New Approach to Generate Ratiometric Signals on Immunochromatographic Strips for Small Molecules

The self-calibration capability of ratiometric signals has been widely considered to enhance the accuracy, sensitivity, and anti-interference ability of immunoassays. Exploring a new approach to generate ratiometric signals can provide more options for various requirements. Herein, we integrated the negative-readout competitive and positive-readout noncompetitive immunoassays into a single assay by employing different color tracers, labeled peptidomimetic and anti-immunocomplex peptides, to create a new unconstrained ratiometric signal approach. Using an immunochromatographic strip (ICS) and a fungicide benzothiostrobin as the analytical platform and analyte, respectively, we showed that this approach can be extensively applied to fluorescence and colorimetry readouts, which have also been proven for strong anti-interference ability to an external light environment. Moreover, the enormous intuitional color changes of ratiometric fluorescent and colorimetric ICSs (RFICS and RCICS) enabled the formation of the color reference cards (like the pH paper) for visual judgment. After adaptation with a portable smartphone, the quantitative detection limits for RFICS and RCICS were 0.17 and 0.44 ng mL–1, respectively. In addition, the ICSs showed good accuracy for the detection of benzothiostrobin in spiked samples.