Study On New Aptamer-Based Assays For Thrombin

Chapter1:This part briefly introduced the significance of protein detection and the commonly used methods for protein detection. The definition and properties of aptamer were also introduced. Aptamer-based assays for protein and the applications of nanomaterials in these assays were reviewed. The research background, main research work, and innovation on the thesis were described.Chapter2:A fluorecent assay for thrombin was developed by using fluorophore-labeled aptamer as a probe and gold nanoparticles (GNPs) as a quencher. A29-mer fluorophore-labeled aptamer against thrombin was adsorbed on gold nanoparticles, leading to fluorescence quenching. With the addition of thrombin, the binding of aptamer to thrombin would change the conformation of the aptamer, and the aptamer was released from GNPs, causing the recovery of fluorescence of the aptamer probe, so the detection of thrombin was achieved. Results showed this method was simple, fast, and specific. The detection limit of thrombin was0.54nM in200μL of solution.Chapter3:Taking advantage of aptamer affinity, preconcentration by magnetic beads, and the sensitive fluorescence detection of quantum dots (QDs), we developed an aptamer-based sandwich assay for thrombin by using a pair of thrombin-binding aptamers (15-mer Apt15and29-mer Apt29), which could bind to two distinct binding sites of thrombin. Either Apt29or Apt15can be used as capture aptamers on magnetic beads or reporter aptamers on the QDs to form the sandwich complex. The choice of capture aptamers and reporter aptamers and the effect of the addition order of the aptamers modified magnetic beads and the aptamers modified QDs on assay performance were investigated. Under optimized experimental conditions, sensitive detection of thrombin was achieved. Three other proteins such as hemoglobin, lysozyme, and transferrin did not cause interference in the sandwich assays, showing a good specificity of the assays. Chapter4:A simple chromogenic assay for thrombin was developed through aptamer affinity capture and a subsequent enzyme reaction. Thrombin was captured on the aptamer-modified magnetic beads, and catalyzed the conversion of chromogenic substrates (T3068,(3-Ala-Gly-Arg-p-nitroanilide) to optically measured products. The measurements of generated products by an absorbance spectrometer allowed for the final quantification of thrombin. This assay showed high sensitivity by taking advantage of sample enrichment and enzyme amplification, and exhibited good specificity by involving the selective aptamer binding and the specific enzyme reaction. A concentration detection limit of2pM was obtained when250μL of thrombin sample was analyzed in24-h enzyme reaction.Chapter5:By using detection principle similar to that in Chapter4, another chromogenic substrate (T1637, N-P-tosyl-Gly-Pro-Arg-p-nitroanilide) of thrombin was applied to further enhance the sensitivity of the assay for thrombin. Thrombin was captured on the aptamer-modified magnetic beads, and catalyzed the conversion of chromogenic substrates T1637to optically measured products. As the turn number of substrate T1637was100times higher than that of substrate T3068under the same conditions, higher velocity of enzyme reaction could be obtained by T1637. A concentration detection limit of thrombin reached40fM when250μL of thrombin sample was analyzed in24-h enzyme reaction, which was lowered by50-fold comparing to that obtained by using substrate T3068. This assay was applied to detect thrombin in dilute human serum.