Lateral Flow Immunoassay Based on Quantum-Dot Nanobeads for Detection of Chloramphenicol in Aquatic Products.

Quantum dot nanobeads (QBs) were used as signal source to develop competitive lateral flow immunoassay (LFIA) for the detection of chloramphenicol (CAP). The quantitative detection of CAP was achieved by calculating the total color difference (∆E) values of the test line (T line) using the images of test strips. QB-based LFIA (QBs-LFIA) allowed the effective dynamic linear detection of CAP in the range of 0.1-1.5 ng/mL. The limit of detection (LOD) was 3.0 ng/mL, which was 50 and 667 times lower than those achieved for two different brands of colloidal gold kits. The recoveries of CAP during real-sample detection were 82.82-104.91% at spiked levels of 0.1, 0.7, and 1.5 ng/mL. These results indicate that the developed QBs-LFIA facilitates the sensitive detection of CAP.

CRISPR-based quantum dot nanobead lateral flow assay for facile detection of varicella-zoster virus.

Varicella-zoster virus (VZV) infects more than 90% of the population worldwide and has a high incidence of postherpetic neuralgia in elderly patients, seriously affecting their quality of life. Combined with clustered regularly interspaced short palindromic repeats (CRISPR) system, we develop a quantum dot nanobeads (QDNBs) labeled lateral flow assay for VZV detection. Our assay allows the identification of more than 5 copies of VZV genomic DNA in each reaction. The entire process, from sample preparation to obtaining the results, takes less than an hour. In 86 clinical vesicles samples, the test shows 100% concordance with quantitative real-time PCR for VZV detection. Notably, when vesicles are present in specific areas, such as the genitals, our method outperforms clinical diagnosis. Compared to traditional detection methods, only a minute amount of blister fluid is required for accurate detection. Therefore, we anticipate that our method could be translated to clinical applications for specific and rapid VZV detection. KEY POINTS: • CRISPR/Cas12a and quantum dot nanobead-based lateral flow assay achieved 5 copies per reaction for VZV detection • Specific identification of VZV in atypical skin lesions • Results read by the naked eye within one hour.

Ratiometric fluorescent immunochromatography for simultaneously detection of two nitrofuran metabolites in seafoods.

Competitive immunochromatographic assay (ICA) has been widely used in food safety on-site monitoring. However, traditional competitive ICA relies on the change of single-signal intensity to discriminate the results, leading to limited sensitivity and accuracy of naked-eye visual inspection. In this study, we developed a sensitive ratiometric fluorescent immunochromatographic assay (RFICA) judging results based on the color changes of fluorescence. Two different color quantum dot nanobeads (QBs) were introduced as signal labels for rapid and accurate detection of 3-amino-2-oxazolidinone (AOZ) and semicarbazide (SEM) in seafoods. Under the optimal conditions, the visual detection limit for 2-NPAOZ and 2-NPSEM reached up to 1 and 0.5 ng/mL, which were 5- and 14-fold lower than that of traditional single-signal response fluorescent ICA strips. In addition, the RFICA strips were applied to real samples with acceptable recoveries of 81.33 âˆ¼ 99.23 %. These results demonstrate that the developed RFICA platform possesses considerable potential in the on-site detection.

Quantum Dot Nanobeads as Multicolor Labels for Simultaneous Multiplex Immunochromatographic Detection of Four Nitrofuran Metabolites in Aquatic Products.

A multicolor immunochromatographic assay platform based on quantum dot nanobeads (QBs) for the rapid and simultaneous detection of nitrofuran metabolites in different aquatic products is documented. These metabolites include 3-amino-2-oxazolidinone (AOZ), 1-aminohydantoin (AHD), semicarbazide (SEM), and 3-amino-5-morpholino-methyl-1,3-oxazolidinone (AMOZ). QBs with emission colors of red, yellow, green, and orange were employed and functionalized with the corresponding antibodies to each analyte to develop a multicolor channel. The visual detection limits (cutoff values) of our method for AOZ, AHD, SEM, and AMOZ reached up to 50 ng/mL, which were 2, 20, 20, and 20 times lower than those of traditional colloidal gold test strips, respectively. The test strip is capable of detection within 10 min in real samples while still achieving good stability and specificity. These results demonstrate that the developed multicolor immunochromatographic assay platform is a promising technique for multiplex, highly sensitive, and on-site detection of nitrofuran metabolites.

An integrated fluorescent lateral flow assay for multiplex point-of-care detection of four respiratory viruses.

This work established a highly sensitive and specific quantum dot nanobeads-based lateral flow assay for multiplex detection of four respiratory virus markers at point of care. The respiratory virus antigens were detected by fluorescent lateral flow strips within 20 min. The limits of detection for SARS-CoV-2 antigen, IAV antigen, IBV antigen, and ADV antigen were 0.01 ng/mL, 0.05 ng/mL, 0.31 ng/mL, and 0.40 ng/mL, respectively, which were superior to that of conventional AuNPs-based colorimetric lateral flow assay. The coefficients of variation of the test strip were 6.09%, 2.24%, 7.92%, and 12.43% for these four antigens, which indicated that the proposed method had good repeatability. The specificity of the detection system was verified by different combinations of these four respiratory viruses and several other respiratory pathogens. These results indicated that this method could simultaneously detect SARS-CoV-2, IAV, IBV and ADV in a short assay time, showing the remarkable potential for the rapid and multiplex detection of respiratory viruses in resource-limited settings.

Dual quantum dot nanobeads-based fluorescence-linked immunosorbent assay for simultaneous detection of aflatoxin B1 and zearalenone in feedstuffs.

A novel dual quantum dot nanobeads-based fluorescence-linked immunosorbent assay (QBs-FLISA) was successfully developed for simultaneously detecting aflatoxin B1 (AFB1) and zearalenone (ZEN) in feedstuffs. Dual CdSe/ZnS quantum dot nanobeads with different diameters that emit red and green fluorescence were conjugated with anti-AFB1 and anti-ZEN monoclonal antibodies to prepare fluorescent probes, which greatly enhance analytical performance. Under the optimal conditions, the limits of detection for AFB1 and ZEN were 9.3 and 102.1 pg mL-1, respectively. The recoveries ranged from 82.50% to 116.21% with relative standard deviation less than 11.3%. Compared with traditional enzyme-linked immunosorbent assay, detection sensitivities of AFB1 and ZEN using QBs-FLISA were increased 20 and 5 folds, respectively. In addition, results of feedstuff samples analyzed by QBs-FLISA and liquid chromatography tandem mass spectrometry showed a good agreement (R2 = 0.99).

Quantum Dot Nanobeads Based Fluorescence Immunoassay for the Quantitative Detection of Sulfamethazine in Chicken and Milk.

Sulfamethazine (SMZ) as a broad antibiotic is widely used in livestock and poultry. However, the abuse of SMZ in livestock feed can lead to SMZ residues in food and the resistance of bacteria to drugs. Thus, a method for the detection of SMZ in food is urgently needed. In this study, quantum dot (QD) nanobeads (QBs) were synthesized by encapsulating CdSe/ZnS QDs using a microemulsion technique. The prepared QBs as signal probes were applied in lateral flow immunoassay (LFIA) for the detection of SMZ in chicken and milk. Our proposed method had limits of detection of 0.1138-0.0955 ng/mL and corresponding linear ranges of 0.2-12.5, 0.1-15 ng/mL in chicken and milk samples, respectively. The recovery of LFIA for the detection of SMZ was 80.9-109.4% and 84-101.6% in chicken and milk samples, respectively. Overall, the developed QBs-LFIA had high reliability and excellent potential for rapid and sensitive screening of SMZ in food.

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.

Development of a quantum dot nanobead-based fluorescent strip immunosensor for on-site detection of aflatoxin B1 in lotus seeds.

Owing to the serious threat of aflatoxin B1 (AFB1) to public health, development of a reliable method for accurate determination of it is extremely necessary and urgent. In this study, a simple, rapid and highly-sensitive quantum dot nanobeads (QBs) based lateral flow fluorescent strip immunosensor was developed for on-site detection of AFB1 in edible and medicinal lotus seeds. Carboxylated QBs were used as the fluorescent markers to prepare the fluorescent probe through coupling QBs with anti-AFB1 antibodies. Bovine serum albumin (BSA)-AFB1 antigens and goat anti-mouse IgG antibodies were coated on the nitrocellulose (NC) membrane to prepare the test (T) and control (C) lines, respectively. Qualitative analysis of AFB1 was realized by naked eye, and the quantitative determination was achieved with a portable strip reader. Results showed that the newly-developed test strip sensor could achieve rapid detection of AFB1 within 15 min, allowing a limit of detection (LOD) of 1 ng/mL (2 µg/kg) and a linear range of 1-19 ng/mL (2-38 µg/kg). Recovery rates from the fortified lotus seeds with low, medium and high spiking concentrations (2.5, 5 and 10 µg/kg) ranged from 94.0% to 116.0% with relative standard deviations less than 10%. All the results were confirmed by a standard LC-MS/MS method. The QBs-based fluorescent strip immunosensor with high sensitivity, easy operation, and low cost provided a preferred solution for rapid, on-site screening and highly-sensitive quantitation of AFB1 in a large number of lotus seed samples.

Sensitive Immunochromatographic Assay Using Highly Luminescent Quantum Dot Nanobeads as Tracer for the Detection of Cyproheptadine Hydrochloride in Animal-Derived Food.

Cyproheptadine hydrochloride (CYP), used as human and veterinary drug, has been used illegally as feed additive for food-producing animals, which could remain in food and jeopardize human health. There is a need for on-site detection of CYP residue in animal-derived food. In this study, a hapten was designed, and a specific monoclonal antibody (mAb) was developed to detect CYP with an IC50 of 1.38 ng/mL and negligible cross-reactivity (CR) for other analogs. Forthermore, a high sensitive immunochromatographic assay (QBs-ICA) was developed using quantum dot nanobeads as reporters. The assay showed the linear detection range (IC20-IC80) of 0.03-0.52 ng/mL, the limit of detection (LOD) and visual detection limit (VDL) reached to 0.01 and 0.625 ng/mL, respectively. Spiked recovery study in pig urine and pork confirmed that the QBs-ICA was applicable for on-site testing. This assay showed better sensitivity and speedy than the reported instrumental analysis and immunoassays.

Comparative Study of Time-Resolved Fluorescent Nanobeads, Quantum Dot Nanobeads and Quantum Dots as Labels in Fluorescence Immunochromatography for Detection of Aflatoxin B1 in Grains.

Label selection is an essential procedure for improving the sensitivity of fluorescence immunochromatography assays (FICAs). Under optimum conditions, time-resolved fluorescent nanobeads (TRFN), quantum dots nanobeads (QB) and quantum dots (QD)-based immunochromatography assays (TRFN-FICA, QB-FICA and QD-FICA) were systematically and comprehensively compared for the quantitative detection of aflatoxin B1 (AFB1) in six grains (corn, soybeans, sorghum, wheat, rice and oat). All three FICAs can be applied as rapid, cost-effective and convenient qualitative tools for onsite screening of AFB1; TRFN-FICA exhibits the best performance with the least immune reagent consumption, shortest immunoassay duration and lowest limit of detection (LOD). The LODs for TRFN-FICA, QB-FICA and QD-FICA are 0.04, 0.30 and 0.80 µg kg-1 in six grains, respectively. Recoveries range from 83.64% to 125.61% at fortified concentrations of LOD, 2LOD and 4LOD, with the coefficient of variation less than 10.0%. Analysis of 60 field grain samples by three FICAs is in accordance with that of LC-MS/MS, and TRFN-FICA obtained the best fit. In conclusion, TRFN-FICA is more suitable for quantitative detection of AFB1 in grains when the above factors are taken into consideration.

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.

[Vancomycin-based fluorescent enzyme-linked immunoabsorbent assay for detection of Staphylococcus aureus].

In the study, fluorescent enzyme-linked immnoabsorbent assay for detection of Staphylococcus aureus was established with IgG from pig as capture antibody and quantum dot nanobeads (QBs) labeled vancomycin (QB-Vans) as testing antibody. Quantum dot of about 100 nm partical size nanobeads were prepared and linked with vancomycin. The optimum concentrations of salt ions were 0.01 mol/L, and the optimum pH was 6.0. Under the optimum conditions, the detection sensitivity for S. aureus was 104 CFU/mL, and there was no cross-reaction with other pathogenic bacteria. Thus, the method could be used for rapid screening of S. aureus, for the clinical monitoring and foodborne pathogens detection.

Vancomycin-based fluorescent enzyme-linked immunoabsorbent assay for detection of Staphylococcus aureus / 生物工程学报

In the study, fluorescent enzyme-linked immnoabsorbent assay for detection of Staphylococcus aureus was established with IgG from pig as capture antibody and quantum dot nanobeads (QBs) labeled vancomycin (QB-Vans) as testing antibody. Quantum dot of about 100 nm partical size nanobeads were prepared and linked with vancomycin. The optimum concentrations of salt ions were 0.01 mol/L, and the optimum pH was 6.0. Under the optimum conditions, the detection sensitivity for S. aureus was 10⁴ CFU/mL, and there was no cross-reaction with other pathogenic bacteria. Thus, the method could be used for rapid screening of S. aureus, for the clinical monitoring and foodborne pathogens detection.