Department of Applied Chemistry, Graduate School of Engineering, Nagoya University, Japan.
Anal Chem. 2011 May 1;83(9):3528-32. doi: 10.1021/ac200236r. Epub 2011 Apr 5.
A solid-state molecular beacon using a gold support as a fluorescence quencher is combined with a polydimethylsiloxane (PDMS) microfluidic channel to construct an optical sensor for detecting single-stranded DNA binding protein (SSBP) and histone protein. The single-stranded DNA-Cy3 probe or double-stranded DNA-Cy3 probe immobilized on the gold surface is prepared for the detection of SSBP or histone, respectively. Due to the different quenching ability of gold to the immobilized single-stranded DNA-Cy3 probe and the immobilized double-stranded DNA-Cy3 probe, low fluorescence intensity of the attached single-stranded DNA-Cy3 is obtained in SSBP detection, whereas high fluorescence intensity of the attached double-stranded DNA-Cy3 is obtained in histone detection. The amounts of SSBP in sample solutions are determined from the degree of fluorescence recovery of the immobilized single-stranded DNA-Cy3 probe, whereas that of histone in sample solutions is determined from the degree of fluorescence quenching of the immobilized double-stranded DNA-Cy3 probe. Using this approach, label-free detection of target proteins at nanomolar concentrations is achieved in a convenient, general, continuous flow format. Our approach has high potential for the highly sensitive label-free detection of various proteins based on binding-induced conformation changes of immobilized DNA probes.
一种使用金作为荧光猝灭剂的固态分子信标与聚二甲基硅氧烷（PDMS）微流道结合，构建了用于检测单链 DNA 结合蛋白（SSBP）和组蛋白的光学传感器。分别将固定在金表面上的单链 DNA-Cy3 探针或双链 DNA-Cy3 探针用于 SSBP 或组蛋白的检测。由于金对固定化的单链 DNA-Cy3 探针和固定化的双链 DNA-Cy3 探针的猝灭能力不同，因此在 SSBP 检测中获得了附着的单链 DNA-Cy3 的低荧光强度，而在组蛋白检测中获得了附着的双链 DNA-Cy3 的高荧光强度。通过固定化的单链 DNA-Cy3 探针的荧光恢复程度来确定样品溶液中的 SSBP 含量，而通过固定化的双链 DNA-Cy3 探针的荧光猝灭程度来确定样品溶液中的组蛋白含量。通过这种方法，可以在方便、通用、连续流动的格式下实现对纳摩尔浓度的目标蛋白质的无标记检测。我们的方法具有基于固定化 DNA 探针的结合诱导构象变化的高灵敏度无标记检测各种蛋白质的潜力。
