pose a great threat to human health. To mornitor these factors, development of a sensitive, reliable, and efficient detection platform is significantly important. Herein, a sensitive indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) was developed, in which bacteria was introduced as natural signal carriers to construct probes. Through the carriers, the ratio between signal and recognition molecules was flexibly adjusted. As is known, in the competitive immunoassay, the limited number of antibodies was key to significantly improved sensitivity. By this protocol, horseradish peroxidase (HRP) was enriched on the bacterial surface to realize signal amplification, extremely reduce the applied quantity of antibody, and finally trigger intense competition between free target analytes and immobilized antigen. Clenbuterol (CL) as a model target was analyzed by the proposed biosensor system, with a linear range of 0.02–1.0 ng mL−1 and a sensitivity (expressed as IC50) of 0.16 ng mL−1. This contribution has opened up a new route to improve analytical performance of ic-ELISA and provided an application prospect in the detection of other small molecule targets.

The rapid monitoring of foodborne pathogens by monoclonal antibody (McAb)-based immunochromatographic tests (ICTs) is desirable but highly challenging as a result of the screening obstacle for a superior performance probe, which will greatly determine the capture efficiency of targets and the sensitivity of the immunoassay. In this work, on the basis of two-dimensional (2D) nanosheets (including MoS

Here, a facile, label-free and sensitive lateral flow strip (LFS) biosensor for foodborne pathogens was established relying on the innovative introduction of Gram staining and the direct immunoreaction. Target bacteria can be directly marked with crystal violet (CV) by one-step staining which is superior to traditional signal marking techniques in LFS assay, and the method’s selectivity can be guaranteed by high-specificity monoclonal antibody. With Salmonella Enteritidis (S. Enteritidis) as a model target, this protocol can selectively detect 80 CFU mL−1 S. Enteritidis within 11 min in the optimized conditions. Moreover, with Listeria monocytogenes as another model target, the biosensor shows a high universality for detections of both gram-negative and gram-positive bacteria. The unexpected applicability of biological dye tracer in strip biosensor reveals that the biological dye can be a potential tool serving as a universal signal tracer for pathogenic microorganisms in food safety monitoring and early clinical diagnosis.

The design of materials that target heavy metal ions with fast and highly effective adsorption performance is imperative for the removal of heavy metal contaminants from water ecosystems. Inspired by the “function follows form” principle, herein, we developed an approach to control the morphologies and phases of zeolitic imidazolate framework-based nanomaterials (denoted as ZIF-8-x, where x represents the methanol/water ratio) to achieve an ultra-high heavy metal adsorption property. By tuning the solution conditions, the transformation of the morphology of various obtained nanocrystals from a typical rhombic dodecahedron to leaf shape, accompanied by a phase transition from the ZIF-8 to the ZIF-L phase and an increase in amorphous regions, was achieved; this benefited the adsorption interaction between heavy metal ions and 2-methylimidazole binding sites that enhanced the adsorption capacities. The optimized ZIF-8-x of ZIF-8-0 (synthesized in pure water) with leaf-like morphology and the ZIF-L phase endows the prepared adsorbent with extra imino groups for the adsorption of heavy metal ions, which exhibits highest adsorption capacity towards Cu(II) (∼1457.87 mg g−1) and quickly achieves adsorption equilibrium in 5 min. Our results suggest that enhanced adsorption can be achieved by engineering the morphology and phase of nanocrystals, and the as-prepared ZIF-8-0 can be a promising candidate for the purification of water.

The nanomaterials based lateral flow immunoassays (LFIAs) have been widely used for on-site detection of small molecules contaminants. Nevertheless, their practical applications are often constrained by the complicated materials synthesis and surface modification process of nanomaterials. Herein, inspired by chemical-staining, a crystal violet (CV) staining based LFIAs was innovatively developed for the first time to analyze small molecules contaminant with significantly simplified behaviour and surpassing repeatability. This nanomaterials-free technique overcomes the intrinsic limitations of nanomaterials, owing to the superior protein dye property of CV. To demonstrate the feasibility of the unique strategy, furazolidone (FZD) was used as a model analyte, results showed detection limits of as low as 7 and 1.53 ng mL−1 by visual observation and quantitative analysis, respectively. Finally, to validate its applicability in actual conditions, the platform is well applied in three kinds of food samples (shrimp, fish and pork). Furthermore, as the detection principle is based on the molecular recognition between antigen and antibody, our method may find general applications by staining other antibodies against other target analytes.

A homogeneous fluorescence quenching immunoassay is described for simultaneous separation and detection of aflatoxin M1 (AFM1) in milk. The novel assay relies on monoclonal antibody (mAb) functionalized Fe3O4 decorated reduced-graphene oxide (rGO-Fe3O4-mAb) as both capture probe and energy acceptor, combined with tetramethylrhodamine cadaverine-labeled aflatoxin B1 (AFB1-TRCA) as the energy donor. In the assay, AFB1-TRCA binds to rGO-Fe3O4-mAb in the absence of AFM1, quenching the fluorescence of TRCA by resonance energy transfer. Significantly, the immunoassay integrates sample preparation and detection into a single step, by using magnetic graphene composites to avoid washing and centrifugation steps, and the assay can be completed within 10 min. Under optimized conditions, the visual and quantitative detection limits of the assay for AFM1 were 50 and 3.8 ng L−1, respectively, which were significantly lower than those obtained by fluorescence polarization immunoassay using the same immunoreagents. Owing to its operation and highly sensitivity, the proposed assay provides a powerful tool for the detection of AFM1.

The two sensitive fluorescence quantitative immunochromatographic assays (FQICAs), background fluorescence quenching immunochromatographic assay (bFQICA) and time-resolved fluorescent immunochromatographic assay (TRFICA), play an important role increasingly in rapid detection technology for food safety. Amantadine (AMD), used extensively in virus infections in livestock and poultry, has been prohibited due to hazard concerns over public human health. Therefore, AMD was used as a model molecule in the FQICAs establishment and comparison based on the same bioreagents. The outstanding performance in technical parameters of the two FQICAs indicated that they could provide rapid, precise, reliable technical support for large-scale on-site screening for AMD detection. What's more, the systematic and comprehensive comparison of the two FQICAs would give useful suggestions for scientists and users in monitoring the harmful compounds.

An innovative lateral flow competition immunoassay (LFCIA) for detecting clenbuterol (CL) was developed by employing the advantages of the coomassie brilliant blue (CBB) staining method. An antibody stained by CBB was used both as a recognition reagent and as a chromogenic probe, enabling the simple but sensitive LFCIA of CL. The CBB-based LFCIA exhibited sensitivity for CL with a detection limit of 2 ng mL-1. Furthermore, this strategy was preliminarily verified by screening for CL in milk, pork tenderloin, and swine liver with recoveries ranging from 81 to 102%. Compared with conventional LFCIAs, the use of CBB as a signal label not only avoided the complicated material synthesis and surface modification process but also simplified the cross-linking with antibodies, meanwhile reducing the steric hindrance and increasing the possibility of immune recognition reactions, which was propitious for the effective utilization of antibodies. Taking advantages of the simplicity, rapidity, and cost-effectiveness, the CBB-based LFCIA may have potential for on-demand monitoring of general harmful small molecules by changing the kind of the staining antibody.

Furazolidone (FZD) and its metabolite called 3-amino-2-oxazolidinone (AOZ) would induce carcinogenic and mutagenic effects to human. In this work, to develop a novel, stable, and simple point of care testing (POCT) with a potential to social applied for FZD detection, we utilized the aspect of protein staining of coomassie brilliant blue (CBB) to exploit a new CBB-LFIA strategy free of NPs. Only one mixing step is needed during the probe manufacturing process, which requires just 2 h and is a great time saving strategy compared with other methods (requiring 4–33 h for probe preparation). Besides, the cost of CBB-LFIA is 300 times lesser than other LFIA with respect to obtaining the label. The developed CBB-LFIA was successfully applied to detect AOZ with a detection limit of 2 ng mL−1, without any influence from other potential interfering compounds. The proposed CBB-LFIA exhibited prominent practical application, and possesses considerable utilization potential in the related field.

Small molecules are immunochemically classified as hapten that lacking of at least two epitopes, usually using competitive format for establishing immunoassays. However, theoretically, noncompetitive immunoassay format is more sensitive and has a wider analytical range. In the present study, a novel hapten of halofuginone was synthesized and used to produce a monoclonal antibody (mAb). By analyzing the binding kinetics, we found that the affinity of analyte-enzyme to mAb was much greater than that of analyte, which could result in a low sensitivity of competitive assay format. Based on this, we established a novel noncompetitive immunoassay by using a replacement approach. The noncompetitive format has obvious advantages in sensitivity and analytical range, which promoted approximately 3.5- and 5-fold, respectively, compared to the competitive immunoassay. Ultimately, the newly designed noncompetitive immunoassay in this work will provide insights as well as alternative method to traditional small molecule competitive assays.

A homogeneous fluorescence quenching immunoassay is described for simultaneous separation and detection of aflatoxin M

Creating universal signal labels from fundamental building blocks with excellent biocompatibility and well-controlled size/uniformity simultaneously for immunochromatographic assay (ICA) is highly desired but extremely challenging. Here, a nano-signal label strategy was reported, in which the amino-terminated zirconium MOFs (NU66) are adopted to construct powerful matrix materials and gold nanoparticles (AuNPs) act as the linker between metal-organic frameworks (MOFs) and antibodies. Particularly, AuNPs were immobilized directly on the surface of NU66, giving NU66 excellent biocompatibility with bright color signal labels and improving the salt ion stability of AuNPs. As a proof of concept, the furazolidone residues was monitored by the developed NU66@AuNPs-ICA in food samples (pork, shrimp and eggs). With 3-[(4-carboxyphenyl) monomethyl] amino-2-oxazolidinone (CPAOZ) as analyte target, the visual limit of detection (vLOD) and cut-off level were 0.6 ng/mL and 3.0 ng/mL, respectively. This work may open a new avenue for the application of MOFs in immunochromatography assays.

Prussian blue nanoparticles (PBNPs), one kind of metal organic frameworks with the general formula of Fe4[Fe(CN)6]3·nH2O, are increasing attractive due to their excellent properties. Here, with PBNPs as labels, a PBNP-based competitive lateral flow assay (LFA) for efficient and sensitive detection of clenbuterol was developed. With unique control structure, PBNPs are quite larger than most of the commonly used labels, meaning much fewer antibodies are needed in the probe to produce clearly visible results, which will bring a more sensitive dose-response relation between the analyte concentration and signal intensity. Under optimal conditions, the PBNP-based LFA showed a great linear relationship over the range of 0.5–5 ng mL−1 for clenbuterol. The visual limit of detection was 1.0 ng mL−1, 5-fold improvement in sensitivity compared with that of the traditional gold nanoparticles based LFA. In our perception, our work opens new possibilities to choose metal organic frameworks as labeling matrices in immunoassays.

Here, to realize the simultaneous, simple, and highly sensitive detection for nitrofurazone and furazolidone, an immune-scaffold relying immunochromatographic assay was proposed. In this multi-detection immunochromatographic assay (MDICA), the immune-scaffold is composed of labeled anti-antibody IgG (Ab2) and two anti-analyte monoclonal antibodies (Abs1). A nanocomposite of MIL-101 (Cr)@AuNPs is used as the signal tag, where the unique optical property and biocompatibility of AuNPs and the protective effect of metal-organic framework can enable the nanocomposite possesses powerful signals, conjugating ability with antibodies and enhanced colloidal stability. In the immune-scaffold platform, the labeled Ab2 is the core trestle, on the one hand to exempt Ab1 from labeling requirement and offer strong detection signals, on the other hand to orient Abs1 to expose their antigen-binding sites as well as improve the identification and binding efficiency between antigen and antibody. Compared with randomly fixed antibody in the conventional probe, a 6-fold reduction of applied Ab1 amount can be achieved to produce the similar signal intensity in the immune-scaffold based method. In brief, the proposed MDICA system can effectively simplify the construction steps of MDICA, improve the Abs1 utilization efficiency, heighten analytical performances, and providing an efficient, valuable reference for the establishment of other MDICAs.

Theoretically, sandwich immunoassay is more sensitive and has a wider working range than that of competitive format. However, it has been thought that small molecules cannot be detected by the sandwich format due to their limited size. In the present study, we proposed a novel strategy for achieving sandwich immunoassay of β-lactams with low molecular weights. Firstly, five β-lactam antibiotics were selected to bind with penicillin binding protein (PBP)2x* to form complexes. Then, monoclonal and polyclonal antibodies against PBP2x*-β-lactams complexes were produced by animal immunization. Subsequently, the optimal pairing antibodies were utilized to establish sandwich immunoassay for detection of 18 PBP2x*-β-lactam complexes. Among them, ceftriaxone could be detected at as low as 1.65 ng/mL with working range of 1–1000 ng/mL in milk. To reveal the detection mechanism, computational chemistry and molecular recognition study were carried out. The results showed that β-lactams with a large size and complex structures maybe conducive to induce conformational changes of PBP2x*, and then exhibit greater possibility of being detected by sandwich immunoassay after combination with PBP2x*. This study provides insights for subsequent investigations of anti-metatype antibody screening and sandwich immunoassay establishment for small-molecule detection.

Conventional gold nanoparticles-based lateral flow immunoassays (AuNPs-LFIA) lack sensitivity. In this work, we developed a graphite-like carbon nitride-laden AuNPs (g-C3N4@Au) assisted LFIA to improve sensitivity for 17β-estradiol (E2) in foods. g-C3N4 nanosheets were applied as carriers, because of their excellent chemical stability, large surface areas and low-cost, loading large numbers of AuNPs to amplify signals and improve the overall response of g-C3N4@Au-based LFIA (g-C3N4@Au-LFIA). The lowest visual limit of detection (vLOD) of E2 is 0.5 ng mL−1 for g-C3N4@Au-LFIA, which exhibit a significantly three-fold improved analytical performance compared with that of AuNPs-LFIA. Additionally, this method was successfully used to the detection of E2 in four spiked food samples, offering a great potential of the g-C3N4@Au based LFIA for its application in food products.

The donor-acceptor distance is a critical factor for the occurrence of chemiluminescent resonance energy transfer (CRET). We herein evaluate the donor-acceptor distance and transfer efficiency of CRET immunoassays of a series of donors which contain different sized antibody fragments, intact monoclonal antibody (IgG), antigen binding fragment (Fab), and single chain fragment antibody (scFv). Core/multishell quantum dots were used as the acceptor in three CRET systems. IgG is the maximum antibody fragment, leading to the longest donor-acceptor distance and the lowest transfer efficiency. Donors with Fab and scFv show significantly decreased distance and increased transfer efficiency. These results suggest an inverse correlation between donor size and transfer efficiency and can be used to provide guidance for the construction of controllable CRET. By combining the controllable CRET with immunoassay, we further develop a tunable sulfamethazine analytical system. Three different sized donors based CRET immunoassay show a markedly different sensitivity and dynamic range. Such adjustable detection provides greater flexibility for contaminant detection in different foodstuffs with different residue limits. This work not only illustrates the effect of donor-acceptor distance on regulating the energy transfer efficiency of CRET system, but also provides a guideline for the construction of a tunable immunoassay.

A method for the rapid, sensitive, and eco-friendly detection of anticoagulant rodenticides (ARs) in emergency clinics and food samples with point-of-care (POC) assay is highly demanded. The gold nanoparticles and cadmium-based quantum dots (QDs) used as probe in POC testing usually suffer from low sensitivity and high toxicity. Hence, here, we synthesized hydrophilic QDs-based polystyrene nanospheres (QNs) by encapsulating cadmium-free and oleylamine-coated InP/ZnS QDs into polystyrene nanospheres via a facile swelling-evaporation strategy. A lateral flow immunoassay (LFIA) adopted the QNs as probes was proposed to detect eight ARs, achieving quantitative limits of detection from 0.01–2.0 μg/L and visual limits of detection from 0.16–32.0 μg/L for warfarin, coumachlor, coumafuryl, bromadiolone, difenacoum, coumatetralyl, diphacinone and chlorophacinone. The accuracy and precision of the QNs-based LFIA were evaluated by ARs-spiked wheat, human serum and urine, and acceptable recoveries were obtained. The sensitivity of QNs-based LFIA was 2.5–6.25 times better than that of the gold nanoparticles-based LFIA. The QNs-based LFIA was applied to clinical human serum and showed good correlation with ultraperformance liquid chromatography-tandem mass spectrometry. In summary, the QNs-based LFIA provides a rapid, sensitive, and quantitative POC testing method of ARs for poisoning diagnosis and food analysis.

Development of sensitive, facile and rapid biosensors is important for widespread applications. Nanozymes can be ideal signal donors for constructing dual-readout lateral flow immunoassays (LFIA) because they are an excellent class of optical reporters. Herein, a magnetic prussian blue nanozyme (MPBN) mediated dual-readout on-demand multiplex lateral flow immunoassay (MLFIA) was established by employing ractopamine (RAC) and clenbuterol (CLE) as the model analytes. The MPBN was synthesized through in-suit shell-growing and introduced as a bifunctional signal tag owing to their darker original color and peroxidase-like activity. Based on the catalytic signal created by catalyzing oxidation of chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB) and colorimetric signal generated by tag's original color, improved precision and broadened detection range were acquired by implementing a dual-readout strategy. And a two-fold increase in the detection range could fulfill different limit requirements of the same target in various regions. The obtained recoveries from 84.01% to 119.94% indicating the repeatability and reliability of the proposed method. This method provides an attractive platform for the detection of a same target with different detection limits, which possesses a considerable potential in monitoring of other targets.

Currently, the low sensitivity and poor binding stability of detection probe prepared via electrostatic adsorption have become the dilemmas of colloidal gold-based lateral flow immunoassays (Au-LFIAs). In this connection, polydopamine nanospheres (PDA NPs) with an eminent covalent connectivity property were introduced as a promising substitute to improve the stability of probe and sensitivity of LFIA. Whereafter, the PDA NPs-based LFIA was applied for the monitoring of furazolidone (FZD) in food samples because of the potential carcinogenic/mutagenic effects to human of its metabolite (3-amino-2-oxazolidinone, AOZ). Compared with electrostatic adsorption, the binding stability of PDA NPs-based probes was superior. And, as expected, the PDA NPs-based LFIA biosensor exhibited higher sensitivity than that of the Au-LFIA with a detection limit of 3.5 ng mL−1 for AOZ by naked-eye readout. Based on the significant enhanced binding stability and sensitivity, the PDA NPs-based LFIA is of certain spreading value for detecting other analytes.

Pathogenic bacterial infection has emerged as a global health threat, causing death, disability, and socioeconomic disruption worldwide. The current detection system is generally based on the various characteristics of pathogenic bacteria themselves. The readout method, which relies on the characteristic change of pathogenic bacteria receptor (normal cells), is rarely used. Herein, inspired by the pathogenesis and mimicking a minimalistic model system of living cells, a biomimetic liposome membrane comprising lecithin and cholesterol and encapsulated with rhodamine B (RhB) was innovatively developed to analyze the virulence factor with significantly simplified behavior. Based on the specific interaction between cholesterol and streptolysin O (SLO), the biomimetic liposome membrane was selectively ruptured, causing RhB emancipation and enabling fluorescent and intelligent colorimetric dual-signal detections of SLO toxin with limits of detection (LODs) as low as 0.05 μg·mL−1 and 0.33 μg·mL−1, respectively. We anticipate that this study will provide new insights into the development of host cells and biomimetic-system-based methods for pathogen detection.

As a rapid and facile means for foodborne bacteria detection in situ, lateral flow immunoassay (LFA) still has intrinsic limitations in the construction of the existing sandwich LFA format, e.g. screening difficulties of paired antibodies (Abs), poor stability of Ab probe, etc. Here, combined the strong affinity of antibiotic with the superior specificity of antibody molecules, a novel and robust LFA based on a dual recognition strategy and magnetic separation was designed to achieve specific and sensitive determination of Salmonella enteritidis (S. enteritidis). In this work, ampicillin (Amp), a broad-spectrum antibiotic against bacteria, was employed as an ideal Ab replacer to anchor cells of target bacteria. By coating Amp on magnetite nanoparticles (MNPs), the Amp-MNPs showed remarkable binding, separation and enrichment capacities toward bacteria even under complex sample matrices. To ensure the selectivity of this protocol, anti-S. enteritidis monoclonal antibody was then adopted as the second anchoring agent to form a sandwich complex with Amp-MNPs. Based on these facts, S. enteritidis, as low as 102–103 CFU/mL, could be detected by naked eyes in food samples. Therefore, this creative antibiotic-bacteria-antibody LFA sandwich pattern shows great application potential in the monitoring of food contamination and infectious diseases caused by pathogenic bacteria. Compared to the common paired Abs based sandwich method, the proposed approach was cost-effective, non-labor intensive, stable, sensitive and efficient.

Small molecules are difficult to detect by conventional gold lateral flow assay (GLFA) sensitively because the test system must satisfy the conflict requirements between enough signal intensity and limited antibody (Ab) amount. In this work, a paired labels recognition (PLR)-based biosensor was designed by utilizing the specific binding of Ab and secondary antibody (anti-Ab) to enhance signal intensity and reduce antibody amount applied in small molecule detection. The PLR amplification system is fabricated by self-assembling the common detection probe, Au-labeled Ab (Au-Ab), and the signal booster, Au-labeled anti-Ab (Au-anti-Ab). Benefiting from this, a powerful network structure can be generated to accumulate numerous gold nanoparticles (GNPs) and thus significantly strengthen the signal intensity of detection. Therefore, a lower Ab amount will be applied to offer adequate signal strength, and further, the limit of detection will be obviously downregulated due to the more effective competition reaction. Using furazolidone (FZD) as a model analyte, we achieve a detection limit of as low as 1 ng mL−1, which was at least fivefold improved over that of the traditional GLFA. Furthermore, the practicality of this strategy was certificated in five different food samples.

We presented a signal amplified lateral flow assay (LFA) based on magnetite nanoparticles (MNPs) labeled dual-probe and applied it in the high sensitive and rapid on-site detection of furazolidone metabolite of 3-amino-2-oxazolidinone (AOZ). The amplified signal benefited from high affinity between two probes of MNPs labeled murine monoclonal antibody (MNPs-MAb) and goat anti-mouse antibody (MNPs-GAMA) and was achieved by the generation of dual-probe network complex. This developed method could realize high sensitive detection of AOZ with a threshold value of 0.88 ng mL−1 and a detection limit of 0.044 ng mL−1, the sensitivity was at least 10-fold improved than that of the traditional gold nanoparticle based LFA. This facile developed assay was successfully applied for rapid detection of AOZ in milk samples. The proposed method paves a new way for on-site screening of other hazardous substances in food and can be referred in all lateral flow assays.