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.

High-performance green-emitting AIE nanoparticles for lateral flow immunoassay applications.

Ultrabright green-emissive AIE nanoparticles (AIENPs) were used as signal-amplification probes to enhance the detectability of lateral flow immunoassay (LFIA). The detection performances of the green-emissive AIENP probes in both sandwich and competitive LFIA formats were systematically evaluated. Benefiting from its remarkable fluorescent brightness, the developed AIENP-LFIA showed versatile applicability for the detection of small molecules and macromolecules by using ochratoxin A (OTA) and procalcitonin (PCT) as model analytes, respectively. Under the optimum conditions, the detection limits (LODs) of the fabricated AIENP-LFIA for OTA and PCT were 0.043 ng mL-1 and 0.019 ng mL-1, respectively. These LOD values are significantly lower than those of conventional LFIA methods using gold nanoparticles as signal reporters. In addition, we demonstrated the practical application potential of AIENP-LFIA for the detection of OTA in real maize samples and PCT in real serum samples. These results indicated that the ultrabright green-emissive AIENPs were promising as signal output materials for building high-performance LFIA platform and broadening the application scenarios of LFIA.

Ultrasensitive dynamic light scattering immunosensing platform for NT-proBNP detection using boronate affinity amplification.

Herein, we reported a new dynamic light scattering (DLS) immunosensing technology for the rapid and sensitive detection of glycoprotein N-terminal pro-brain natriuretic peptide (NT-proBNP). In this design, the boronate affinity recognition based on the interaction of boronic acid ligands and cis-diols was introduced to amplify the nanoparticle aggregation to enable highly sensitive DLS transduction, thereby lowering the limit of detection (LOD) of the methodology. After covalently coupling with antibodies, magnetic nanoparticles (MNPs) were employed as the nanoprobes to selectively capture trace amount of NT-proBNP from complex samples and facilitate DLS signal transduction. Meanwhile, silica nanoparticles modified with phenylboronic acid (SiO2@PBA) were designed as the crosslinking agent to bridge the aggregation of MNPs in the presence of target NT-proBNP. Owing to the multivalent and fast affinity recognition between NT-proBNP containing cis-diols and SiO2@PBA, the developed DLS immunosensor exhibited charming advantages over traditional immunoassays, including ultrahigh sensitivity with an LOD of 7.4 fg mL-1, fast response time (< 20 min), and small sample consumption (1 µL). The DLS immunosensor was further characterized with good selectivity, accuracy, precision, reproducibility, and practicability. Collectively, this work demonstrated the promising application of the designed boronate affinity amplified-DLS immunosensor for field or point-of-care testing of cis-diol-containing molecules.

Quantum dot bead-based competitive immunochromatographic assay for enterotoxin aureus A detection in pasteurized milk.

Staphylococcal enterotoxin A (SEA) is an important biotoxin, produced by Staphylococcus aureus under appropriate conditions, and often contaminates milk and dairy products. Herein, an anti-SEA monoclonal antibody (anti-SEA mAb) was prepared by injecting the SEA protein in BALB/c mice, and a novel immunochromatographic assay (ICA) was developed for the rapid and sensitive determination of SEA in pasteurized milk by using highly luminescent quantum dot beads (QB) as signal amplification probe. Given the 1020-fold enhancement in the photoluminescence intensity of QB to the original quantum dot, the proposed QB-ICA exhibits high sensitivity for SEA determination in real milk samples with a limit of detection of 1.89 ng/mL, and shows good dynamic linearity for SEA quantitative detection from 2 to 150 ng/mL within 15 min of test time. The proposed QB-ICA also shows good selectivity to SEA detection with a negligible cross-reaction to common analogs, including staphylococcal enterotoxins B, C, D, and E. In addition, the accuracy and precision of QB-ICA were assessed by analyzing SEA-fortified milk samples. The average recoveries of intra- and interassays range from 85.5 to 128.1%, and the coefficients of variation range from 4.6 to 14.2%, indicating an acceptable accuracy for the quantitative detection of SEA in real milk samples. In summary, this work provides a powerful and rapid analysis tool for the sensitive monitoring of SEA contamination in pasteurized milk samples.

I2/I--mediated fluorescence quenching of an Ag+-doped gold nanocluster-based immunoassay for sensitive detection of Escherichia coli O157:H7 in milk.

Escherichia coli O157:H7 is a type of hazardous bacteria in the field of food safety. A sensitive and effective method is urgently needed to detect it, avoiding enormous harm for the human health. In this study, we synthesized stable Ag+-doped gold nanoclusters (Ag-AuNC) with a fluorescence intensity 4.8 times stronger than that of AuNC. It was further demonstrated that Ag0 existing in the AuNC core and a fraction of Ag+ anchored on the AuNC shell eliminated the surface defects and improved the luminescent properties of AuNC. A combination of I2 and I- was used to quench fluorescence-enhanced Ag-AuNC, which was first applied in ELISA for detecting E. coli O157:H7 to improve the sensitivity. In the presence of E. coli O157:H7, the biotinylated anti-E. coli O157:H7 mAb and streptavidin-alkaline phosphatase would be immobilized and catalyze l-ascorbic acid 2-phosphate sesquimagnesium salt hydrate to produce ascorbic acid. After addition of KIO3, I2/I- were generated. The I2 could trigger oxidative etching of Ag-AuNC and I- could combine with Ag+ to decrease the Ag+ concentration of Ag-AuNC, which resulted in fluorescence quenching of Ag-AuNC. Under optimal conditions, the linear range of I2/I--mediated fluorescence quenching of Ag-AuNC-based immunoassay for detecting E. coli O157:H7 was 3.3 × 103 to 106 cfu/mL, with a detection limit of 9.2 × 102 cfu/mL, 10.7-fold lower than that of the traditional ELISA. The proposed immunoassay exhibits excellent sensitivity, specificity, recovery, and accuracy, which is useful for quantitative detection of E. coli O157:H7 in food safety.

A Universal Boronate-Affinity Crosslinking-Amplified Dynamic Light Scattering Immunoassay for Point-of-Care Glycoprotein Detection.

Herein, we report a universal boronate-affinity crosslinking-amplified dynamic light scattering (DLS) immunoassay for point-of-care (POC) glycoprotein detection in complex samples. This enhanced DLS immunoassay consists of two elements, i.e., antibody-coated magnetic nanoparticles (MNP@mAb) for target capture and DLS signal transduction, and phenylboronic acid-based boronate-affinity materials as crosslinking amplifiers. Upon the addition of targets, glycoproteins are first captured by MNP@mAb and amplified by target-induced crosslinking stemming from the selective binding between the boronic acid ligand and cis-diol-containing glycoprotein, thereby resulting in a remarkably increased DLS signal in the average nanoparticle size. Benefiting from the multivalent binding and fast boronate-affinity reaction between glycoproteins and crosslinkers, the proposed immunosensing strategy has achieved the ultrasensitive and rapid quantitative assay of glycoproteins at the fM level within 15 min. Overall, this work provides a promising and versatile design strategy for extending the DLS technique to detect glycoproteins even in the field or at POC.

Point-of-care COVID-19 diagnostics powered by lateral flow assay.

Since its first discovery in December 2019, the global coronavirus disease 2019 (COVID-19) pandemic caused by the novel coronavirus (SARS-CoV-2) has been posing a serious threat to human life and health. Diagnostic testing is critical for the control and management of the COVID-19 pandemic. In particular, diagnostic testing at the point of care (POC) has been widely accepted as part of the post restriction COVID-19 control strategy. Lateral flow assay (LFA) is a popular POC diagnostic platform that plays an important role in controlling the COVID-19 pandemic in industrialized countries and resource-limited settings. Numerous pioneering studies on the design and development of diverse LFA-based diagnostic technologies for the rapid diagnosis of COVID-19 have been done and reported by researchers. Hundreds of LFA-based diagnostic prototypes have sprung up, some of which have been developed into commercial test kits for the rapid diagnosis of COVID-19. In this review, we summarize the crucial role of rapid diagnostic tests using LFA in targeting SARS-CoV-2-specific RNA, antibodies, antigens, and whole virus. Then, we discuss the design principle and working mechanisms of these available LFA methods, emphasizing their clinical diagnostic efficiency. Ultimately, we elaborate the challenges of current LFA diagnostics for COVID-19 and highlight the need for continuous improvement in rapid diagnostic tests.

Development of a rapid and sensitive quantum dot nanobead-based double-antigen sandwich lateral flow immunoassay and its clinical performance for the detection of SARS-CoV-2 total antibodies.

Owing to the over-increasing demands in resisting and managing the coronavirus disease 2019 (COVID-19) pandemic, development of rapid, highly sensitive, accurate, and versatile tools for monitoring total antibody concentrations at the population level has been evolved as an urgent challenge on measuring the fatality rate, tracking the changes in incidence and prevalence, comprehending medical sequelae after recovery, as well as characterizing seroprevalence and vaccine coverage. To this end, herein we prepared highly luminescent quantum dot nanobeads (QBs) by embedding numerous quantum dots into polymer matrix, and then applied it as a signal-amplification label in lateral flow immunoassay (LFIA). After covalently linkage with the expressed recombinant SARS-CoV-2 spike protein (RSSP), the synthesized QBs were used to determine the total antibody levels in sera by virtue of a double-antigen sandwich immunoassay. Under the developed condition, the QB-LFIA can allow the rapid detection of SARS-CoV-2 total antibodies within 15 min with about one order of magnitude improvement in analytical sensitivity compared to conventional gold nanoparticle-based LFIA. In addition, the developed QB-LFIA performed well in clinical study in dynamic monitoring of serum antibody levels in the whole course of SARS-CoV-2 infection. In conclusion, we successfully developed a promising fluorescent immunological sensing tool for characterizing the host immune response to SARS-CoV-2 infection and confirming the acquired immunity to COVID-19 by evaluating the SRAS-CoV-2 total antibody level in the crowd.

An Auto-Focus Method of Microscope for the Surface Structure of Transparent Materials under Transmission Illumination.

This paper is concerned with auto-focus of microscopes for the surface structure of transparent materials under transmission illumination, where two distinct focus states appear in the focusing process and the focus position is located between the two states with the local minimum of sharpness. Please note that most existing results are derived for one focus state with the global maximum value of sharpness, they cannot provide a feasible solution to this particular problem. In this paper, an auto-focus method is developed for such a specific situation with two focus states. Firstly, a focus state recognition model, which is essentially an image classification model based on a deep convolution neural network, is established to identify the focus states of the microscopy system. Then, an endpoint search algorithm which is an evolutionary algorithm based on differential evolution is designed to obtain the positions of the two endpoints of the region where the real focus position is located, by updating the parameters according to the focus states. At last, a region search algorithm is devised to locate the focus position. The experimental results show that our method can achieve auto-focus rapidly and accurately for such a specific situation with two focus states.

Functional DNA Regulated CRISPR-Cas12a Sensors for Point-of-Care Diagnostics of Non-Nucleic-Acid Targets.

Beyond its extraordinary genome editing ability, the CRISPR-Cas systems have opened a new era of biosensing applications due to its high base resolution and isothermal signal amplification. However, the reported CRISPR-Cas sensors are largely only used for the detection of nucleic acids with limited application for non-nucleic-acid targets. To realize the full potential of the CRISPR-Cas sensors and broaden their applications for detection and quantitation of non-nucleic-acid targets, we herein report CRISPR-Cas12a sensors that are regulated by functional DNA (fDNA) molecules such as aptamers and DNAzymes that are selective for small organic molecule and metal ion detection. The sensors are based on the Cas12a-dependent reporter system consisting of Cas12a, CRISPR RNA (crRNA), and its single-stranded DNA substrate labeled with a fluorophore and quencher at each end (ssDNA-FQ), and fDNA molecules that can lock a DNA activator for Cas12a-crRNA, preventing the ssDNA cleavage function of Cas12a in the absence of the fDNA targets. The presence of fDNA targets can trigger the unlocking of the DNA activator, which can then activate the cleavage of ssDNA-FQ by Cas12a, resulting in an increase of the fluorescent signal detectable by commercially available portable fluorimeters. Using this method, ATP and Na+ have been detected quantitatively under ambient temperature (25 °C) using a simple and fast detection workflow (two steps and <15 min), making the fDNA-regulated CRISPR system suitable for field tests or point-of-care diagnostics. Since fDNAs can be obtained to recognize a wide range of targets, the methods demonstrated here can expand this powerful CRISPR-Cas sensor system significantly to many other targets and thus provide a new toolbox to significantly expand the CRISPR-Cas system into many areas of bioanalytical and biomedical applications.

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.

Gold nanoparticle-decorated metal organic frameworks on immunochromatographic assay for human chorionic gonadotropin detection.

Gold nanoparticle-decorated metal organic frameworks (MOF@AuNPs) with significantly enhanced color signal intensity were synthesized through in situ growth of AuNPs on the MOF skeleton. The resultant MOF@AuNP nanocomposites were characterized with 16.7-fold higher absorbance than conventional 40 nm AuNPs (AuNP40). Thus, for the first time, we applied it as a signal amplification label to improve the immunochromatographic assay (ICA) of human chorionic gonadotropin (HCG). The detection limit of our enhanced ICA was 1.69 mIU/mL, which is ca. 10.6-fold improvement in sensitivity compared to traditional AuNP40-ICA. The recoveries of this MOF@AuNPs-ICA ranged from 86.03 to 119.22%, with coefficients of variation of 3.05 to 13.74%. The reliability and practicability were further validated by the clinically used chemiluminescence immunoassay method. Given their excellent signal amplification ability, the proposed MOF@AuNPs could serve as an ideal ICA label for rapid and sensitive detection of disease biomarkers. Graphical abstract.

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.

Urease-induced metallization of gold nanorods for the sensitive detection of Salmonella enterica Choleraesuis through colorimetric ELISA.

We applied urease-induced silver metallization on the surface of gold nanorods (AuNR) to improve colorimetric ELISA for the rapid and sensitive detection of Salmonella enterica Choleraesuis. To this end, we introduced a biotin-streptavidin system as a bridge to determine the correlation between urease and S. enterica Choleraesuis concentrations. The captured urease can catalyze the hydrolysis of urea into carbon dioxide and ammonia, and the generated ammonia can then induce the deposition of silver shell on the surface of AuNR in the presence of silver nitrate and glucose. With the decreased aspect ratio (length divided by width) of AuNR, a multicolor change of AuNR solution and a significant blue shift in the longitudinal localized surface plasmon resonance absorption peak (Δλmax) of AuNR were obtained at the target concentrations of 1.21 × 101 to 1.21 × 108 cfu/mL. Consequently, the detection limits of our proposed colorimetric ELISA were as low as 1.21 × 102 cfu/mL for qualitative detection with naked eyes, and 1.21 × 101 cfu/mL for quantitative detection, in which changes in Δλmax of AuNR were recorded with a microplate reader. These values were at least 2 to 3 orders of magnitude lower than those obtained with conventional horseradish peroxidase-based ELISA. The analytical performance of our developed colorimetric ELISA in terms of selectivity, accuracy, reliability, and practicability were investigated by analyzing S. enterica Choleraesuis-spiked pasteurized whole milk samples.

Invited review: Advancements in lateral flow immunoassays for screening hazardous substances in milk and milk powder.

Dairy-related food safety outbreaks, such as food-borne pathogen contamination, mycotoxin contamination, and veterinary drug contamination, sometimes happen and have been reported all over the world, affecting human health and, in some cases, leading to death. Thus, rapid yet robust detection methods are needed to monitor milk and milk powder for the presence of hazardous substances. The lateral flow immunoassay (LFI) is widely used in onsite testing because of its rapidity, simplicity, and convenience. In this review, we describe some traditional LFI used to detect hazardous substances in milk and milk powder. Furthermore, we discuss recent advances in LFI that aim to improve sensitivity or detection efficiency. These advances include the use of novel label materials, development of signal amplification systems, design of multiplex detection systems, and the use of nucleic acid-based LFI.

Quantum bead-based fluorescence-linked immunosorbent assay for ultrasensitive detection of aflatoxin M1 in pasteurized milk, yogurt, and milk powder.

Herein, we reported a novel direct competitive fluorescence-linked immunosorbent assay (dcFLISA) for the ultrasensitive detection of aflatoxin M1 (AFM1) in pasteurized milk, yogurt, and milk powder using 150-nm quantum dot beads (QB) as the carrier of competing antigen. Large QB were applied to decrease the binding affinity of the competing antigen to antibody and enhance the fluorescent signal intensity. The aflatoxin B1 molecule was used as the surrogate of AFM1 to label with BSA on the surface of QB because of its 63% cross reaction to anti-AFM1 mAb. The binding affinity of the competing antigen to mAb was tuned by changing the labeled molar ratios of aflatoxin B1 to BSA. Through combining the advantages of QB as the carrier of the competing antigen, including low binding affinity to mAb and highly fluorescent signal output, the proposed dcFLISA exhibited an ultrahigh sensitivity for AFM1 detection, with a half-maximal inhibitory concentration of 3.15 pg/mL in 0.01 M phosphate-buffered saline solution (pH 7.4), which is substantially lower than that of the traditional horseradish peroxidase-based ELISA. The proposed method also exhibited very low detection limitations of 0.5, 0.6, and 0.72 pg/mL for real pasteurized milk, yogurt, and milk powder, respectively. These values are considerably below the maximum permissible level of the European Commission standard for AFM1 in dairy products. In summary, the proposed dcFLISA offers a novel strategy with an ultrahigh sensitivity for the routine monitoring of AFM1 in various dairy products.