Institute of Quality Standards and Testing Technology for Agro-products, Key Laboratory of Agro-product Quality and Safety, Chinese Academy of Agricultural Sciences, Beijing 100081, China; Key Laboratory of Agri-food Quality and Safety, Ministry of Agriculture, Beijing 100081, China; College of Food Science, Sichuan Agricultural University, Ya'an 625014, China.
Institute of Quality Standards and Testing Technology for Agro-products, Key Laboratory of Agro-product Quality and Safety, Chinese Academy of Agricultural Sciences, Beijing 100081, China; Key Laboratory of Agri-food Quality and Safety, Ministry of Agriculture, Beijing 100081, China.
Biosens Bioelectron. 2015 Feb 15;64:530-4. doi: 10.1016/j.bios.2014.09.095. Epub 2014 Oct 5.
Highly sensitive detection of proteins is of great importance for effective clinical diagnosis and biomedical research. However, so far most detection methods rely on antibody-based immunoassays and are usually laborious and time-consuming with poor sensitivity. Here, we developed a simple and ultra-sensitive method to detect a biomarker protein-thrombin by taking advantage of the fluorescent probe Perylene tetracarboxylic acid diimide (PTCDI) derivatives and thrombin aptamer. The water-soluble dye PTCDI shows strong fluorescence in buffer solution for the existence of free dye monomer, but becomes weak after aggregation through self-assembly on nucleic acid aptamer. In the presence of thrombin, it specifically binds to thrombin aptamer which causes the conformational transition between aptamer and PTCDI and results in a significant fluorescence recovery. The results showed that as low as 40 pM of thrombin could be detected by this method. The high sensitivity of the developed sensing system mainly attributes to the ultra-sensitivity of the fluorescence intensity changes of PTCDI. With the specificity of aptamer, the assay exhibited high selectivity for thrombin against three other proteins (bovine serum albumin, lysozyme, mouse IgG) and 1% diluted fetal bovine serum. The detection method might be extended to sensitive detection of a variety of proteins for its advantages of isothermal conditions required, simple and rapid without multiple separation and washing steps.
高灵敏度检测蛋白质对于有效的临床诊断和生物医学研究非常重要。然而，到目前为止，大多数检测方法都依赖于基于抗体的免疫测定法，通常繁琐且耗时，并且灵敏度较差。在这里，我们利用荧光探针苝四羧酸二酰亚胺（PTCDI）衍生物和凝血酶适体开发了一种简单且超灵敏的方法来检测生物标志物蛋白-凝血酶。水溶性染料 PTCDI 在缓冲溶液中因游离染料单体的存在而显示出强荧光，但在通过核酸适体自组装聚集后变得很弱。在凝血酶存在下，它特异性地与凝血酶适体结合，导致适体与 PTCDI 之间的构象转变，从而导致荧光显著恢复。结果表明，该方法可以检测低至 40 pM 的凝血酶。所开发的传感系统的高灵敏度主要归因于 PTCDI 的荧光强度变化的超高灵敏度。由于适体的特异性，该测定法对凝血酶对其他三种蛋白质（牛血清白蛋白、溶菌酶、小鼠 IgG）和 1%稀释的胎牛血清具有高选择性。由于该检测方法不需要恒温条件，且简单快速，无需进行多次分离和洗涤步骤，因此可能会扩展到对各种蛋白质的灵敏检测。
