Fluorescent Immunochromatographic Assay For Quantitative Detection Of CK-MB And Mycotoxins

Immunochromatographic assay（ICA）is a promising technology for on-site detection,and has been widely used in clinical diagnosis,food safety,animal health and environmental monitoring because of its outstanding advantages,including rapidity,low cost,and easy to use.Nonetheless,the colloidal gold（30-40 nm）based ICA commonly suffers from its inherent disadvantages,including poor repeatability,low sensitivity,single-target detection,and weak resistance to complex sample matrix.Improving the sensitivity and realizing the multiple target detection are the future direction of ICA.This study explored the effects of three fluorescent microsphere probes on the detection performance of sandwich ICA method,and a reliable multiple ICA using streptavidin（SA）-biotin system as the control line for the simultaneous quantitative detection of three kinds of mycotoxins.In recent years,fluorescent microspheres/beads have been used an alternative of colloidal gold for improving the sensitivity of ICA because of their high sensitivity and stability.In this study,three kinds of fluorescence microspheres/beads including quantum dot beads（QBs）and fluorescein isothiocyanate microspheres（FMs）with the average sizes of 300 nm and 500 nm were used as the labeling probes to establish the ICA strip for the quantitative detection of creatine kinase isoenzyme（CK-MB）.Under the optimum conditions,the QBs-ICA for CK-MB quantitative detection showed a good linear range from 0.048 ng/mL to 100 ng/mL with a limit of detection（LOD）of0.04 ng/mL,the FM₃₀₀-ICA for CK-MB was 0.78 ng/mL to 100 ng/mL with a LOD of0.7 ng/mL,while that of FM₅₀₀-ICA was 0.19 ng/mL to 100 ng/mL with a LOD of0.18 ng/mL,respectively.The antibody consumptions for QBs-ICA,FM₃₀₀-ICA and FM₅₀₀-ICA were 0.375,4.48 and 0.448μg per ICA strip,respectively.The recoveries of intra-and inter-assays for low CK-MB spiked concentration were in the range of111.27%-115.27%,86.11%-87.03%and 6.77%-9.42%,with the variation coefficients（CV）of 3.15%-4.72%,3.59%-12.32%and 17.81%-35.25%,respectively.Although the sensitivity of FM₅₀₀-ICA was achieved at 0.18 ng/mL,the recoveries for low CK-MB spiked concentration was only between 6.77%to 9.42%,and CV was more than 15%.The recoveries of FM₃₀₀-ICA in the inter-and intra-assays were reached at86.11%-87.03%,whereas the LOD was lower 17.5-folds than that of QBs-ICA.All in all,the QBs-ICA exhibited more advantages for the CK-MB detection due to its high sensitivity,low antibody consumption,good precision and accuracy.In addition,a novel multiplexed QBs-based ICA（QBs-ICA）multiple test lines was established for the simultaneous quantitative detection of three mycotoxins,namely,aflatoxin B₁（AFB₁）,fumonisin B₁（FB₁）and ochratoxin A（OTA）.The SA-biotin system was introduced as the signal output of control line,and further compared with the traditional QBs-ICA using the goat anti-mouse IgG as control line in the quantitative accuracy for multiple target detection.The results showed that the accuracy of the traditional QBs-ICA using goat anti-mouse IgG as control line could not meet the quantitative requirements of for multiple target detection（recoveries<50%）.Using the SA-biotin system as the signal output of the control line,the proposed QBs-ICA showed a good performance for simultaneous quantitative detection of three mycotoxins.The corresponding regression equations of the calibration curves for AFB₁,FB₁ and OTA detection are expressed as follows:y=-21.99 ln（x）+133.52（R²=0.9838,10 pg/mL≤x≤160 pg/mL）,y=-21.31 ln（x）+99.728（R²=0.989,3 ng/mL≤x≤50 ng/mL）and y=-10.4 ln（x）+41.352（R²=0.9841,25 pg/mL≤x≤6400 pg/mL）,respectively.The proposed QBs-ICA demonstrated high sensitivity for the simultaneous detection of AFB₁,FB₁ and OTA with the half-maximal inhibitory concentrations of 46.94 pg/mL,10.31 ng/mL and0.44 ng/mL,respectively.The average recoveries of the intra-and inter-assays ranged from 84.66%to 116.97%,and the variation coefficients were less than 12%.Furthermore,the large amounts of actual cereal samples were detected,and the results of QBs-ICA were consistent with those of ultra-performance liquid chromatography,indicating that the proposed method is highly accurate and robust.