An evaluation assay for thymine-mercuric-thymine coordination in the molecular beacon-binding system based on microscale thermophoresis

•By introducing microscale thermophoresis technology, we provided a simple and institutional analysis for molecular binding system.•A relatively complete and convictive discussion about the Hg2+ detecting principle of molecular binding system was presented.•Based on T-Hg2+-T coordination chemistry and molecular beacon, a sensitive and convenient detection method for Hg2+ has been established.

A novel evaluation assay for molecular beacon (MB)-binding system was developed by microscale thermophoresis (MST) and thymine-mercuric-thymine coordination chemistry which was applied for the detection of mercury (II) ion (Hg2+) upon opening the hairpin and facilitating fluorescence recovery through binding with suitable mismatched target. The affinities between the loop of MBs and the complementary ssDNAs in the binding system were varied, determined and optimized by MST in the presence or absence of Hg2+. It suggested that the relative low background could be obtained when the number of mismatches of ssDNA was four and in the present of Hg2+ displayed strong recovery of fluorescence. The lowest detection limit of Hg2+ was 3 nM which was below the maximum contaminant level defined by U.S. Environmental Protection Agency (10 nM). The working range was 5-320 nM with negligible possible interference from the different background composition of water samples by optimization of the binding conditions. Through evaluating by MST, the molecular interaction mechanism was elaborated and the rapid, sensitive and selective detection of Hg2+ was achieved by the designed MB-binding system. It herein has great potential for appraising and analyzing molecular interaction among the interactants.

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