Ultrasensitive Detection Of Mercury Ions In Environmental Water Based On Molecular Beacon Fluorescent Technology And Interaction Analysis

In recent years, with the rapid development of industry, agriculture and economy, all kinds of heavy metal pollution in environmental water are more and more serious, the perniciousness covers from the ecological environment to human themselves. Because of persistence, easy mobility and bio-concentration action, mercury is one of the biggest concerns among environmental pollutants. Mercury ion(Hg²⁺) has high chemical activity and stability, and is the main form of mercury in environmental water. Daily monitoring and evaluation of Hg²⁺ in environmental water?i.e. rivers, sea? is very important. Therefore it is imperative to establish a rapid sensitive method for monitoring the Hg²⁺ in environmental water.Molecular beacon?MB? has become a kind of powerful research tools in molecular biology and biotechnology. The structural stability of MB is an important aspect of factors affecting its performance. The thermodynamic equilibrium relationship between the stem-loop structure and double strands after hybridized with target molecules is the key that its hybridization specificity significantly higher than that of conventional linear probes. We set up a novel evaluation method based on microscale thermophoresis?MST? for MB probes, which can directly reflected the affinity and thermodynamic parameters of the reaction system. And the designed MB-binding system was optimized for the detection of trace Hg²⁺ in Tris-HCl buffer.The MB-based fluorescence detection technology and T-Hg²⁺-T coordination chemistry were combined together. There were different numbers of T-T mismatch pairs between complementary single-stranded DNA?Single standed DNA, ssDNA? and the loop of MB, Hg²⁺ in water was the targets. MB?0.2 ?M? and Hg²⁺?1 ?M? were mixed together, ssDNAs were diluted gradiently from 10 ?M to 16 different concentrations. The optimal MB-ssDNA system for capturing Hg²⁺ was achevied through measuring the affinity among the three-interactant system(MB/ssDNA/Hg²⁺) by MST.The interaction between MB and different mismatched number of ssDNAs with or without Hg²⁺ was compared. The optimal system for the detection of Hg²⁺ was achieved when the number of T-T mismatch was 4. The affinity constant?Kd? was 128±4.1 nM between MB and N4, but decreased to 53.9±2.89 nM after Hg²⁺ was joined. The lowest detection limit of the developed MB-binding system for Hg²⁺ in Tris-HCl buffer was determined to be 3 nM, and detection range covered from 5 nM to 320 nM.The MB-Nx system was combined with the heterogeneous technique-magnetic beads by the technique of flow cytometry and suspended on-chip. ssDNA was fixed through the reaction of avidin-biotin or amino-carboxyl. The heterogeneous detection method for Hg²⁺ was groped for.A new method for the evaluation of MB probe was set up based on MST, this method was free of complicated sample preparation, only needs traces of samples, it can rapidly and directly reflects the real thermodynamic and affinity parameters between MB and its targets. The method could be used for the detection of Hg²⁺ in environmental water samples, and had strong specificity, high sensitivity. The groping for the heterogeneous detection method makes it possible to achieve the detection of multiple metal ions at the same time.