A Novel Aptamer-based Suspension Array Technology For The Detection Of Thrombin And The Interaction Analysis

Due to its many advantages, such as low molecular weight, simple preparation, high affinity and good specificity, aptamer gradually replaces antibody as a novel recognition element and is expected to widely use in fields of biological science research, analysis, detection, clinical diagnosis and so on. Suspension array technology, a biochip technology, is high-throughput and low-background for rapid and sensitive detection, regarding fluorescence-coding beads as the carrier of probes. In the present work, we combined the aptamer and suspension array to establish a novel detection technology with high affinity, good specificity and high throughput via the detection of the typical model target – thrombin.Amination of 15-mer aptamer(thrombin binding aptamer 1, TBA1) was modified on the surface of magnetic beads(MBs) as the probe beads, which could capture thrombin in solution and blood. Biotinylated 29-mer aptamer(thrombin binding aptamer 2, TBA2) acted as the reporter probe bound thrombin to form stable sandwich complex on the MBs. Through magnetic separator the complex was separated from unbound substance. Fluorescent reporter protein, streptavidin R–phycoerythrin(SAPE) was coupled with bio-TBA2 via avidin-biotin system. Then the median fluorescence intensity(MFI) was obtained by suspension array. The optimum interaction conditions and coupling protocol were optimized. Under the best conditions, the standard curve for the detection of thrombin was built. The dynamic quantitative working range was 18.37–554.31 n M, and the lowest detection limit of thrombin was 5.4 nM. It suggested that the detection technology had high sensitivity and accuracy. The specific test illustrated the good specificity of the aptamer-suspension array detection technology. Thrombin was added in the 100-fold diluted human serum to determine the recovery. The recoveries of the spiked thrombin were 82.6% to 114.2% with relative standard deviations(RSDs) below 10%, which was satisfied. It proved the method was expected to be successfully applied in clinical diagnosis with high-throughput detection.Meanwhile, the interaction mechanism between TBAs and thrombin was discussed. Microscale thermophoresis(MST) technology was used to analyze. It could be demonstrated that two aptamers rendered high binding affinities with thrombin. MST thermophoresis signal reflected the binding mode between TBA1 and thrombin, suggesting that TBA1 could simultaneously bind the fibrinogen and heparin sites of thrombin.We firstly combined the new recognition element – aptamer with sensitive suspension array technology to build a novel aptamer-suspension array detection technology. Thrombin in the serum sample was preliminarily analyzed. This method was sensitive, specific and rapid, and had a good prospect of application and development. MST technology was used to analyze the interaction between aptamer and thrombin. It had laid a solid foundation for deep study on bind mechanism between aptamer and its target, and specially supported data for research on drug targeting and interaction mechanism.