New Aptamer-Based Assays For Enzyme

Chapter 1:This part briefly introduced aptamer, including the significance and features of nucleic acid aptamer and peptide aptamer. Assays for proteins based on aptamers were reviewed. We described the research background, main research work, and innovation on the thesis.Chapter 2:Taking advantage of strong binding affinity of aptamer, and signal amplification via an enzymatic reaction, we developed a fluorogenic assay for activated protein C (APC). APC is specifically captured by the DNA aptamers on magnetic beads, and the APC then catalyzes the conversion of a fluorogenic substrate (D-Val-Pro-Arg-ANSNH-C4H9) of APC to a fluorescent product. Detection of APC is achieved by measuring the generated product. This method is simple, sensitive, and specific. APC can be detected at 0.4 pM concentration level in a sample volume of 250 uL, corresponding to 0.1 femtomole of APC, when 2-h enzymatic reaction is employed. The proteins thrombin, trypsin, proteinase K, chymotrypsin, and elastase do not interfere.Chapter 3:We present a sensitive and specific assay for thrombin through the affinity capture of thrombin with peptide aptamers and the subsequent thrombin-involved enzymatic reaction. Thrombin is specific-ally captured by the peptide aptamers on magnetic beads, then thrombin catalyzes the conversion of a fluorogenic substrate(N-p-tosyl-Gly-Pro-Arg-7-amido-4-methylcoumarin) of thrombin to a fluorescent product. Detection of thrombin is achieved by measuring the generated product. Thrombin can be detected at 50 fM concentration level in a sample volume of 100 uL. This method is simple, sensitive, and specific. In addition, this method can be used for the detection of thrombin in human plasma samples.Chapter 4:By using detection principle similar to that in Chapter 3, we used a chromogenic substrate (N-P-tosyl-Gly-Pro-Arg-p-nitroanilide) to develope a chromogenic assay for thrombin by using aptamer modified magnetic beads. Thrombin is specifically captured by the peptide aptamers on magnetic beads, and the concentrated thrombin then catalyzes the conversion of a chromogenic substrate of thrombin to optically measured products. Detection of thrombin is achieved by measuring the absorbance of generated products. The detection limit of thrombin is 1 pM. Taking advantage of the specificity of peptide aptamer to thrombin and the specificity of enzymatic reaction, this method is specific. In addition, this method can be used for the detection of thrombin in real samples.