Simultaneous or separate detection of heavy metal ions Hg²⁺ and Ag⁺ based on lateral flow assays

A lateral flow assay (LFA) was developed for the simultaneous or separate detection of mercury ion and silver ion based on isothermal nucleic acid amplification. T-Hg²⁺-T and C-Ag⁺-C were utilized in the isothermal nucleic acid amplification strategy to form specific complementary base pairs. Under the action of KF polymerase and endonuclease Nt.BbvCl, trace amounts of Hg²⁺ and Ag⁺ were converted to Product-Hg²⁺ and Product-Ag⁺ as bridges. Biotin-labeled capture strands (Biotin-DNA1, Biotin-DNA1, and Biotin-DNA3) immobilized on the test strips could capture the Au NPs-DNA nanoprobes by hybridization with the generated bridge products for monitoring of two heavy metal ions simultaneously or separately. The assembly method of DNAs on the nanoprobes was explored, and the DNA sequences on the nanoprobes were designed so that only one kind of DNA strand was used to bind to all three capture DNA strands on the C, T1, and T2 bands. Under optimal detection conditions, the limits of detection for Hg²⁺ and Ag⁺ were 2.19 and 5.41 pM, respectively, with desired selectivity and reproducibility.