Gold nanoparticle-decorated covalent organic frameworks as amplified light-scattering probes for highly sensitive immunodetection of Salmonella in milk.

Traditional immunoassays exhibit insufficient screening sensitivity for foodborne pathogens due to their low colorimetric signal intensities. Herein, we propose an ultrasensitive dynamic light scattering (DLS) immunosensor for Salmonella based on a "cargo release-seed growth" strategy enabled by a probe, namely gold nanoparticle-decorated covalent organic frameworks (COF@AuNP). Large amounts of AuNPs in COF@AuNP can be released by acid treatment-induced decomposition of the imine-linked COF, and then they are enlarged via gold growth to generate a dramatically enhanced light-scattering signal, leading to a vast improvement in detection sensitivity. Based on an immunomagnetic microbead carrier, the proposed DLS immunosensor is capable of detecting trace Salmonella in milk in the range of 2.0 × 102-2.0 × 105 CFU mL-1, with a limit of detection of 60 CFU mL-1. The immunosensor also demonstrated excellent selectivity, good accuracy and precision, and high reliability for detecting Salmonella in milk.

Integrated gold superparticles into lateral flow immunoassays for the rapid and sensitive detection of Escherichia coli O157:H7 in milk.

Escherichia coli O157:H7 is a common harmful foodborne pathogen that can cause severe diseases at low infectious doses. Traditional lateral flow immunoassay (LFIA) for the rapid screening of E. coli O157:H7 in food suffers from low sensitivity due to its dependence on 20- to 40-nm gold nanoparticles (AuNP) with insufficient brightness as labels. To address this issue, we reported for the first time the successful synthesis of gold superparticles (GSP) by encapsulating numerous small AuNP into a polymer nanobead using an evaporation-induced self-assembly method. Results indicated that the resultant GSP exhibited remarkably enhanced absorbance compared with the most widely used 40 nm AuNP in LFIA. In addition, the absorbance of GSP could be easily tuned by varying GSP sizes. Under optimized conditions, we achieved a rapid and sensitive determination of E. coli O157:H7 in milk with a detection limit of 5.95 × 102 cfu/mL when using the GSP with a size of 342 nm as LFIA signal reporters, exhibiting improvement of approximately 32-fold relative to the conventional 40 nm AuNP-LFIA method. We further demonstrated the selectivity, accuracy, reliability, and practicality of the proposed GSP-LFIA strip. In summary, this work offers a new strategy for improving LFIA sensitivity using assembled GSP as markers and demonstrates huge potential in rapidly and sensitively detecting foodborne pathogens.

AIEgens: An emerging fluorescent sensing tool to aid food safety and quality control.

As a global public health problem, food safety has attracted increasing concern. To minimize the risk exposure of food to harmful ingredients, food quality and safety inspection that covers the whole process of "from farm to fork" is much desired. Fluorescent sensing is a promising and powerful screening tool for sensing hazardous substances in food and thus plays a crucial role in promoting food safety assurance. However, traditional fluorphores generally suffer the problem of aggregation-caused quenching (ACQ) effect, which limit their application in food quality and safety inspection. In this regard, luminogens with aggregation-induced emission property (AIEgens) showed large potential in food analysis since AIEgens effectively surmount the ACQ effect with much better detection sensitivity, accuracy, and robustness. In this contribution, we review the latest developments of food safety monitoring by AIEgens, which will focus on the molecular design of AIEgens and their sensing principles. Several examples of AIE-based sensing applications for screening food contaminations are highlighted, and future perspectives and challenges in this emerging field are tentatively elaborated. We hope this review can motivate new research ideas and interest to aid food safety and quality control, and facilitate more collaborative endeavors to advance the state-of-the-art sensing developments and reduce actual translational gap between laboratory research and industrial production.

Magnetic Quantum Dot Nanobead-Based Fluorescent Immunochromatographic Assay for the Highly Sensitive Detection of Aflatoxin B1 in Dark Soy Sauce.

Herein, we synthesized bifunctional magnetic fluorescent beads (MFBs) with a distinct core/shell structure by encapsulating octadecylamine-coated CdSe/ZnS QDs (OC-QDs) and oleic acid-modified iron oxide nanoparticles (OA-IONPs) into two polymer matrixes with different hydrophobic properties. The OC-QDs and OA-IONPs were mainly distributed in the outer layer of MFBs. The resultant MFBs displayed ca. 226-fold stronger fluorescence emission relative to the corresponding OC-QDs and retained ca. 45.4% of the saturation magnetization of the OA-IONPs. The MFBs were used to purify and enrich aflatoxin B1 (AFB1) from dark soy sauce and then utilized as a fluorescent reporter of immunochromatographic assay (ICA) for the sensitive detection of AFB1. Under the optimal detection conditions, the MFB-based ICA (MFB-ICA) displayed a dynamic linear detection of AFB1 in sauce extract over the range of 5-150 pg/mL with a half maximal inhibitory concentration of 27 ± 3 pg/mL ( n = 3). The detection limits for AFB1 in sauce extract and real dark soy sauce were 3 and 51 pg/mL, respectively, which are considerably better than those of the previously reported fluorescent bead-based ICA methods. The analytical performance of the proposed MFB-ICA in terms of selectivity and accuracy was investigated by analyzing AFB1-spiked dark soy sauce samples. The reliability of the proposed method was further confirmed by ultraperformance liquid chromatography with fluorescence detection. With the combined advantages of QDs and IONPs, the resultant MFBs offer great potential as reporters of ICA for the sensitive detection of trace pollutants in complex matrix samples.

Supramolecular Recognition-Mediated Layer-by-Layer Self-Assembled Gold Nanoparticles for Customized Sensitivity in Paper-Based Strip Nanobiosensors.

Herein, a smart supramolecular self-assembly-mediated signal amplification strategy is developed on a paper-based nanobiosensor to achieve the sensitive and customized detection of biomarkers. The host-guest recognition between ß-cyclodextrin-coated gold nanoparticles (AuNPs) and 1-adamantane acetic acid or tetrakis(4-carboxyphenyl)porphyrin is designed and applied to the layer-by-layer self-assembly of AuNPs at the test area of the strip. Thus, the amplified platform exhibits a high sensitivity with a detection limit at subattogram levels (approximately dozens of molecules per strip) and a wide dynamic range of concentration over seven orders of magnitude. The applicability and universality of this sensitive platform are demonstrated in clinically significant ranges to measure carcinoembryonic antigen and HIV-1 capsid p24 antigen in spiked serum and clinical samples. The customized biomarker detection ability for the on-demand needs of clinicians is further verified through cycle incubation-mediated controllable self-assembly. Collectively, the supramolecular self-assembly amplification method is suitable as a universal point-of-care diagnostic tool and can be readily adapted as a platform technology for the sensitive assay of many different target analytes.

Amphiphilic ligand modified gold nanocarriers to amplify lanthanide loading for ultrasensitive DELFIA detection of Cronobacter.

Conventional dissociation-enhanced lanthanide (Ln3+) fluoroimmunoassays (DELFIAs) using Ln3+ chelate-labeled antibodies as molecular probes exhibit limited sensitivity because of their relatively low Ln3+ labeling ratio per biomolecule. Herein, we applied gold nanoflowers (AuNFs) as amplified nanocarriers to increase the Ln3+ labeling ratio in a single molecular binding event for improving the sensitivity of traditional DELFIA. Two thiolated amphiphilic ligands (thiolated ethylenediaminetetraacetic acid (EDTA) and thiolated acylhydrazine-terminated ligands), consisting of a hydrophobic alkane chain, oligo(ethylene glycol) unit, and functional terminal of the EDTA or acylhydrazine group, were designed for the surface modification of AuNFs. The resultant ligand-coated AuNFs exhibited dual functions of Ln3+ chelation via the EDTA group and oriented attachment of antibodies via the acylhydrazine group. By utilizing 80 nm AuNFs as amplified carriers, we demonstrated that the maximum Eu3+ loading amount reached 1.07 × 104 Eu3+ ions per AuNF, which is approximately two to three orders of magnitude higher than that of traditional molecular probes, thereby amplifying the luminescence signal and enhancing the sensitivity of DELFIA. By combining a magnetic-mediated sandwich-type DELFIA method, the designed amplified AuNF nanoprobes achieved an ultrasensitive luminescence detection of Cronobacter muytjensii with a limit of detection (LOD) of 1.2 × 102 cfu mL-1 in a powdered infant formula. This LOD value was ca. 230-fold lower than that of the traditional colorimetric immunoassay. The designed signal amplification strategy using bifunctional ligand-modified AuNFs as enhanced Ln3+ nanocarriers provided a huge potential for building various ultrasensitive luminescence immunoassays for in vitro biodetection.

Plasmonic ELISA based on DNA-directed gold nanoparticle growth for Cronobacter detection in powdered infant formula samples.

The traditional gold nanoparticle (AuNP) growth-based plasmonic ELISA (pELISA) strictly and directly controlled by reducing reagents can achieve high sensitivity, but it remains fragile toward the surrounding environment. This work developed a sandwich pELISA for Cronobacter detection in powdered infant formula samples by mediating AuNP growth through DNA. In this assay, DNA adsorbed on the surface of gold nanoseeds guided the anisotropic crystal growth with hydroxylamine as a reducing reagent, and the catalase-hydrogen peroxide (Cat-H2O2) system was introduced to bridge the DNA-directed AuNP growth and pELISA, as such DNA can be cleaved into fragments by the hydroxyl radical generated from oxidation of H2O2 through Fenton reagents. Under optimized conditions, the proposed pELISA can qualitatively detect Cronobacter species (Cronobacter muytjensii ATCC 51329) by the naked eye with a cut-off limit of 3 × 105 cfu/mL. This method also revealed a good linear range (3 × 102 to 3 × 107 cfu/mL) for quantitative detection of C. muytjensii ATCC 51329 with a limit of detection of 1.6 × 102 cfu/mL, which is approximately 162.5 times lower than that of horseradish peroxidase-based conventional ELISA (2.6 × 104 cfu/mL). By taking advantage of highly stable DNA-directed AuNP growth, the proposed method shows a good performance in powdered infant formula samples spiked with different concentrations of C. muytjensii ATCC 51329 with average recoveries ranging from 90.79 to 119.09% and coefficient of variation ranging from 4.24 to 9.55%. These values corresponded to an acceptable accuracy and precision for the proposed method. In brief, this work shows potential for screening other analytes in food safety, clinical diagnostics, and environmental monitoring.

Establishment of a novel quantum dots-encoded microbead-based flow cytometric method for quantification of soluble FcεRIα in serum.

The soluble form of the transmembrane glycoprotein, FcεRIα which corresponds to the high-affinity receptor for IgE, is found in serum. Growing evidence suggests the pathogenic role of IgE and FcεRI in systemic lupus erythematosus (SLE). The goal of this study is to develop a sensitive and standardized cytometric assay for quantification of sFcεRIα. A membrane emulsification technique was utilized to incorporate CuInS2 /ZnS quantum dots and Fe3 O4 nanoparticles into poly (styrene-co-maleic anhydride) microbeads. The beads were then carboxylated and coated with capture antibody monoclonal anti-human FcεRIα. This antibody binds to FcεRIα but does not block the binding of FcεRIα to IgE. After incubation with standards or serum samples, the microbeads were incubated with excessive native human IgE, followed by incubation with Phycoerythrin (PE) conjugated anti-human IgE. The resulting quantum dot microbeads were gated, and sFcεRIα quantification was analyzed based on PE fluorescence intensity. The method exhibited good linearity (R2 > 0.99), and the limit of detection was established at 0.29 ng/mL with the dynamic range of up to 200 ng/mL. The precision of the assay validated by intra- and inter-assay variability met the acceptance criteria with the mean recovery falling within 80-110% of the theoretical concentration and a corresponding CV < 20%. We tested 149 serum samples which 89 were from SLE patients and 60 were from healthy volunteers. For the first time, we detected an increased sFcεRIα level in the serum of SLE patients, which was confirmed by a commercial ELISA kit. Compared to ELISA, this novel method is more sensitive and efficient. It allows for the simple comparative analysis of sFcεRIα levels in health and disease. © 2017 International Society for Advancement of Cytometry.

Suspension arrays based on nanoparticle-encoded microspheres for high-throughput multiplexed detection.

Spectrometrically or optically encoded microsphere based suspension array technology (SAT) is applicable to the high-throughput, simultaneous detection of multiple analytes within a small, single sample volume. Thanks to the rapid development of nanotechnology, tremendous progress has been made in the multiplexed detecting capability, sensitivity, and photostability of suspension arrays. In this review, we first focus on the current stock of nanoparticle-based barcodes as well as the manufacturing technologies required for their production. We then move on to discuss all existing barcode-based bioanalysis patterns, including the various labels used in suspension arrays, label-free platforms, signal amplification methods, and fluorescence resonance energy transfer (FRET)-based platforms. We then introduce automatic platforms for suspension arrays that use superparamagnetic nanoparticle-based microspheres. Finally, we summarize the current challenges and their proposed solutions, which are centered on improving encoding capacities, alternative probe possibilities, nonspecificity suppression, directional immobilization, and "point of care" platforms. Throughout this review, we aim to provide a comprehensive guide for the design of suspension arrays, with the goal of improving their performance in areas such as multiplexing capacity, throughput, sensitivity, and cost effectiveness. We hope that our summary on the state-of-the-art development of these arrays, our commentary on future challenges, and some proposed avenues for further advances will help drive the development of suspension array technology and its related fields.

NIR-emitting quantum dot-encoded microbeads through membrane emulsification for multiplexed immunoassays.

NIR-emitting CdSeTe/CdS/ZnS core/shell/shell QD-encoded microbeads are combined with common flow cytometry with one laser for multiplexed detection of hepatitis B virus (HBV). A facile one-pot synthetic route is developed to prepare CdSeTe/CdS/ZnS core/shell/shell QDs with high photoluminescence quantum yield and excellent stability in liquid paraffin, and a Shirasu porous glass (SPG) membrane emulsification technique is applied to incorporate the QDs into polystyrene-maleic anhydride (PSMA) microbeads to obtain highly fluorescent QD-encoded microbeads. The relatively wide NIR photoluminescence full width half maximum of the CdSeTe/CdS/ZnS QDs is used to develop a 'single wavelength' encoding method to obtain different optical codes by changing the wavelengh and emission intensity of the QDs incorporated into the microbeads. Moreover, a detection platform combining NIR-emitting CdSeTe/CdS/ZnS QD-encoded microbeads and Beckman Coulter FC 500 flow cytometry with one laser of 488 nm is successfully used to conduct a 2-plex hybridization assay for hepatitis B surface antigen (HBsAg), hepatitis B e antigen (HBeAg), and a 3-plex hybridization assay for hepatitis B surface antibody (HBsAb), hepatitis B e antibody (HBeAb), and hepatitis B core antibody (HBcAb), which suggests the promising application of NIR QD-encoded microbeads for multiplex immunoassays.

Ultrasensitive Lateral Flow Immunoassay for Fumonisin B1 Detection Using Highly Luminescent Aggregation-Induced Emission Microbeads.

Lateral flow immunoassay (LFIA) based on fluorescent microbeads has attracted much attention for its use in rapid and accurate food safety monitoring. However, conventional fluorescent microbeads are limited by the aggregation-caused quenching effect of the loaded fluorophores, thus resulting in low signal intensity and insufficient sensitivity of fluorescent LFIA. In this study, a green-emitting fluorophore with an aggregation-induced emission (AIE) characteristic was encapsulated in polymer nanoparticles via an emulsification technique to form ultrabright fluorescent microbeads (denoted as AIEMBs). The prepared AIEMBs were then applied in a competitive LFIA (AIE-LFIA) as signal reporters for the rapid and highly sensitive screening of fumonisin B1 (FB1) in real corn samples. High sensitivity with a detection limit of 0.024 ng/mL for FB1 was achieved by the developed AIE-LFIA. Excellent selectivity, good accuracy, and high reliability of the AIE-LFIA were demonstrated, indicating a promising platform for FB1 screening.

A label-free fiber ring laser biosensor for ultrahigh sensitivity detection of Salmonella Typhimurium.

The rapid detection of low concentrations of Salmonella Typhimurium (S. Typhimurium) is an essential preventive measure for food safety and prevention of foodborne illness. The study presented in this paper addresses this critical issue by proposing a single mode-tapered seven core-single mode (STSS) fiber ring laser (FRL) biosensor for S. Typhimurium detection. The experimental results show that the specific detection time of S. Typhimurium is less than 20 min and the wavelength shift can achieve -0.906 nm for an S. Typhimurium solution (10 cells/mL). Furthermore, at a lower concentration of 1 cell/mL applied to the biosensor, a result of -0.183 nm is observed in 9% of samples (1/11), which indicates that the proposed FRL biosensor has the ability to detect 1 cell/mL of S. Typhimurium. In addition, the detection results in chicken and pickled pork samples present an average deviation of -27% and -23%, respectively, from the measured results in phosphate buffered saline. Taken together, these results show the proposed FRL biosensor may have potential applications in the fields of food safety monitoring, medical diagnostics, etc.

Bifunctional M13 Phage as Enzyme Container for the Reinforced Colorimetric-Photothermal Dual-Modal Sensing of Ochratoxin A.

"Point of care" (POC) methods without expensive instruments and special technicians are greatly needed for high-throughput analysis of mycotoxins. In comparison, the most widely used screening method of the conventional enzyme-linked immunosorbent assay (ELISA) confronts low sensitivity and harmful competing antigens. Herein, we develop a plasmonic-photothermal ELISA that allows precise readout by color-temperature dual-modal signals based on enzymatic reaction-induced AuNP aggregation for highly sensitive detection of ochratoxin A (OTA). The bifunctional M13 phage carrying OTA that mimics the mimotope on the end of p3 proteins and abundant biotin molecules on the major p8 proteins is adopted as an eco-friendly competing antigen and enzyme container for amplifying the signal intensity. Under optimal conditions, both colorimetric and photothermal signals enable good dynamic linearity for quantitative OTA detection with the limits of detection at 12.1 and 8.6 pg mL-1, respectively. Additionally, the proposed ELISA was adapted to visual determination with a cutoff limit of 78 pg mL-1 according to a vivid color change from deep blue to red. The recoveries of OTA-spiked corn samples indicate the high accuracy and robustness of the proposed method. In conclusion, our proposed strategy provides a promising method for eco-friendly and sensitive POC screening of OTA. Moreover, it can be easily applied to other analytes by changing the involved specific mimotope sequence.

"Three-in-One" Multifunctional Nanohybrids with Colorimetric Magnetic Catalytic Activities to Enhance Immunochromatographic Diagnosis.

Colorimetric lateral flow immunoassay (LFIA) with gold nanoparticles (AuNPs) as signal reporters has been widely used in point-of-care testing. Nonetheless, the potential of traditional AuNP-based LFIA for the early diagnosis of disease is often compromised by limited sensitivity due to the insufficient colorimetric signal brightness of AuNPs. Herein, we develop a "three-in-one" multifunctional catalytic colorimetric nanohybrid (Fe3O4@MOF@Pt) composed of Fe3O4 nanoparticles, MIL-100(Fe), and platinum (Pt) nanoparticles. Fe3O4@MOF@Pt displays enhanced colorimetric signal brightness, fast magnetic response, and ultrahigh peroxidase-mimicking activity, which are beneficial to the enhancement of the sensitivity of LFIA by coupling with magnetic separation and catalytic amplification. When integrated with the dual-antibody sandwich LFIA platform, the developed Fe3O4@MOF@Pt can achieve an ultrasensitive immunochromatographic assay of procalcitonin with a sensitivity of 0.5 pg mL-1, which is approximately 2280-fold higher than that of conventional AuNP-based LFIA and superior to previously published immunoassays. Therefore, this work suggests that the proposed catalytic colorimetric nanohybrid can act as promising signal reporters to enable ultrasensitive immunochromatographic disease diagnostics.

M13 bacteriophage as biometric component for orderly assembly of dynamic light scattering immunosensor.

The ordered assembly of nanostructure is an effective strategy used to manipulate the hydrodynamic diameter (DH) of nanoparticles. Herein, a versatile dynamic light scattering (DLS) immunosensing platform is presented to sensitively detect small molecules and biomacromolecules by using the M13 phage as the building module to order the assembly of gold nanoflowers and gold-coated magnetic nanoparticles, respectively. After the directional assembly of M13 phage, the DH of the probes was significantly increased due to its larger filamentous structure, thus improving the detection sensitivity of the DLS immunosensor. The designed M13 assembled DLS immunosensor with competitive and sandwich formats showed high sensitivities for ochratoxin A and alpha-fetoprotein in real corn and undiluted serum samples, with the detection limits of 1.37 and 57 pg/mL, respectively. These values are approximately 15.8 and 164.9 times lower than those of traditional phage-based enzyme-linked immunosorbent assays. Collectively, this work provides a promising strategy to manipulate the DH of nanoparticles by highly evolved biomaterials such as engineered M13 phages and opens upon a new direction for developing DLS immunosensors to detect various targets by the fusion expression of special peptide or nanobody on the pIII or pVIII protein of M13 phage.

Recent advances in colorimetry/fluorimetry-based dual-modal sensing technologies.

Tailored to the increasing demands for sensing technologies, the fabrication of dual-modal sensing technologies through combining two signal transduction channels into one method has been proposed and drawn considerable attention. The integration of two sensing signals not only promotes the analytical efficiency with reduced assumption, but also improves the analytical performances with enlarged detection linear range, enhanced accuracy, and boosted application flexibility. The two top-rated output signals for developing dual-modal sensors are colorimetric and fluorescent signals because of their outstanding merits for point of care applications and real-time sensitive sensing. Given the rapid development of material chemistry and nanotechnology, the recent decade has witnessed great advance in colorimetric/fluorimetric signal based dual-modal sensing technologies. The new sensing strategy leads to a broad avenue for various applications in disease diagnosis, environmental monitoring and food safety because of the complementary and synergistic effects of the two output signals. In this state-of-the-art review, we comprehensively summarize different types of colorimetric/fluorimetric dual-modal sensing methods by highlighting representative research in the last 5 years, digging into their sensing methodologies, particularly the working principles of the signal transduction systems. Then, the challenges and future prospects for boosting further development of this research field are discussed.

Integrated nanoparticle size with membrane porosity for improved analytical performance in sandwich immunochromatographic assay.

The use of luminescent nanobeads to improve the sensitivity of sandwich immunochromatographic assay (ICA) has obtained increasing concern. Illustrating the relationship among luminescent intensity, nanobead size, nitrocellulose membrane aperture, and ICA sensitivity is important for achieving the optimal target detection. Thus, we synthesized six differently sized quantum dot beads (QBs) (95, 140, 180, 235, 325, and 405 nm) as ICA labels and applied them in three aperture membranes (10, 15, and 25 µm). Results indicate that increasing the QB size to less than an appropriate size of 235 nm is beneficial for ICA sensitivity because of the increased fluorescence. However, oversized QBs result in reduced sensitivity due to the decreased diffusion or settlement of the QB on the membrane that causes obvious background signal. The small aperture membrane perfectly matching the QB size contributes to ICA sensitivity by decreasing the migration velocity of the QB probe for increased binding of the QB@analyte complex at the T zone. Consequently, the best detection of hepatitis B surface antigen with a sensitivity of 0.156 ng/mL is achieved using 235 nm QBs in 15 µm membrane. Further performance evaluation of our QB235-ICACN95 demonstrates excellent accuracy, selectivity, and practicability. This work provides a new idea to manipulate the sensitivity of sandwich ICA by tuning the QB size and the membrane aperture, and a theoretical guidance for selecting the probe and membrane to achieve the best detection of target analytes.

Gold Nanobeads with Enhanced Absorbance for Improved Sensitivity in Competitive Lateral Flow Immunoassays.

BACKGROUND: Colloidal gold based lateral flow immunoassay (LFIA) commonly suffers from relatively low detection sensitivity due to the insufficient brightness of conventional gold nanoparticles (AuNPs) with the size of 20-40 nm. METHODS: Herein, three kinds of gold nanobeads (GNBs) with the size of 94 nm, 129 nm, and 237 nm, were synthesized by encapsulating numerous hydrophobic AuNPs (10 nm) into polymer matrix. The synthesized GNBs exhibited the enhanced colorimetric signal intensity compared with 20-40 nm AuNPs. The effects of the size of GNBs on the sensitivity of LFIA with competitive format were assessed. RESULTS: The results showed that the LFIA using 129 nm GNBs as amplified signal probes exhibits the best sensitivity for fumonisin B1 (FB1) detection with a cut-off limit (for visual qualitative detection) at 125 ng/mL, a half maximal inhibitory concentration at 11.27 ng/mL, and a detection limit at 1.76 ng/mL for detection of real corn samples, which are 8-, 3.82-, and 2.89-fold better than those of conventional AuNP40-based LFIA, respectively. The developed GNB-LFIA exhibited negligible cross-reactions with other common mycotoxins. In addition, the accuracy, precision, reliability, and practicability were demonstrated by determining real corn samples. CONCLUSIONS: All in all, the proposed study provides a promising strategy to enhance the sensitivity of competitive LFIA via using the GNBs as amplified signal probes.

Dramatically Enhancing the Sensitivity of Immunoassay for Ochratoxin A Detection by Cascade-Amplifying Enzyme Loading.

Enzyme-linked immunosorbent assay (ELISA) is widely used in the routine screening of mycotoxin contamination in various agricultural and food products. Herein, a cascade-amplifying system was introduced to dramatically promote the sensitivity of an immunoassay for ochratoxin A (OTA) detection. Specifically, a biotinylated M13 bacteriophage was introduced as a biofunctional competing antigen, in which a seven-peptide OTA mimotope fused on the p3 protein of M13 was used to specifically recognize an anti-OTA monoclonal antibody, and the biotin molecules modified on capsid p8 proteins were used in loading numerous streptavidin-labeled polymeric horseradish peroxidases (HRPs). Owing to the abundance of biotinylated p8 proteins in M13 and the high molar ratio between HRP and streptavidin in streptavidin-polyHRP, the loading amount of HRP enzymes on the M13 bacteriophage were greatly boosted. Hence, the proposed method exhibited high sensitivity, with a limit of detection of 2.0 pg/mL for OTA detection, which was 250-fold lower than that of conventional ELISA. In addition, the proposed method showed a slight cross-reaction of 2.3% to OTB, a negligible cross-reaction for other common mycotoxins, and an acceptable accuracy for OTA quantitative detection in real corn samples. The practicability of the method was further confirmed with a traditional HRP-based ELISA method. In conclusion, the biotinylated bacteriophage and polyHRP structure showed potential as a cascade-amplifying enzyme loading system for ultra-trace OTA detemination, and its application can be extended to the detection of other analytes by altering specific mimic peptide sequences.

Eco-Friendly Fluorescent ELISA Based on Bifunctional Phage for Ultrasensitive Detection of Ochratoxin A in Corn.

Conventional enzyme-linked immunosorbent assay (ELISA) is commonly used for Ochratoxin A (OTA) screening, but it is limited by low sensitivity and harmful competing antigens of enzyme-OTA conjugates. Herein, a bifunctional M13 bacteriophage with OTA mimotopes fused on the p3 protein and biotin modified on major p8 proteins was introduced as an eco-friendly competing antigen and enzyme container for enhanced sensitivity. Mercaptopropionic acid-modified quantum dots (MPA-QDs), which are extremely sensitive to hydrogen peroxide, were chosen as fluorescent signal transducers that could manifest glucose oxidase-induced fluorescence quenching in the presence of glucose. On these bases, a highly sensitive and eco-friendly fluorescent immunoassay for OTA sensing was developed. Under optimized conditions, the proposed method demonstrates a good linear detection of OTA from 4.8 to 625 pg/mL and a limit of detection (LOD) of 5.39 pg/mL. The LOD is approximately 26-fold lower than that of a conventional horse radish peroxidase (HRP) based ELISA and six-fold lower than that of a GOx-OTA conjugate-based fluorescent ELISA. The proposed method also shows great specificity and accepted accuracy for analyzing OTA in real corn samples. The detection results are highly consistent with those obtained using the ultra-performance liquid chromatography-fluorescence detection method, indicating the high reliability of the proposed method for OTA detection. In conclusion, the proposed method is an excellent OTA screening platform over a conventional ELISA and can be easily extended for sensing other analytes by altering specific mimic peptide sequences in phages.