Dual-mode immunochromatographic assay based on dendritic gold nanoparticles with superior fluorescence quenching for ultrasensitive detection of E. coli O157:H7.

We presented a colorimetric/fluorescent dual-mode immunochromatographic assay (ICA) for the sensitive detection of Escherichia coli O157:H7. The use of polydopamine (PDA)-modified gold nanoparticles (AuNPs) with broadband absorption allowed for excellent colorimetry signals for the ICA detection. Moreover, the absorption spectrum of PDA-AuNPs significantly overlaps with the excitation and emission spectra of ZnCdSe/ZnS quantum dots (QDs), resulting in effective quenching of the QDs fluorescence due to the inner filter effect. The fluorescence intensity changes induced by PDA-AuNPs were utilized for the sensitive detection of E. coli O157:H7, achieving a detection limit of 9.06 × 101 CFU/mL, which was 46-fold lower than that of traditional AuNPs-based immunoassay. The proposed immunosensor exhibited the recovery rate between 80.12% and 114.69% in detecting actual samples, indicating its reliability and satisfactory accuracy. This study provides insights into dual-mode signal outputs and the ICA development for food safety applications.

Synthesizing Submicron Polyelectrolyte Capsules to Boost Enzyme Immobilization and Enhance Enzyme-Based Immunoassays.

Polyelectrolyte capsules (PCs) exhibit attractive superiorities in enzyme immobilization, including providing a capacious microenvironment for enzyme conformational freedom, highly effective mass transfer, and protecting enzymes from the external environment. Herein, we provide the first systemic evaluation of submicron PCs (SPCs, 500 nm) for enzyme immobilization. The catalytic kinetics results show that SPC encapsulation affected the affinities of enzymes and substrates but significantly enhanced their catalytic activity. The stability test indicates that SPC-encapsulated horseradish peroxidase (HRP) exhibits ultrahigh resistance to external harsh conditions and has a longer storage life than that of soluble HRP. The proposed encapsulation strategy enables 7.73-, 2.22-, and 11.66-fold relative activities when working at a pH as low as 3, at a NaCl concentration as high as 500 mM, and at a trypsin concentration as high as 10 mg/mL. We find that SPC encapsulation accelerates the cascade reaction efficiency of HRP and glucose oxidase. Owing to SPCs enhancing the catalytic activity of the loaded enzymes, we established an amplified enzyme-linked immunosorbent assay (ELISA) for the detection of Escherichia coli O157:H7 using HRP-loaded SPCs. The detection sensitivity of SPC-improved ELISA was found to be 280 times greater than that of conventional HRP-based ELISA. Altogether, we provide an elaborate evaluation of 500 nm SPCs on enzyme immobilization and its application in the ultrasensitive detection of foodborne pathogens. This evaluation provides evidence to reveal the potential advantage of SPCs on enzyme immobilization for enzyme-based immunoassays. It has excellent biological activity and strong stability and broadens the application prospect in urine, soy sauce, sewage, and other special samples.

Controlled PAH-mediated method with enhanced optical properties for simple, stable immunochromatographic assays.

Despite their potential for signal amplification in immunochromatographic assays (ICAs) with Au nanoparticles (AuNPs) as probes, metal growth methods are of limited practical applicability given their complex non-specificity and lack of robust growth schemes. Here, we propose a novel method of polyallylamine hydrochloride (PAH)-mediated metal growth for the detection of Escherichia coli O157:H7 by AuNP-ICA. The developed method relies on the highly controlled growth of Cu shells on the AuNP core and allows one to achieve highly enhanced colorimetric signals by controlling PAH as the growth framework. The introduction of PAH eliminates the non-specific adsorption of Cu ions on the nitrocellulose membrane and thus provides maximized and effective signal-to-noise ratios to achieve a detection limit of 9.8 CFU/mL for E. coli O157:H7. Moreover, the newly developed detection method exhibits good reproducibility (coefficient of variation <13%), remarkable stability, and practical applicability. The PAH-mediated signal enhancement system paves the way to the realization of stable metal growth methods based on Au, Ag, and other metals and is well suited for the rapid, stable, and sensitive detection of food-borne pathogens using the AuNP-ICA platform.

Development of a high resolution melting method based on a novel molecular target for discrimination between Bacillus cereus and Bacillus thuringiensis.

Delimitation within the Bacillus cereus group is confusing due to the highly similar genetic background of its constituent bacteria. This study aimed to develop a rapid and efficient method for the identification of Bacillus cereus and Bacillus thuringiensis, two closely related species within the B. cereus group. Using average nucleotide identity analysis (ANI) and ribosomal multilocus sequence typing (rMLST), the authenticity of the genomes of B. cereus and B. thuringiensis was determined. Emetic B. cereus and Bacillus bombysepticus were also included to provide novel genomic insights into the boundaries within the B. cereus group. Using pan-genome analysis, ispD, a novel core and single-copy molecular target, was identified for the differentiation between B. cereus and B. thuringiensis. Based on the single nucleotide polymorphism within ispD, a high resolution melting (HRM) method for the determination of B. cereus and B. thuringiensis was developed. This method can not only distinguish B. cereus and B. thuringiensis, but can also separate B. cereus from other foodborne pathogenic bacteria. The detection limit of this method could reach 1 pg of pure genomic DNA and 3.7 × 102 cfu/mL of pure culture. Moreover, this new method could effectively differentiate B. cereus and B. thuringiensis in spiked, mixed, and real food samples. Collectively, the established HRM method can provide a new reference paradigm for the sensitive and specific nucleic acid detection of pathogens with identical genomes.

Recent advances in enzyme-enhanced immunosensors.

Among the products for rapid detection in different fields, enzyme-based immunosensors have received considerable attention. Recently, great efforts have been devoted to enhancing the output signals of enzymes through different strategies that can significantly improve the sensitivity of enzyme-based immunosensors for the need of practical applications. In this manuscript, the significance of enzyme-based signal transduction patterns in immunoassay and the central role of enzymes in achieving precise control of reaction systems are systematically described. In view of the rapid development of this field, we classify these strategies based on the combination of immune recognition and enzyme amplification into three categories, namely enzyme-based enhancement strategies, combination of the catalytic amplification of enzymes with other signal amplification methods, and substrate-based enhancement strategies. The current focus and future direction of enzyme-based immunoassays are also discussed. This article is not exhaustive, but focuses on the latest advances in different signal generation methods based on enzyme-initiated catalytic reactions and their applications in the detection field, which could provide an accessible introduction of enzyme-based immunosensors for the community with a view to further improving its application efficiency.

Quantitative detection of aflatoxin B1 using quantum dots-based immunoassay in a recyclable gravity-driven microfluidic chip.

To achieve rapid and sensitive detection of aflatoxin B1 (AFB1), we developed a polydimethylsiloxane gravity-driven cyclic microfluidic chip using the two-signal mode strategy. The structural design of the chip, together with the two-wavelength quantum dot ratio fluorescence, effectively eliminates the influence of environmental factors, improves the signal stability, and ensures that the final detection result positively correlates with the target concentration. Moreover, the theoretical analysis performed for the established physical model of the three-dimensional reaction interface inside the chip confirmed the improved reaction rate of immune adsorption in the microfluidic strategy. Overall, the method exhibited a wide analytic range (0.2-500 ng mL-1), low detection limit (0.06 ng mL-1), high specificity, good precision (coefficient of variation < 5%), excellent reusability (20 times, 89.1%) and satisfactory practical sample analysis capacity. Furthermore, the reusability and designability of this chip provide a reliable scheme for field detection of AFB1, analysis of other small molecules, and establishment of high-throughput detection systems under different conditions.

Hydrazide-assisted directional antibody conjugation of gold nanoparticles to enhance immunochromatographic assay.

The analytical performance of immunochromatographic assay (ICA) is usually determined by the biological activity of antibody and gold nanoparticle conjugates (AuNP probes). However, conventional probes are constructed using the nondirectional coupling method that can cause the improper orientation of antibodies with the poor accessibility of antigen-binding sites. To address these issues, we report a site-specific directional coupling strategy to enhance the bioactivity of AuNP probes through the specific covalent binding of the aldehyde group in the Fc domain of antibodies with the hydrazide group modified on the surface of AuNPs. Through this design, the antibodies can be erected on the AuNP surface to fully expose the Fab domain and achieve the maximized functional availability. Leveraging these AuNP probes as ICA labels, we demonstrate an improved detection of the hepatitis B surface antigen with less used amount of labeled antibody (0.2 mg/pmol AuNPs), shorter reaction time (10 min), better antibody bioactivity, and higher detection sensitivity (2 ng/mL) compared with the carbodiimide method. Overall, this work provides great promise for the design and the construction of high-performance probes to enhance the detection performance of ICA sensors.

Biotin-Streptavidin System-Mediated Ratiometric Multiplex Immunochromatographic Assay for Simultaneous and Accurate Quantification of Three Mycotoxins.

The quantitative multiplex immunochromatographic assay (mICA) has received an increasing amount of attention in multitarget detection. However, the quantitative results in the reported mICAs were obtained by recording the signals on the test lines that with which various analyte-independent factors readily interfere, resulting in inaccurate quantitation. The ratiometric strategy using the T/C value (ratios of signals on the test line to those of the control line) for signal correction can effectively circumvent these issues to enable more accurate detection. Herein, we present for the first time a novel ratiometric mICA strip with multiple T lines for the simultaneous quantitative detection of aflatoxin B1 (AFB1), fumonisin B1 (FB1), and ochratoxin A (OTA) using highly luminescent quantum dot nanobeads (QBs) as enhanced signal reporters. To achieve reliable ratiometric signal output, a biotin-streptavidin system was introduced to replace the conventional anti-mouse IgG antibody for reliable reference signals on the control line that are completely independent of the signal probe and analyte. By using stable T/C values as quantitative signals, our proposed QB-mICA method can successfully detect three mycotoxins with concentrations as low as 1.65 pg/mL for AFB1, 1.58 ng/mL for FB1, and 0.059 ng/mL for OTA. The detection performance of the developed QB-mICA strip, including precision, specificity, and reliability, was further evaluated using artificially contaminated cereal samples. The results demonstrate the improved accuracy and reliability of quantitative determination by comparison with the anti-mouse IgG antibody. Thus, this work provides a promising strategy for developing a ratiometric mICA method for accurately quantifying multiple analytes using the biotin-SA system, opening up a new direction in quantitative mICAs.

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.

An amphiphilic-ligand-modified gold nanoflower probe for enhancing the stability of lateral flow immunoassays in dried distillers grains.

An amphiphilic ligand-capped gold nanoflower (AuNF) was proposed as a novel lateral flow immunoassay (LFA) reporter for zearalenone (ZEN) detection in distillers dried grains solubles (DDGS). The amphiphilic ligand consists of a thiol-terminated hydrophobic alkane chain, a tetra (ethylene glycol) unit, and a terminal carboxyl group. The novel AuNF probe (N-AuNF-Abs) was prepared by coupling the amino group of anti-ZEN antibodies with the AuNF carboxyl group via an amido covalent linkage. For comparison, a traditional AuNF probe (Tr-AuNF-Abs) was prepared by labeling antibodies on the surface of citrate capped AuNFs via an electrostatic adsorption method. The detection performance of the two probes in LFA was systematically investigated, including the half maximal inhibitory concentration (IC50), robustness and reproducibility for ZEN quantitative detection in DDGS samples, and shelf life. The N-AuNF-Ab based LFA (N-LFA) had a lower IC50 value (15.97 ng mL-1) for ZEN detection in phosphate buffered saline than that of the Tr-AuNF-mAb based LFA (Tr-LFA, 31.06 ng mL-1). The IC50 value of N-LFA in DDGS extract was 17.46 ng mL-1, whereas the Tr-LFA showed poor robustness and reproducibility in DDGS samples, resulting in a failed determination. The intra- and inter-assays of N-LFA for ZEN-spiked DDGS samples indicated that the average recoveries ranged from 93.0% to 125.9%, with coefficients of variation ranging from 2.8% to 21.9%. These results indicated that the N-LFA strip exhibits good robustness and an acceptable accuracy for ZEN quantitative detection in complex DDGS samples. In accelerated aging studies, N-LFA showed a longer shelf life (5 years) than that of Tr-LFA (1 year). In summary, the proposed method provided a novel strategy to prepare a super-stable probe for enhancing the detection performance of LFA for small molecular detection in complex sample matrices such as DDGS.

Quantum dot nanobead-based multiplexed immunochromatographic assay for simultaneous detection of aflatoxin B1 and zearalenone.

Immunochromatographic assay (ICA) is a promising technology for on-site detection. Nonetheless, the wide-scale application of ICA is hindered by several disadvantages, such as poor reproducibility, low sensitivity, and single-target detection. Thus, a novel quantum dot nanobead (QB)-based multiplexed ICA (QB-ICA) with multiple test lines was developed in this study for the simultaneous quantitative detection of aflatoxin B1 (AFB1) and zearalenone (ZEN), where QBs with high luminescence were used as labels to enhance the analytical sensitivity of the ICA. Moreover, a streptavidin (SA)-biotin system, which was undisturbed by the target mycotoxins, was introduced as the signal output for the control line. Consequently, stable and reliable T/C values (ratios of signals on the test line to that of the control line) were obtained as quantitative signals. The proposed QB-ICA demonstrated high sensitivity for the simultaneous detection of AFB1 and ZEN, of which the half-maximal inhibitory concentrations reached as low as 38.98 pg mL-1 and 1.23 ng mL-1, respectively. At 10% competitive inhibition concentration, the limit detections (LOD) were 1.65 and 59.15 pg mL-1 for AFB1 and ZEN, respectively. The average recoveries of the intra- and inter-assays ranged from 81.77% to 119.70% and from 94.18% to 111.4% for AFB1 and ZEN quantification, respectively, and the variation coefficients were less than 12%, thereby indicating that the proposed method is highly accurate and robust. These findings suggest that QB-ICA using SA-biotin system as the signal output of control line is an excellent point-of-care platform for the rapid screening of mycotoxins.

Size-Dependent Immunochromatographic Assay with Quantum Dot Nanobeads for Sensitive and Quantitative Detection of Ochratoxin A in Corn.

Fluorescent microspheres are a novel luminescent nanomaterial proposed as an alternative probe to improve the detection sensitivity of competitive immunochromatographic assay (ICA). Quantum dot nanobeads (QBs) possess strong luminescence and resistance to matrix interference. Theoretically, large-sized QBs exhibit stronger luminescent intensity than small-sized QBs and are beneficial to ICA sensitivity. However, oversized QBs may reduce the sensitivity of competitive ICA. Thus, the relationship between the size and luminescent intensity of QBs and the competitive ICA sensitivity must be elucidated. In this study, QBs of different sizes (58, 124, 255, 365, and 598 nm) were synthesized. Ochratoxin A (OTA) was selected as the model analyte for competitive ICA. The effects of QB size on the detection performance of competitive ICA were then evaluated. The cutoff limit of QB-ICA for naked eye detection was used for qualitative analysis, and the half-maximal inhibitory concentration (IC50) and LOD were employed for quantitative analysis. Results indicated that 124 nm QBs used as labeling probes for competitive ICA showed the optimal detection performance and the lowest cutoff value of 5 ng/mL for qualitative detection and IC50 (0.39 ng/mL) for quantitative detection. Similar to commercial ELISA, QB124-ICA displayed good accuracy, specificity, reproducibility, and practicability. In summary, 124 nm QBs can be used as a new labeling probe for competitive ICA.