Quantum Dot Nanobead-Based Fluorescence-Linked Immunosorbent Assay for Detection of Glycinin in Soybeans and Soy Products.

Soybean glycinin, as a major soybean allergen, is difficult to accurately quantify due to its large molecular weight and complex structure. CdSe/ZnS quantum dot nanobead (QB) is a core/shell fluorescent nanomaterial with strong fluorescent signals and high sensitivity at 630 nm. An immunosorbent assay based on CdSe/ZnS quantum dot nanobeads (QBs-FLISA) was developed for the glycinin quantification in soybean and soybean products. Here, the purified glycinin was coated on the microporous plate to serve as the coating antigen, and CdSe/ZnS nanobead conjugated with anti-glycinin polyclonal antibodies was used as fluorescent detection probe. The target glycinin in the sample and the coated antigen on the plate competitively adsorbed the antibody labeled the CdSe/ZnS QBs probes. The limits of detection and quantitation for glycinin were 0.035 and 0.078 µg mL-1, respectively. The recoveries of the spiked samples ranged from 89.8% to 105.6%, with relative standard deviation less than 8.6%. However, compared with ELISA, the sensitivities of QBs-FLISA for the detection of glycinin were increased by 7 times, and the detection time was shortened by two-thirds. This QBs-FLISA method has been effectively applied to the detection of soybean seeds with different varieties and soy products with different processing techniques, which will provide a rapid screening method for soybean and soybean products with low allergens.

Ultrabright plasmonic fluor nanolabel-enabled detection of a urinary ER stress biomarker in autosomal dominant tubulointerstitial kidney disease.

Autosomal dominant tubulointerstitial kidney disease (ADTKD)-uromodulin (UMOD) is the most common nonpolycystic genetic kidney disease, but it remains unrecognized due to its clinical heterogeneity and lack of screening test. Moreover, the fact that the clinical feature is a poor predictor of disease outcome further highlights the need for the development of mechanistic biomarkers in ADTKD. However, low abundant urinary proteins secreted by thick ascending limb cells, where UMOD is synthesized, have posed a challenge for the detection of biomarkers in ADTKD-UMOD. In the CRISPR/Cas9-generated murine model and patients with ADTKD-UMOD, we found that immunoglobulin heavy chain-binding protein (BiP), an endoplasmic reticulum chaperone, was exclusively upregulated by mutant UMOD in the thick ascending limb and easily detected by Western blot analysis in the urine at an early stage of disease. However, even the most sensitive ELISA failed to detect urinary BiP in affected individuals. We therefore developed an ultrasensitive, plasmon-enhanced fluorescence-linked immunosorbent assay (p-FLISA) to quantify urinary BiP concentration by harnessing the newly invented ultrabright fluorescent nanoconstruct, termed "plasmonic Fluor." p-FLISA demonstrated that urinary BiP excretion was significantly elevated in patients with ADTKD-UMOD compared with unaffected controls, which may have potential utility in risk stratification, disease activity monitoring, disease progression prediction, and guidance of endoplasmic reticulum-targeted therapies in ADTKD.NEW & NOTEWORTHY Autosomal dominant tubulointerstitial kidney disease (ADTKD)-uromodulin (UMOD) is an underdiagnosed cause of chronic kidney disease (CKD). Lack of ultrasensitive bioanalytical tools has hindered the discovery of low abundant urinary biomarkers in ADTKD. Here, we developed an ultrasensitive plasmon-enhanced fluorescence-linked immunosorbent assay (p-FLISA). p-FLISA demonstrated that secreted immunoglobulin heavy chain-binding protein is an early urinary endoplasmic reticulum stress biomarker in ADTKD-UMOD, which will be valuable in monitoring disease progression and the treatment response in ADTKD.

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.

Fluorescence-linked immunosorbent assay for detection of phenanthrene and its homolog.

A competitive fluorescence-linked immunosorbent assay (FLISA) was developed using rhodamine B isothiocyanate (RBITC) as the model fluorescent dye conjugate monoclonal antibody (McAb) for detection of Phe and its homolog (acenaphthene, fluorene, fluoranthene, pyrene and indeno [1,2,3-cd] pyrene) in water samples. The detection range of the assay for Phe was from 2.10 to 91.95 ng/mL. The limit of detection was 1.05 ng/mL, which was approximately 2-fold lower than that of traditional ic-ELISA. Compared with traditional ic-ELISA, more than 70 min was saved because of only one immunoreaction step was needed to accomplish the assay. The average recoveries of Phe and its homolog from domestic water, contaminated water and natural water were 100.7%, 100.8% and 101.2% respectively. The accuracy and precision of the developed FLISA were validated with GC-MS/MS. There were good correlation between the two methods from tap water, contaminated water and river water samples were 0.9994, 0.9935 and 0.9967, respectively. The results suggested that the proposed FLISA could be a potential alternative format for rapid, sensitive, and quantitative detection of Phe and its homolog in environmental water.

Cross-sensitization to Artemisia and Ambrosia pollen allergens in an area located outside of the current distribution range of Ambrosia.

INTRODUCTION: The role of long-distance transported (LDT) Ambrosia pollen in inducing new sensitization and affecting sensitization rates in Artemisia-sensitized patients is unclear. AIM: The aim of this study was to estimate the degree of cross-sensitization to Ambrosia/Artemisia allergens in citizens of Poznan (Western Poland). This area is covered by extensive Artemisia populations but does not currently have local Ambrosia populations. MATERIAL AND METHODS: Sera of 119 patients were tested by fluoroenzyme immunoassay (CAP-FEIA system) against pollen allergen extracts of Artemisia vulgaris and Ambrosia artemisiifolia, an allergenic component of A. vulgaris (nArt v 1), and an allergenic component of A. artemisiifolia (nAmb a 1). Skin prick tests (SPTs, n = 86) were performed with pollen allergen extracts of A. vulgaris and A. artemisiifolia. Artemisia and Ambrosia pollen in ambient air was collected (1996-2013) by a Hirst type volumetric trap sited at roof level (33 m). RESULTS: The SPT showed that the prevalence of sensitization to Ambrosia and Artemisia pollen exceeded 3.5%, and 10.5%, respectively. The measurements of IgE in blood serum (CAP-FEIA) revealed that among Ambrosia-sensitized patients 90.1% (20/22 patients) were concomitantly sensitized to Artemisia. 59.1% (13/22) of these patients reacted to nArt v 1, suggesting primary sensitization to Artemisia pollen. Only 2 (9.1%) patients were mono-sensitized to Ambrosia pollen extract, but surprisingly not to nAmb a 1. CONCLUSIONS: The LDT Ambrosia pollen had a negligible effect on the rate of sensitization to Ambrosia allergens in Poznan and did not increase the prevalence of sensitization to Artemisia pollen in this region. However, the majority of patients showing hypersensitization to Artemisia pollen might also present symptoms during elevated episodes of LDT of Ambrosia pollen.

Development of Fluorescence-Linked Immunosorbent Assay for Icariin.

We have been looking for a faster and simpler method for traditional Chinese medicine and natural product assay. In this study, we developed a fluorescent immunoassay approach to detect icariin (ICA) using a fluorescently labelled monoclonal antibody. The ICA-specific antibody was purified by the caprylic acid-ammonium sulphate method and then labelled with rhodamine B isothiocyanate (RBITC). Subsequently, an indirect competitive fluorescence-linked immunosorbent assay (icFLISA) was developed to detect ICA using RBITC-labelled anti-ICA MAbs. The RBITC-labelled monoclonal antibody was highly specific for ICA. The fluorescence assay demonstrated an effective ICA measurement range of 1.28 ng/mL to 20 µg/mL (R2 = 0.9946) with relative standard deviations below 10% for both intra-assay and inter-assay repeatability and precision. This icFLISA for ICA is simple, rapid, and sensitive, with a 20-fold greater linear range and a 10-fold lower limit of detection than with the previously developed indirect competitive enzyme-linked immunosorbent assay (ELISA). Thus, this study establishes a useful method for detecting ICA, enabling in vivo visualization research. In the future, FLISA can be also used to assay the concentrations of ICA in biological samples, as well as to investigate the pharmacokinetics of ICA in different tissues to explore the targets of ICA in vivo.

Highly sensitive and accurate detection of C-reactive protein by CdSe/ZnS quantum dot-based fluorescence-linked immunosorbent assay.

BACKGROUND: The conventional and widely used enzyme-linked immunosorbent assays (ELISA), due to specificity and high-sensitivity, were suitable in vitro diagnosis. But enzymes are vulnerable to the external conditions, and the complex operation steps limit its application. Semiconductor quantum dots have been successfully used in biological and medical research due to the high photoluminescence and high resistance to photobleaching. In this study, we have developed a novel quantum dot-labeled immunosorbent assay for rapid disease detection of C-reactive protein (CRP). RESULTS: The assay for the detection of CRP can provide a wide analytical range of 1.56-400 ng/mL with the limit of detection (LOD) = 0.46 ng/mL and the limit of quantification = 1.53 ng/mL. The precision of the assay has been confirmed for low coefficient of variation, less than 10% (intra-assay) and less than 15% (inter-assay). The accuracy of assay meets the requirements with the recoveries of 95.4-105.7%. Furthermore, clinical samples have been collected and used for correlation analysis between this FLISA and gold standard Roche immunoturbidimetry. It shows excellent accurate concordance and the correlation coefficient value (R) is as high as 0.989 (n = 34). CONCLUSIONS: This in vitro quantum dot-based detection method offers a lower LOD and a wide liner detection range than ELISA. The total reaction time is only 50 min, which is much shorter than the commercialization ELISA (about 120 min). All of the results show that a convenient, sensitive, and accurate fluorescence-linked immunosorbent assay method has been well established for the detection of CRP samples. Therefore, this method has immense potential for the development of rapid and cost-effective in vitro diagnostic kits.

Development of a Fluorescence-Linked Immunosorbent Assay for Baicalin.

Previously, we developed an indirect competitive enzyme-linked immunosorbent assay (icELISA) for baicalin (BAL) and used this assay to investigate the pharmacokinetics of BAL in mice. In this study, an anti-BAL monoclonal antibody (MAb) was purified by the caprylic acid method and then labelled with fluorescein isothiocyanate (FITC). Subsequently, an indirect competitive fluorescence-linked immunosorbent assay (icFLISA) was developed to detect baicalin (BAL) using FITC-labelled anti-BAL MAbs. Characterization of the assay demonstrated an effective BAL measurement range of 6.4 ng/mL to 500 µg/mL (R(2) = 0.997). The relative standard deviations (RSDs) for both intra-assay and inter-assay repeatability and precision were below 10 %. This icFLISA for BAL is simple, rapid and sensitive, with a 390-fold larger linear range and a 2-fold lower limit of detection (LOD) compared with the previously developed icELISA. We observed a strong correlation between the results determined by the icFLISA and icELISA methods. Overall, this study provides a useful method for detecting BAL in medicines, enabling in vivo visualization research.

Quantification of full and empty particles of adeno-associated virus vectors via a novel dual fluorescence-linked immunosorbent assay.

The adeno-associated virus (AAV) vector is one of the most advanced platforms for gene therapy because of its low immunogenicity and non-pathogenicity. The concentrations of both AAV vector empty particles, which do not contain DNA and do not show any efficacy, and AAV vector full particles (FPs), which contain DNA, are important quality attributes. In this study, a dual fluorescence-linked immunosorbent assay (dFLISA), which uses two fluorescent dyes to quantify capsid and genome titers in a single analysis, was established. In dFLISA, capture of AAV particles, detection of capsid proteins, and release and detection of the viral genome are performed in the same well. We demonstrated that the capsid and genomic titers determined by dFLISA were comparable with those of analytical ultracentrifugation. The FP ratios determined by dFLISA were in good agreement with the expected values. In addition, we showed that dFLISA can quantify the genomic and capsid titers of crude samples. dFLISA can be easily modified for measuring other AAV vector serotypes and AAV vectors with different genome lengths. These features make dFLISA a valuable tool for the future development of AAV-based gene therapies.

Establishment of a Ca(II) ion-quantum dots fluorescence signal amplification sensor for high-sensitivity biomarker detection.

Quantum dots (QDs) have been considered as the promising fluorescent labeling material, which is expected to meet the requirement of high-sensitivity detection in clinical diagnostics. Some common metal ions are known to affect the stability and fluorescence properties of QDs, but scarcely any systematic research has been done about their impacts on QD-based bio-detection. By evaluating the effect of Ca2+ metal ions on the properties of aqueous QDs, a new metal ion-QD fluorescence signal amplification sensor (i.e., Ca2+-QD-fluorescence-linked immunosorbent assay, Ca2+-QD-FLISA) has been developed for the detection of inflammatory biomarkers with high sensitivity. Compared with the common QD-FLISA, the detection sensitivity for CRP of Ca2+-QD-FLISA was improved by a 4-fold of magnitude to 0.23 ng/mL, and this assay showed good selectivity, high accuracy, and excellent repeatability. The versatility of the QD-FLISA method were also validated by using different metal ion-QD probes (Ca2+, Mg2+, Ba2+, Fe2+, and Mn2+) to detect CRP, serum amyloid A (SAA), and procalcitonin (PCT). The significant improvement in detection sensitivity was achieved due to the crosslinking of aqueous QDs by Ca2+ ions to enhance fluorescence and at the same time promote antigen-antibody binding efficiency. The present study illustrates the versatility of metal ion-QD-FLISA as a simple and effective method to detect a wide range of biomarkers with high sensitivity and accuracy.

Establishment of an Immunological Method for Detection of Bluetongue Virus by Fluorescence-Linked Immunosorbent Assay.

Bluetongue (BT) is a severe noncontagious infectious disease that occurs in sheep and wild ruminants but occasionally also in cattle and camels. The worldwide BT pandemic has had a significant impact on global livestock production. Rapid detection helps prevent outbreaks of bluetongue disease. Fluorescence-linked immunosorbent assay (FLISA) labeled with quantum dots (QDs) is typically used for detection due to its high sensitivity. There has been no reported detection of BT virus (BTV) using QD-based fluorescence immunoassays. In this study, monoclonal antibodies (MAbs) against BT were prepared by immunizing BALB/c mice with recombinant VP7 protein. Two MAbs with high sensitivity and specificity were selected as the detection antibody (2F11) and capture antibody (11B7). Then, the detection antibody was coupled with QDs to prepare QD-MAb fluorescence probes. Fluorescence-linked immunosorbent assay is highly specific, detecting only VP7 protein/BTV, and did not show any nonspecific reactions with other reoviruses. The detection limit of VP7 protein was 3.91 ng/mL using fluorescence-linked immunosorbent assay, with a coefficient of variation (CV) of less than 15%. The establishment of rapid, sensitive direct FLISA has potential for bluetongue virus detection and control of BT vaccine quality. IMPORTANCE Bluetongue virus causes the severe infectious disease BT. BTV has many serotypes, and there is no cross-protection among different serotypes. BT is listed as a notifiable animal infectious disease by the World Organisation for Animal Health (OIE) and occurs throughout the world, causing significant economic losses. The establishment of a fast and effective detection method is the key to controlling and preventing this disease. Current methods for detecting BTV mainly include reverse transcription-PCR (RT-PCR), enzyme-linked immunosorbent assays (ELISA), and immunochromatographic strips that are based on antigen-antibody recognition. Immunoassays are most commonly used because of their low cost, high specificity, and fast analysis, making them particularly useful for routine monitoring. These conventional detection strategies for BTV have some drawbacks. Recently, FLISA has been drawing attention due to its sensitivity, which is higher than traditional immunoassays. Fluorescence-linked immunosorbent assays (FLISA) using fluorescent materials as labels overcome ELISA's disadvantage of being time-consuming.

Polydopamine-Functionalized Copper Peroxide/ZIF-8 Nanoparticle-Based Fluorescence-Linked Immunosorbent Assay for the Sensitive Determination of Carcinoembryonic Antigen by Self-Supplied H2O2 Generation.

Copper peroxide/zeolitic imidazolate framework/polydopamine nanoparticles (CP/ZIF-8/PDA)-based fluorescence-linked immunosorbent assay (FLISA) was designed for the sensitive and high-throughput determination of carcinoembryonic antigen (CEA) by self-supplied H2O2 generation. Specifically, the CEA aptamer was modified on the surface of CP/ZIF-8/PDA to form an immunoprobe. The structures of CP and ZIF-8 could be broken under acidic conditions, and produced the Cu2+ and H2O2 due to the dissociation the CP. A subsequent Fenton-type reaction of Cu2+ and H2O2 generated hydroxyl radical (·OH). o-phenylenediamine (OPD) was oxidized by the ·OH to form 2, 3-diaminophenazine (DPA) with a significant fluorescence signal. CP/ZIF-8/PDA could be used as an efficient Fenton-type reactant to generate a large amount of ·OH to promote OPD oxidation. The sensitive detection of CEA could be realized. Under optimal conditions, the FLISA platform displayed a linear detection range from 0.01 to 20 ng mL-1 with a detection limit of 7.6 pg mL-1 for CEA. This strategy has great application potential for sensitive and high-throughput determination for other biomarkers in the field of biomedicine.

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).

Development of a canine blood C-reactive protein-measuring device using a flow-type immunosensor.

This study aimed to construct a measurement system with the same performance as a measurement system using an automated analyzer and immunoturbidimetric reagents (comparative method) using a flow-type immunosensor (FIS) based on the fluorescence-linked immunosorbent assay technology. In the FIS constructed in this study, all control samples were within the indicated values. The coefficient of variation of repeatability and intermediate precision were less than 2.4% and less than 4.4%, respectively. The lower limit of quantification in this measurement system was 3.9 mg/L, and linearity was confirmed for quantification values, ranging from 3.9 to 465 mg/L. Canine plasma samples (N = 39) were used to measure C-reactive protein (CRP) levels using the comparative method (x) and FIS (y). The regression equation between the measurements was y = 1.035 × - 0.002, with a correlation coefficient of 0.9809, indicating a significantly high correlation. Although the Brandt-Altman analysis suggested the possibility of a proportional systematic error between the two measurements, 38 of the 39 canine plasma samples measured fell within the acceptable range of error, indicating that the measurements are highly consistent. These results suggest that the analytical accuracy of the FIS constructed in this study and the quantitative value of canine CRP are equivalent to those of measurement systems using automated analyzers and immunoturbidimetric reagents.

VEGF Detection via Simplified FLISA Using a 3D Microfluidic Disk Platform.

Fluorescence-linked immunosorbent assay (FLISA) is a commonly used, quantitative technique for detecting biochemical changes based on antigen-antibody binding reactions using a well-plate platform. As the manufacturing technology of microfluidic system evolves, FLISA can be implemented onto microfluidic disk platforms which allows the detection of trace biochemical reactions with high resolutions. Herein, we propose a novel microfluidic system comprising a disk with a three-dimensional incubation chamber, which can reduce the amount of the reagents to 1/10 and the required time for the entire process to less than an hour. The incubation process achieves an antigen-antibody binding reaction as well as the binding of fluorogenic substrates to target proteins. The FLISA protocol in the 3D incubation chamber necessitates performing the antibody-conjugated microbeads' movement during each step in order to ensure sufficient binding reactions. Vascular endothelial growth factor as concentration with ng mL-1 is detected sequentially using a benchtop process employing this 3D microfluidic disk. The 3D microfluidic disk works without requiring manual intervention or additional procedures for liquid control. During the incubation process, microbead movement is controlled by centrifugal force from the rotating disk and the sedimentation by gravitational force at the tilted floor of the chamber.

Photoluminescence-based immunochemical methods for determination of C-reactive protein and procalcitonin.

Modern, sensitive, rapid, and selective analytical methods for the detection of inflammatory markers are a crucial tool for the assessment of inflammation state, efficacy of medical intervention, and the prediction of future diseases. Their development requires understanding of current state for point-of-care testing of inflammatory markers and identification of their crucial drawbacks. This review summarizes the progress in the application of luminescent labels for immunoassays. The luminescent labels became more popular in the latest decade due to their high sensitivity, selectivity, and robustness. This review presents a constructive analysis of different luminescent labels such as fluorescent organic dyes, quantum dots, long-lived emissive nanoparticles, and up-converting nanocrystals, as well as a range of the strategies for inflammatory markers determination. The advantages and disadvantages of all classes of luminescent labels are demonstrated, and the strategies of labels modification for their improvement are discussed. The current approaches for the creation of luminescent probes and robust assays are also highlighted.

Open sandwich fluorescence-linked immunosorbent assay for detection of soy isoflavone glycosides.

To detect major soy isoflavone glycosides, namely daidzin (DZ) and genistin (GEN), novel open sandwich fluorescence-linked immunosorbent assay (os-FLISA) was developed by taking advantage of enhanced interactions between variable regions of heavy (VH) and light chain (VL) domains in the presence of an antigen. The VH and VL genes were expressed in Escherichia coli as a chimera protein with green fluorescence protein (AcGFP1) and maltose-binding protein (MBP), respectively. Comprehensive characterization of os-FLISA displayed nearly the same specificity as parental DZ- and GEN-specific monoclonal antibody, demonstrating the potential of the developed assay for detection of both DZ and GEN. Their detectable range in this system exhibited at 0.1-12.5 µg mL-1. Subsequent validation analysis revealed that os-FLISA was reliable and accurate system for detection of total soy isoflavone glycosides. Notably, this is the first FLISA based on an open sandwich system, which can be employed for the detection of small molecules.

Highly sensitive fluorescence-linked immunosorbent assay based on aggregation-induced emission luminogens incorporated nanobeads.

Aggregation-induced emission luminogens (AIEgens) have attracted considerable interest for application towards the development of various biosensors due to their unique optical properties. However, the major challenge associated with generating a suitable fluorescent signal for constructing an AIEgens-based immunoassay platform, is the complex surface modification and additional chemical reaction required to activate the AIE process. This work reports a novel AIEgens nanobeads-based fluorescence-linked immunosorbent assay (FLISA) platform wherein the fluorescent labels are hexaphenylsilole (HPS) nanobeads, which were synthesized through Shirasu porous glass (SPG) membrane emulsification method and could provide a strong, direct fluorescent signal without any pretreatment. Moreover, the particle-based signal amplification effect affords this platform significantly improved detection sensitivity for carcinoembryonic antigen (CEA) quantitation. Compared to FLISA which uses R-phycoerythrin (PE) or commercial green QDs nanobeads as fluorescent labels, this AIEgens nanobeads-based FLISA platform exhibits detection sensitivity improved up to 45-fold and 12-fold, respectively. Clinical validation experiments applying this AIEgens nanobeads-based FLISA immunoassay platform to analyze human serum samples produce results consistent with those obtained by the clinical gold-standard method, electrochemiluminescence immunoassay (ECLIA). The strong photobleaching resistance and excellent fluorescent stability of the HPS nanobeads negate the need for light shielding, which improves the efficiency and makes the operating conditions more comfortable. Thus, this AIEgens nanobeads-based FLISA platform, with attractive features including direct fluorescent signal generation and significant signal amplification, creates a new, versatile route for the application of AIEgens in biosensors and clinical diagnosis.

Development of monoclonal antibody-based ultrasensitive enzyme-linked immunosorbent assay and fluorescence-linked immunosorbent assay for 1-aminohydantoin detection in aquatic animals.

Monitoring and rapid evaluation of nitrofurantoin metabolite, 1-aminohydantoin (AHD), are important for food safety and human health. Herein, we established the monoclonal antibody-based indirect competitive enzyme-linked immunosorbent assay (ic-ELISA) and quantum dots (QDs)-fabricated fluorescence-linked immunosorbent assay (FLISA). Monoclonal antibody specific to nitrophenyl derivative of AHD was derived from hybridoma cell lines 3.2.4/5A8. For another, CdTe core QDs with emission wavelength of 605nm were also synthesized. The performances of the proposed ic-ELISA and FLISA were further examined and the corresponding results were also validated by standard LC-MS/MS analysis. The obtained results indicated that both ic-ELISA and FLISA exhibited good dynamic linear detection for NPAHD over the range from 0.1 to 3.0ngmL-1. Meanwhile, proposed immunosorbent assays are characterized by satisfactory recovery rates of 81.5-113.7%. The experimental data suggested these two immunoassays could be facile, cost-effective and rapid tools for the prospective quantitative method for AHD analysis in food matrix.

Ultrasensitive direct competitive FLISA using highly luminescent quantum dot beads for tuning affinity of competing antigens to antibodies.

Herein, for the first time we report a novel direct competitive fluorescence-linked immunosorbent assay (dcFLISA) for the ultrasensitive detection of ochratoxin A (OTA) by introducing a large size polymer beads loaded with quantum dots (QBs) as carrier of competing antigen for decreasing binding affinity to antibody and enhancing the fluorescent signal intensity. When using 255 nm QBs as carrier of competing antigen, the equilibrium dissociation constant of QB based competing antigen to antibodies can be tuned to 100 times higher than that of the horseradish peroxidase (HRP) based competing antigen by controlling labeled amounts of antigen on the surface of QBs. Various parameters that influenced the sensitivity of dcFLISA were investigated and optimized. Under optimum detection parameters, the dynamic linear range of developed dcFLISA for detecting OTA was established at 0.05 pg/mL to 1.56 pg/mL with a half maximal inhibitory concentration at 0.14 ± 0.04 pg/mL (n = 5), which is three orders of magnitude lower than that of conventional HRP-based dcELISA (0.24 ng/mL). The developed FLISA is also highly accurate, reliable, and shows no cross reaction to other mycotoxins. In summary, the proposed method offers a straightforward approach to improve the sensitivity of direct competitive immunoassay for trace small chemical molecule detection in food quality control, environmental monitoring, and clinical diagnosis.