Long Wu,Luyao Jiao,Danni Xue,Yueqing Li,Yu Han,Wei Ouyang,Qi Chen
Microcystin-LR (MC-LR), a potent cyanotoxin in freshwater, poses a risk of severe liver damage and other health issues, making its detection vital. However, the detection capabilities of conventional antibodies are constrained, which limited their use in immunoassays. In this work, we designed a new bifunctional nanobody, named A2.3-SBP (comprised of nanobody and streptavidin binding peptide), capable of binding with MC-LR and streptavidin. Based on A2.3-SBP and Fe 3 O 4 @Au-Pt nanozyme, we introduced an enzyme-free immunosensor that operated in microplate with colorimetric and surface-enhanced Raman scattering (SERS) detection modes. The dual-mode assay showed color changes and SERS intensity directly correlating to MC-LR concentrations with a range from 1.0 to 500 ng/mL and a limit of detection of 0.26 and 0.032 ng/mL, respectively. This strategy eliminated the need for complex enzymatic reactions and realized dual-signal detection of MC-LR in 96 water samples (0.03 μg/kg) within 30 min, suggesting its potential in drinking water detection.
A Raman scattering and nanozyme-linked immunosorbent assay (NLISA) dual modal immunosensor, was constructed by mesoporous SiO 2 /Au-Pt nanozymes (m-SAP) and nanobodies (A2.3-SBP). Oxidized TMB served as Raman and ELISA signals in a competitive binding assay. Under optimized conditions, an inverse correlation was established between the Microcystin-LR (MC-LR) concentration and the signals, spanning Raman and ELISA ranges of 0.1–100 μg L −1 and 1.0–500 μg L −1 , with limit of detections (LODs, 3σ/S) of 0.015 μg L −1 and 0.12 μg L −1 , respectively. The LODs showed over 90 times and 11 times higher sensitivity than that of traditional ELISA (t-ELISA, LOD, 1.36 μg L −1 ). The immunosensor exhibited excellent accuracy in practical samples, can be integrated together for the detection of MC-LR within 45 min, which greatly short the detection time of t-ELISA (>2 h). This method displayed potential for detecting other toxins by simply changing the nanobodies.
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