Study On The Detection Of Thrombin Based On Aptamers

As a kind of novel specific molecule tools, aptamers inevitably are competed against antibodies with much more advantages such as easily synthesis, modification and stability in the protein assay. The aptamer-based methods for protein detection are easier to manipulated by transducing recognition events to detectable signals with efficiency and high sensitivity .On the contrary, traditional methods for protein detection based on antibody technologies exist many problems such as complex procedure and denature of protein with labelling. Here, new methods based on aptamer probes were presented and thrombin was studied as a model. Combining the qualities of enzyme and fluorescence, aptamer probes without labelling were designed for protein detection and promised with well ability for other protein detection. This thesis is composed of the following three parts:1. Aptamer-based thrombin detction by exonuclease protection assay with conjugated polymersHere, aptamer probes were used to specifically bind with thrombin that would protect the probes from the hydrolyzed by the exonulceases I. However, the free aptamer probes were easily hydrolyzed by the exonucleases without any protection. When the thrombin was denatured, the protected aptamer probe was freed and quantified by the conjugated polymers which can offered an fluorescent signal change with and without binding with the single-strand probes. This work presented the possibility of applying the fluorescent polymers to the detection of various proteins with simplicity and low cost by aptamer probes.2. Aptamer-based thrombin detection with polymerization assay by steric-hindrance effectAptamer probes prolonged with a complementary tip with primer were used for thrombin detection. DNA polymerases can copy the aptamer probe as the templates to the double nucleic acid strand which will be quantified with the fluorescence Sybr Greeen I. The velocity of the polymerization was hampered by the steric-hinderance of binding between aptamer probes and thrombin. Consequently, the rate of fluorescenc change refered the concentration of thrombin. This method, trancducing the detection of protein to the polymerization reaction rate, took the advantage of the polymerization and fluorescene to avoid the modification of the aptamer probes, with promising application in protein detection because of its simplicity and specifity. 3. Thrombin detection based on proximity extension with dual DNA aptamersHere we reported a simple, sensitive and specific proximity-dependent protein assay with dual DNA aptamers. Thrombin that involves two aptamers recognizing epitopes was used as a model protein. This assay depends on the coassociation of two aptamers by the simultaneous and proximate recognition of thrombin, which gives rise to a polymerase reaction with short tip complementarity attached to the aptamers. The velocity of extension determined by the concentration of thrombin was monitored in real-time using the fluorescence dye Sybr Green I. The dual-aptamer-based approach for protein detection demonstrated in this work promises a simple and agile design of aptamer probes as well as considerable sensitivity with a detection limit of 6.9 pmol/L, Compared with the proximity-based methods for protein detection, this work had shown many advantages involving easier simply probe denign, more sensitivity and convenient assay.