Highly Sensitive Detection For Thrombin Using Graphene Oxide-DNA Based Sensors

Thrombin is an important multifunctional protease, which transforms fibrinogen into fibrin in coagulation. The change of the concentrations of thrombin will lead to some diseases such as thrombosis, disseminated, diffuse intravascular coagulation(DIC), etc. Traditional methods can not satisfy clinical requirements with complex technology and consuming time. Therefore, rapid and effective detection for thrombin is especially important in clinical diagnosis. In recent years, biosensors for thrombin detection as tools in molecular biology have been reported. However, most of them should be further improved in the limit of detection. Therefore, this thesis developed a few GO-DNA based sensors for the detection of thrombin, in which the aptamers were the specific DNA sequence that can specially bind to thrombin.1. Highly sensitive detection for thrombin using PEG modified graphene oxide-DNA based sensors. GO was used for the quencher and FAM modified aptamer was used as recognition elements. Polyethylene glycol(PEG) protected GO was used to prevent the protein from nonspecific adsorption on the surface of GO. After optimizing the conditions of PEG, the detection limit for thrombin was 4.8 pM, which was improved 10 times comparing with 51 pM based on bare graphene oxide.2. Highly sensitive detection for thrombin using graphene oxide-DNA based sensors combing with gold nanoparticle(GNPs). Tween 80 not only can speed up the reaction between GNPs and aptamer modified with SH, but also can prevent nonspecific adsorption on the surface of the GO. Therefore, this can improve the detection sensitivity. A GO-DNA sensor based on the values of GNPs ultraviolet absorption and the change of colour was developed. The concentrations of thrombin were calculated by comparing the change of the solution components and absorption value after thrombin was added. Therefore, it was a promising technique for detection of thrombin with free-label, rapid, and simply.The detection limit was as low as 0.68 pM.3. Highly sensitive detection for thrombin using graphene oxide-DNA by covalent linking. The FAM modified aptamer was covalently connected to the surface of activated GO by amino and removed those that physical adsorption on the surface of activated GO in order to avoid the false positive signals; On the other hand, PEG was also used to prevent the proteins using thrombin as an example from nonspecific binding to the surface of the GO. Therefore, the accuracy and sensitive detection of thrombin was improved. The detection limit was 0.034 pM.4. Highly sensitive detection for thrombin using exonuclease-assisted amplifilication graphene oxide-DNA based sensor. In this study, we fixed capture-DNA to the surface of the GO by covalent linking. It hybridized with fluorescence modified aptamer. Thrombin was recycled with the catalysis of exonuclease, The complex of TBA aptamer–thrombin was continually got, and the fluorescent signal was gradually enhanced, which realized highly detection of thrombin. We develop a method of detection for thrombin. The detection limit in this method was 0.024 pM.