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The widespread use of bromadiolone and brodifacoum poses a threat to public health and initiates poisoning incidents. Conventional colorimetric enzyme-linked immunosorbent assays (ELISAs) suffer from poor sensitivity, making it difficult to meet the trace detection of analytes. In this work, we constructed a sensitive and label-free fluorescence immunoassay based on the in situ formation strategy, for the quantification of bromadiolone and brodifacoum in biological and food samples. Alkaline phosphatase, an essential enzyme in ELISA, catalyzed the substrate sodium ascorbyl phosphate to L -ascorbic acid, which reacted with ο-phenylenediamine and triggered an in situ fluorescence signal with excitation/emission wavelengths of 350/430 nm. To reduce the reaction time, Fe3+ was utilized for the in situ immunoassay system. The fluorescence signal system was introduced to conventional ELISA without any label procedures, thus resulting in higher sensitivity with a 3.8-fold improvement in the half-maximal inhibitory concentration for bromadiolone, compared with colorimetric ELISA. The calculated limits of detection for bromadiolone and brodifacoum were 0.45–0.76 and 0.34–0.58 ng/mL (ng/g) in the human serum and corn samples, respectively, with recovery values ranging from 82.0 % to 93.1 %, and a coefficient of variation lower than 13.4 %. Due to its advantages of high sensitivity, cost-effectiveness, and easy substrate fabrication, this immunoassay may offer promise for applications in poisoning diagnosis and food analysis of bromadiolone and brodifacoum. • The signal transfer mechanism of in situ fluorescence immunoassay is explored. • The assay is applied for the simultaneous detection of bromadiolone and brodifacoum. • Oxidation action of Fe3+ reduces reaction time by 30 min in fluorescence immunoassay. • A 3.8-fold improvement for IC 50 is observed compared with colorimetric ELISA. [ABSTRACT FROM AUTHOR]

Colloidal quantum dots (QDs) have been widely used as fluorescent labeling materials in the field of in vitro diagnostics (IVD). In spite of the significant development of direct single-signal response fluorescent immunosensors, it is still a challenge to obtain the high accuracy and sensitivity of quantitative results of these sensors due to the influence of several factors unrelated to the analysis. Herein, we present a delicate design and synthesis of dual-color QDs bilayer nanobeads (red and green QDs nanobeads, R&G-QD nanobeads), achieving the precisely control of growth of the silicon layer with different proportions of ammonia, and inhibiting fluorescence resonance energy transfer (FRET) successfully. The dual-color QDs bilayer nanobeads have realized dual-channel detection (the acquisition of two emission wavelengths) of the target, and avoided the problems of low analytical accuracy and imprecise sensitivity of single-signal response. A proof-of-concept demonstration of a fluorescence-linked immunosorbent assay (FLISA) platform is conducted for the quantitative detection of prostate-specific antigen (PSA) with a sensitivity of 49 pg/mL and a linear range of 0.2–800 ng/mL. The dual-color QDs bilayer nanobeads FLISA platform with high sensitivity and accuracy holds great potential for early diagnosis of cancer or other diseases and provides a new approach for IVD. [Display omitted] • Dual-color QDs bilayer nanobeads were designed to synthesize by layer-by-layer assembly method. • Controlled growth of alternate red and green QD layers on pre-synthesized SiO 2 beads can suppress energy transfer. • Such dual-color QDs nanobeads deliver dual single calibration and can avoid errors caused from the single signal response. • A proof-of-concept demonstration of a FLISA platform is conducted for detecting PSA with high sensitivity and accuracy. [ABSTRACT FROM AUTHOR]

Abstract: A multiplexed immunoassay biosensor for detecting serum biomarkers was developed by using photoluminescent (PL) aqueous CdSe/ZnS quantum dots (QDs) with high efficiency and stability as fluorescence labels and carboxyl functionalized microporous nylon membrane as a matrix. Two main biomarkers of Down Syndrome (DS), β-human chorionic gonadotrophin (β-HCG) and α-fetal protein (AFP), were selected for detection in this biosensor system. For specific detection of DS, antibody-immobilized microporous nylon membrane first captured the targeted antigens from the physiological samples and formed immunocomplexes (antibody–antigen) on the membrane surface. Then the immunocomplexes continued to react with QDs conjugated antibodies (labeled antibodies), and formed triplex “sandwich” structure, which was confirmed by AFM and confocal microscopy. Quantitative detection of β-HCG and AFP was also realized, the detectable level of this new biosensor was as low as 1×10−6 IU/L for β-HCG and 1ng/mL for AFP in a relatively short assay time without any enrichment. [Copyright &y& Elsevier]

• A novel 3D vertical-flow paper-based device (3VPD) integrated with a sandwich-type fluorescent immunoassay is developed. • The 3VPD can be applied for multiplexed detection of cancer biomarkers simultaneously based on the principle of origami. • The proposed 3VPD shows high sensitivity and selectivity for detecting CEA, AFP and CA199 in human serum, respectively. A novel three-dimensional (3D) vertical-flow paper-based device (3VPD), integrated with a sandwich-type fluorescent immunoassay, was developed for multiplexed detection of cancer biomarkers simultaneously based on the principle of origami. The developed 3VPD, composed of a sample layer, a splitting layer, and a test layer, could be fabricated simply by wax-printing on paper at a large scale. Herein, a vertical-flow immunoassay was introduced into the device, in which fluorescein isothiocyanate labeled antibodies were coated on the splitting layer and mouse monoclonal capture antibodies were functionalized on the test layer. The proposed 3VPD showed high sensitivity and selectivity for detecting three cancer biomarkers, carcinoembryonic antigen (CEA), a-fetoprotein (AFP), and cancer antigen 199 (CA199) with detection limits of 0.03 ng/mL, 0.05 ng/mL, and 0.09 U/mL in human serum, respectively. The results obtained in human serum were compatible with the reference values which were acquired from parallel single-analyte. The developed 3VPD provides a new strategy to develop an inexpensive and sensitive approach for simultaneous detection of multiple analytes and comprehensive clinical diagnosis by a single test. [ABSTRACT FROM AUTHOR]