基于发卡型DNA循环杂交放大技术检测海产品中的Hg^(2+)含量

Determination of Mercury(Ⅱ) Content in Seafood by Cycle DNA Amplification Based on Hybridization Chain Reaction

利用发卡型DNA的循环杂交放大作用和碱基T与Hg^(2+)之间的稳定结构,设计了一种高灵敏性的表面增强拉曼生物传感器用于海产品中痕量汞的检测。首先制备了携带有大量拉曼信号分子的纳米金生物条码作为拉曼信号探针。然后通过酰胺键将捕获DNA固载在磁珠表面上,利用T-Hg^(2+)-T形成的稳定结构和链式循环杂交反应放大技术,将含有大量拉曼信号DNA分子的纳米金颗粒通过生物素和链霉亲和素的特异性结合到磁珠上,最后通过SERS技术实现了溶液中Hg^(2+)的检测。最佳实验条件下,当固定磁珠捕获DNA浓度为1.0×10^(-7) mol/L,Tris-HCl缓冲溶液为p H 7.4,37℃下杂交反应3 h后,Hg^(2+)的浓度与拉曼信号强度呈良好的线性关系,测定线性范围为1.0×10^(-7)~1.0×10^(-13) mol/L,检测限1.0×10^(-13) mol/L(S/N=3)。该传感器用于海产品中Hg^(2+)的测定,测定值与ICP-AES的测定值基本一致。

A highly sensitive biosensor based on surface enhanced Raman scattering （SERS） for detecting trace amounts of mercury （II） in seafood was designed by cycle amplification based on hybridization chain reaction of DNA hairpins and the stable structure formed between the nucleotide base thymine （T） and mercury （II）. Firstly, the nano-Au bio-barcode with numerous Raman signal DNA molecules was prepared as the Raman signal probe, and then the captured DNA was immobilized on magnetic beads by amide bonds. The nano-Au particles containing numerous Raman signal DNA molecules were combined specifically on the magnetic beads by the stable structure formed by T-mercury （II）-T and the cycle amplification based on hybridization chain reaction. Finally, the detection of mercury （II） in the solution was achieved by the SERS technique. The optimum experimental conditions were also investigated. When the concentration of DNA captured by magnetic beads was 1.0× 10^-7 mol/L and the hybridization reaction proceeded for three hours at 37℃ in pH 7.4 Tris-HCl buffer solution, the mercury （II） concentration and the Raman signal intensity had good linear relationship, with a linear range of 1.0×10^-7-1.0×10^-13 mol/L and a detection limit of 1.0×10^-13 mol/L （S/N=3）. The biosensor was applied to determine the mercury （II） content in the seafood samples, and the measured results were consistent with those obtained by inductively coupled plasma atomic emission spectroscopy （ICP-AES）.