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.