Novel Aptamer-based Techniques For The Chemiluminescent Detection Of Thrombin

DNA aptamers are synthetic single-strand nucleic acids discriminated by screening in vitro, which are of high affinity and specificity to many given targets ranging from small molecules to large proteins and even cells. New methods for efficient and fast detection in biochemical and biomedical fields are being developed based on the molecular recognition of aptamers. Aptamers have been demonstrated to have advantages over antibodies with regard to chemical stability, readily availability, simple modifiability, and high flexibility in biosensor design for analyte detection. As a novel functional molecule, aptamer have gained considerable interest in bio-analytical application in many fields such as clinical diagnosis, environmental supervision, pharmaceutical study, etc.Chemiluminescence has been exploited within a wide range of applications in various fields, due to their extremely high sensitivity along with their extra advantages such as needless special light, simple instrument, wide calibration ranges, and suitability for automatical operation in analytical chemistry. Nowadays, based on cross-research with other disciplines, CL has been successfully served for different applications. Our study developed an aptasensor on the strength of the high specifically binding between aptamers and thrombin in the thesis. Description of research in the thesis is presented as follows:Chapter 1:This chapter is composed of three phases. The first phase involves the source, traits and advantages of aptamers and outlines aptamer-based detection techniques. With thrombin as the object of this research, thus in the second phase of this chapter reviews thrombin analysis of current studies based on aptamer in detail, including electrochemical aptasensors, optical aptasensors, chromatographic aptasensors and other aptasensors. Following that, the third phase of this chapter reviews CL detection technology, introduces objectives, significance, contents of this CL research based on aptamer.Chapter 2:Thrombin is an important enzyme in physiology and it palys an important role in some physiological and pathological process, such as blood coagulation, wound cicatrization and inflammation. Therefore, thrombin detection is very important in medicine. A novel CL-biosensor based on aptamer was designed and thrombin detection with sandwich type based on magnetic enrichment and separation was proposed. Employing this aptasensor, thrombin could be specifically detected and the lowest detection concentration (0.01 nM) as well as a wide linear dynamic range (0.5-100 nM) could be achieved. The experiments results demonstrate that the proposed - method possesses the feasibility, high specificity and sensitivity.Chapter 3:A novel CL-biosensor based on aptamer was designed and thrombin detection with sandwich form, employed 96 microplate as high throughput carrier, was proposed. On this ground, an amplified CL technique was investgated taking advantange of 490 nm polystyrene microsphere as the amplification carrier; the biotin-streptavidin system was introduced as the amplification platform to form a superhigh sensitivity CL approach. Nonamplified method acchived a good linear correlation from 0.9375 nM to 30 nM with the detection limit of 0.46 nM and the correlation coefficient is 0.99035. In the amplified approach, an excellent linearity was found the thrombin range from 7.8 pM to 250 pM with the lowest detection concentration of 3.9 pM, which was more than 100-fold improvement on nonamplified technique.