A label-free colorimetric aptasensor for rapid multiplex detection of leuco-malachite green and leuco-crystal violet

Leuco-malachite green (LMG) and leuco-crystal violet (LCV) are widespread co-pollutants in aquatic products that pose a severe threat to human health. Therefore, it is urgent and challenging to develop rapid multiplex detection of LMG and LCV. Herein, the bispecific aptamer (A5b) for LMG and LCV was characterized. Its dissociation constant (K_d) was 2.03 ± 0.13 μM for LMG and 0.97 ± 0.06 μM for LCV. Then, we used A5b as a sensing probe to develop a label-free colorimetric aptasensor using gold nanoparticles (AuNPs) as an indicator and poly-diallyl dimethylammonium chloride (PDDA) as an aggregation inducer. In the absence of LMG and LCV, PDDA hybridized A5b via electrostatic interaction to form a "duplex" structure, which failed to induce the aggregation of AuNPs. However, in the presence of LMG or LCV, A5b specifically bound to LMG or LCV, releasing PDDA. The free PDDA then induced the aggregation of AuNPs. Consequently, a visible color change from red to blue was observed with increasing LMG/LCV concentration. This sensor exhibited excellent sensitivity and bispecificity, and a limit of detection (LOD) of 77.9 nM for LCV and 123.1 nM for LMG, with a linear concentration range of 1-6 μM for LCV and 1-8 μM for LMG. Additionally, the potential of this sensor for real-world applications was well demonstrated in crucian samples. Overall, this work provided a bispecific affinity probe for LMG and LCV for the first time and reported a label-free colorimetric aptasensor for multiplex detection of LMG and LCV in aquatic products.