A cheaper substitute for HRP: ultra-small Cu-Au bimetallic enzyme mimics with infinitesimal steric hindrance to promote catalytic lateral flow immunodetection of clenbuterol.

A highly sensitive lateral flow immunoassay (LFIA) is developed for the enzyme-catalyzed and double-reading determination of clenbuterol (CLE), in which a new type of probe was adopted through the direct electrostatic adsorption of ultra-small copper-gold bimetallic enzyme mimics (USCGs) and monoclonal antibodies. In the assay, based on the peroxidase activity of USCG, the chromogenic substrate TMB-H2O2 was introduced to trigger its color development, and the results were compared with those before catalysis. The detection sensitivity after catalysis is 0.03 ng mL-1 under optimal circumstances, which is 6-fold better than that of the traditional Au NPs-based LFIA and 2-fold greater than that before catalysis. This approach was successfully applied to the detection of CLE in milk, pork and mutton samples with an optimum assay time of 7 min and best catalytic time of 80 s, after which satisfactory recoveries of 98.53-117.79% were obtained. Cu-Au nanoparticles as a signal tag and the use of their nanozyme properties are the first applications in the field of LFIA. This work can be a promising exhibition for the application of a cheaper substitute for HRP, ultra-small bimetallic enzyme mimics, in LFIAs.

Dual-Readout Ultrasensitive Lateral Flow Immunosensing of Salmonella typhimurium in Dairy Products by Doping Engineering-Powered Nanoheterostructure with Enhanced Photothermal Performance.

The photothermal lateral flow immunoassay (LFIA) is of great significance to suitable for on-site semiquantitative detection, which has the upper hand in further constructing detection methods for low-concentration targets. Herein, we presented a doping engineering-powered nanoheterostructure with an enhanced photothermal performance strategy, employing bimetallic nanocuboid Pt3Sn (PSNCs) as a proof of concept. With the help of finite element simulation analysis, the contrast of direct temperature experiment, and the evaluation of photothermal conversion efficiency (η), the distinguished and enthusiastic photothermal feedback of PSNCs is proved. Based on steady bright black of colorimetric and superior photothermal performance, the PSNCs were employed to construct an ultrasensitive model LIFA for detecting Salmonella typhimurium (S. typhimurium), which achieved the double-signal semiquantitative detection, the detection limit reached 103 cfu mL-1 (colorimetric mode) and 102 cfu mL-1 (photothermal mode), which is 100 times higher than that of the traditional colloidal gold method. In addition, the method was effective for the detection of targets in dairy samples only through a simple dilution treatment, which was completed within 15 min. Meanwhile, this PSNCs dual-signal LFIA demonstrated the sensitive detection of S. typhimurium due to the excellent colorimetric signal and significant photothermal performance, which provides a broad spectrum for the future detection of foodborne pathogens.

Enhanced sandwich immunoassay based on bivalent nanobody as an efficient immobilization approach for foodborne pathogens detection.

BACKGROUND: Nanobodies (Nbs), which consist of only antigen-binding domains of heavy chain antibodies, have been used in a various range of applications due to their excellent properties. Nevertheless, the size of Nbs is so small that their antigen binding sites may be sterically hindered after random fixation as capture antibodies, thus leading to poor detection performance in immunoassays. To address this problem, we have focused on the multivalent modification of Nbs, wanted to retain the advantage of good stability through enlarging the size of Nbs to a certain extent, while improve its affinity and reduce its influence by spatial orientation. RESULTS: Here, we designed homo- and heterodimeric Nbs based on Nb413 and Nb422 which recognize different epitopes of Salmonella. The affinity of engineered bivalent nanobodies for S. Enteritidis were 2 orders of magnitude higher compared to monovalent Nbs and low to sub-nM KD, as calculated by Scatchard analysis. To further explore the potential of bivalent Nbs for the detection of Salmonella, we established a sandwich ELISA based on bivalent and phage-displayed Nbs (BNb-ELISA) for multiplex Salmonella determination. Compared with monovalent Nb-based ELISA, the limit of detection (LOD) of the BNb-ELISA was shown to increase 7.5-fold to 2.364 × 103 CFU mL-1 for S. Enteritidis. In addition, the feasibility of this approach for S. Enteritidis detection in real samples was evaluated, with recoveries ranging from 73.0 % to 125.6 % and coefficients of variation (CV) below 7.68 %. SIGNIFICANCE AND NOVELTY: In this study, we developed for the first time bivalent Nbs against Salmonella and examined their improved affinity and impact on the performance of ELISA assay. It confirmed the high binding affinity and good ability of dimeric Nbs to reduce the occupation of the binding sites of immobilized antibodies. Thus, the multivalent modification of Nbs was demonstrated to be a promising means to enhance the performance of Nbs-based immunoassays for foodborne pathogens.

A dual-mode lateral flow immunoassay by ultrahigh signal-to background ratio SERS probes for nitrofurazone metabolites ultrasensitive detection.

In this work, an ultra-sensitive lateral flow immunoassay (LFIA) with SERS/colorimetric dual signal mode was constructed for the detection of nitrofurazone metabolites, an antibiotic prohibited in animal-origin foods. Au@4-MBN@AgNRs nano-sandwich structural signal tag integrates the unique advantages of high signal-to-background ratio and anti-matrix interference through geometric control of SERS tag and nanoengineering adjustment of chemical composition. Under the optimal conditions, the detection limits of nitrofurazone metabolites by SERS/colorimetric dual-mode LFIA were 20 pg/mL (colorimetric mode) and 0.08 pg/mL (SERS mode). Excitingly, the vLOD of the colorimetric signal improved by a factor of 100 compared to Au NPs-based LFIA. In this study, the proposed dual-mode LFIA was successfully applied to the on-site real-time detection of honey, milk powder, and chicken. It is anticipated that with low background interference and anti-matrix interference output signal, our proposed dual-mode strategy can pave an innovative pathway for the fabrication of a powerful biosensor.

Joint-detection of Salmonella typhimurium and Escherichia coli O157:H7 by an immersible amplification dip-stick immunoassay.

To explore the superiority of multifunctional nanocomposites and realize the joint-detection of foodborne pathogens, an immersible amplification dip-stick immunoassay (DSIA) was exploited for the sensitive detection of Salmonella typhimurium (S. typhi) and Escherichia coli O157:H7 (E. coli O157:H7). Saving for the basic colorimetric performance, the reporter molecule of CoFe2O4 (CFO) possesses multivalent elements (Co2+/3+, Fe2+/3+) as well as multifunction of superior catalase-like activity and magnetic properties. By dint of the catalytic activity of CFO, a directly immersible amplification can be simply achieved to endure the DSIA with an intensive signal and a dual-visible mode for the determination of S. typhi and E. coli O157:H7. In virtue of the magnetic separation and enrichment capability of the CFO, the DSIA can perform a matrix-interference-free detection and obtain a dynamic detection range of 102-108 CFU/mL and a low assay limit of 102 CFU/mL. Moreover, the DSIA has reasonable recovery rates for contamination monitoring of two target bacteria in milk and beef samples. Our research provides a persuasive supplement for the application of multifunctional nanocomposites in the ongoing dip-stick immunoassay and an alternative strategy for the efficient detection of foodborne pathogens.

Highly photothermal and biodegradable nanotags-embedded immunochromatographic assay for the rapid monitoring of nitrofurazone.

Immunochromatographic assay platforms are up-and-coming detection tools for disease diagnosis and harmful substances monitoring in food. Herein, by combining photothermal imaging and immunochromatographic analysis, a photothermal immunoassay is developed for the rapid and ultrasensitive detection of nitrofurazone. The nickel disulfide nanosphere with excellent biocompatibility, biodegradability, and high photothermal conversion efficiency, is introduced to offer straightforward readout by color and temperature based on the nature of the crystal, without advanced equipment. It is demonstrated that the nitrofurazone metabolite of semicarbazide can be qualitatively detected by colorimetric signals with a visual limit of 2 µg kg-1. And the quantitative detection limit of photothermal signals is 0.01 µg kg-1, improving the detection sensitivity by about 200 times. Furthermore, recovery rates of the proposed method in food samples were 93 %-120 %. This photothermal immunoassay not merely provides straightforward, rapid, simultaneous qualitative/quantitative detection tactics but may also be valuable in automated and portable diagnostic applications.

A lateral flow immunoassay based on chemisorbed probes in virtue of hydrogen bond receptors on the Bi2S3 NPs.

Improving detection sensitivity is still a major research emphasis for lateral flow immunoassay (LFIA). Increasing the binding efficiency and stability of the probe is an achievable and effective solution. In this work, we developed a highly sensitive lateral flow immunoassay for clenbuterol detection by using bismuth sulfide nanoparticle (Bi2S3) nanoparticles (NPs) as a novel marker. Here, Bi2S3 NPs can link with the antibody by hydrogen bonding to improve the performance of the probe, e.g., stability and sensitivity. Benefiting from the direct hydrogen bonding between Bi2S3 NPs and the monoclonal antibody (mAb), high sensitivity is obtained by the proposed LFIA with a lower visible detection limit of 0.1 ng mL-1 and a cut-off value of 4 ng·mL-1 for CLE detection, which is 5-fold and 7.5-fold improved than the conventional Au NPs based LFIA. In addition, the encouraging practical application results in milk, pork, and beef show that the bismuth sulfide nanoparticle has a great popularizing potential in the performance promotion of LFIAs for food safety monitoring.

Vanadium Disulfide Nanosheet Boosts Optical Signal Brightness as a Superior Enzyme Label to Improve the Sensitivity of Lateral Flow Immunoassay.

The color-enzyme lateral flow immunoassay (LFIA) has attracted widespread attention to expand the detection range and improve sensitivity via amplifying the color signal after catalyzing the substrate. As a kind of layered transition-metal dichalcogenide (TMD), the vanadium disulfide nanosheet (VS2NS) possesses superior peroxidase-like catalytic activity. Here, a VS2NS was applied as an enzyme label in the LFIA to detect 17ß-estradiol (E2). Compared to natural horseradish peroxidase, the VS2NS expresses a more prominent enzyme catalytic performance, stability, and adsorption ability. Under optimal conditions, the calculated limit of detection (cLOD) of the VS2NS-based LFIA is 0.065 ng mL-1 for E2, which is sixfold lower than that of the optimized colloidal nanoparticle-based LFIA (cLOD = 0.406 ng mL-1). Besides, the detection linear range of the VS2NS-based LFIA can be widened by 1.5 times after the catalytic reaction. Moreover, the VS2NS-based LFIA exhibits excellent practicability in real sample detection. Simultaneously, this study helps open up the application of the VS2NS in the trace analysis of LFIAs, which can broaden TMDs' scope of application and better show their properties of color enzymes.

Multiplex immunochromatographic platform based on crystal violet tag for simultaneous detection of streptomycin and chloramphenicol.

Antibiotic abuse has caused serious health risks for human beings for long. To address the problem, novel and facile detection techniques are highly desired. Here, an effective multiplex immunochromatographic platform (MICP) with synthesis-free and cost-effective merits is established for simultaneous detection of antibiotics on a single immunochromatographic assay (ICA) strip. Adopting crystal violet (CV) as a signal tag for multiplex ICA allows for direct coupling with multiple antibodies in several minutes. By combining CV and ICA perfectly, this convenient strategy offers improvements in convenience, speed, flexibility, and portability, eventually ensuring the optimized effectiveness of this approach. As a result, the established platform is successfully used to detect streptomycin (STR) and chloramphenicol (CAP) with visual detection mode, and the obtained total recoveries of milk and honey real samples changed from 83.82 to 113.38% with total RSD values of 0.48 to 4.15%.

Nature-inspired nanozymes as signal markers for in-situ signal amplification strategy: A portable dual-colorimetric immunochromatographic analysis based on smartphone.

Owing to the lack of a universal descriptor to predict the nanozymes as signal markers (SM) of immunochromatographic analysis (ICA), the present exploitation of nanozymes as SM heavily relies on trial-and-error strategies, which obstructs the rational design of nanozymes with monoclonal antibodies (mAbs) recognition activity. Herein, inspired by the structure of the active center of natural multi-iron peroxidases and polyphenol-protein interactions, a rational design of an artificial peroxidase with mAbs recognition activity by utilizing gallic acid (GA) to chelate with multivalent iron was successfully proposed by utilizing gallic acid (GA) to chelate with multivalent iron. The most essential features of peroxidase-like Fe-GA nanozymes (FGN) were investigated, showing high catalytic performance and good stability. Subsequently, FGN was employed as SM for mAbs in ICA, which played the following triple roles in the ICA sensor: (i) the direct recognizer of mAbs; (ii) the generator of original colorimetric signal; (iii) the generator of catalytic in-suit amplification colorimetric signal. To make the ICA more portable, we have employed a smartphone and principal component analysis (PCA) to assist this on-site detection. As a proof-of-concept, clenbuterol (CLL) was analyzed by the proposed nanozymes-mediated dual-colorimetric ICA based on a smartphone. Notably, the proposed dual-colorimetric ICA exhibits high analytical performance for the quantification of CLL in the detection range of 0-6 ng mL-1 with a limit of detection (LOD) as low as 0.172 ng mL-1. Meanwhile, the proposed dual-colorimetric ICA exhibits remarkable feasibility and was successfully employed for the detection of CLL in pork and chicken matrixes.

Bioresource-derived tannic acid-supported immuno-network in lateral flow immunoassay for sensitive clenbuterol monitoring.

Although lateral flow immunoassay (LFIA) as a point-of-care (POC) diagnostic tool has been widely used because of numerous merits, it remains challenging to realize higher sensitivity, easier labeling, and fewer antibodies consumption. Herein, a bioresource-derived tannic acid (TA)-supported immuno-network based LFIA for clenbuterol (CLE) monitoring was proposed by employing poly TA nanospheres (PTAN) as the new tracer. Attributing to the effective protein-enrichment ability of TA, plenty of goat anti-mouse immunoglobulins (GAMI) were first absorbed on the surface of PTAN and then monoclonal antibodies (AbCLE) were added to form immuno-network. Compared with directly coupling antibodies, the utilization efficiency of AbCLE was significantly improved. Thus, the detection sensitivity (take the cut-off value as an example) was enhanced 5-fold and 10-fold at least compared with PTAN-AbCLE and colloidal gold-based LFIA, respectively. Additionally, the proposed method was successfully verified in beef and pork liver and provided an effective strategy for food safety monitoring.

Self-Assembling Antibody Network Simplified Competitive Multiplex Lateral Flow Immunoassay for Point-of-Care Tests.

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 (Ab2) as a scaffold to noncovalently combine with various monoclonal antibodies (mAbs), thus permitting each mAb to act as an independent functional unit to maintain bioactivity. Furthermore, the NAN was colored by simple one-step staining using coomassie brilliant blue R-250 (CBBR) to form a chromogenic probe, eliminating the need for complex nanomaterials to improve reproducibility and precision. Under optimal conditions, a satisfactory detection performance (the visual limit of detection (v-LOD) of 3 ng mL-1 for CAP and 20 ng mL-1 for STR) was obtained for whole milk analysis, which met the basic requirement of detection and had good specificity, reproducibility (relative standard deviation (RSD) < 15%), and robustness. In addition, the precision of the detection results was improved usefully since the test procedure was simplified. Overall, the developed system enables fast, simple, and reliable point-of-care assays of multiple analytes.

Expanded detection range of lateral flow immunoassay endowed with a third-stage amplifier indirect probe.

Here, a third-stage amplifier indirect probe (TsAIP) based lateral flow immunoassay (LFIA) was proposed to detect furazolidone (FZD) with Prussian blue nanoparticles (PBNPs) as carrier to label the goat anti-mouse antibody-horseradish peroxidase conjugation [GAMA(HRP)]. In this strategy, owing to the fact that one monoclonal antibody (mAb) can combine several GAMA molecules simultaneously, the indirect probe can generate primary signal amplification, then realize second-stage amplification attributing to PBNPs, and finally achieve third-stage amplification because of the conjugated HRP. The TsAIP-based LFIA shows improved performance for FZD metabolite derivative with a detection limit of 1 ng mL-1. The detection range is expanded about 2-fold compared with the original outcome. Besides, the proposed sensor could be successfully applied in food samples. This method provides a platform for broadening the detection range and application of PBNPs based LFIAs.

Controllable assembly metal-organic frameworks and gold nanoparticles composites for sensitive immunochromatographic assay.

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.

Lateral flow immunoassay for furazolidone point-of-care testing: Cater to the call of saving time, labor, and cost by coomassie brilliant blue labeling.

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.

Antibiotic and mammal IgG based lateral flow assay for simple and sensitive detection of Staphylococcus aureus.

Lateral flow assay (LFA), performed with simple devices and short detection time, is popular in field applications. Herein, a novel sandwich type-based LFA was constructed for high sensitivity and selectivity detection of Staphylococcus aureus (S. aureus). Vancomycin-immobilized gold nanoparticles (VAN-Au NPs) were utilized as the first identifier to capture S. aureus and the specificity was guaranteed by the second recognition agent of pig immunoglobulin G (IgG). In addition, gold growth was adopted for signal amplification to further improve the detection sensitivity. S. aureus could be directly assayed by this LFA within the concentration range of 1.0 × 103-1.0 × 108 cfu mL-1 with a detection limit of 1.0 × 103 cfu mL-1. Furthermore, the novel sandwich LFA realized S. aureus detection in food samples with admissible recoveries and established a rapid, simple, cost-effective and sensitive platform, could meet the demand for on-site testing of S. aureus.

Nanozyme amplification mediated on-demand multiplex lateral flow immunoassay with dual-readout and broadened detection range.

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.

Small size nanoparticles-Co3O4 based lateral flow immunoassay biosensor for highly sensitive and rapid detection of furazolidone.

Lateral flow immunoassay (LFIA) biosensor is a paper-based tool and widely utilized in various fields. Here, we developed a novel LFIA biosensor by introducing Co3O4 nanoparticles (NPs) as signal labels for highly sensitive detection of 3-amino-2-oxazolidinone (AOZ), a metabolite of furazolidone. The characteristic brown color of Co3O4 NPs enabled AOZ to be visually detected by the LFIA. Significantly, the size of Co3O4 NPs is relatively small compared with most of other signal labels, which could remarkably reduce steric hindrance, increase immunoreaction probability and shorten the analysis time. Under optimal conditions, the novel Co3O4 NPs-LFIA could possess high sensitivity for the detection of AOZ with a detection limit of 0.4 ng mL-1 by naked eyes, which was at least 3-fold improved than that of the conventional gold nanoparticles (GNPs) based LFIA. Moreover, the detection could be achieved within 6 min and without cross-reactions with other analogue small molecules. Taking merits of convenience, rapid and sensitivity, the proposed Co3O4 NPs-LFIA may be readily adapted for the detection of other small molecules.