The New Methods For Thrombin Detection Based On Aptamer Sensor

Protein is one of the most important components of organisms,and it participates in every process of life activities.The change of protein content directly affects the biological function of human body,even if with the extremely trace amounts,it will also trigger physiological and pathological reactions,especially the protein molecules related to diseases,therefore,the determination of protein expression level is of great significance for the prevention,early diagnosis and treatment of diseases.With the continuous development of protein detection technology,it is no longer limited to the use of antibodies as biomolecular recognition elements.Because the aptamer has some advantages that antibodies cannot match,such as a wide range of target molecules,high stability,easy modification,easy preparation,no immunogenicity,the sensor using aptamer as recognition element exhibits the characteristics of high sensitivity,high specificity and easy operation,which makes the protein detection method based on aptamer sensor become the current research hotspot and the most promising method for protein detection.In this paper,thrombin protein was used as the detection object,and different types of new sensors were constructed for the quantitative analysis of thrombin,in the meanwhile,the performance of the sensor was studied and discussed.The detection performance of these new methods has been greatly improved on the basis of the original types,and the main contents of this paper are as follows:In this study,three types of aptamer sensors were constructed with different methods,and the performance of the constructed sensors were verified and discussed by taking thrombin protein as the classical analysis object.（1）A type of switch aptamer electrochemical sensor was constructed to detect thrombin using the host-guest recognition mechanism ofβ-cyclodextrin（β-CD）and ferrocene（Fc）.The aptamer that modified with Fc at it’s terminal was used as detection probe,and fixed on the surface of the gold electrode via the host-guest recognization ofβ-CD and Fc,after binding to thrombin,the conformation of the aptamer undergoes a change from vertical linear to a"G-tetramer"and then detached from the surface of the electrode,which result in a decrease in the redox current of the probe and product"signal-off"effect.The detection results indicated that the thrombin concentration had a consideration linear relationship to the response signal between 5.0×10^-13 mol/L⁵.0×10^-9 mol/L,and as low as 2.0×10^-13 mol/L thrombin has been detected（S/N=3）.The sensor constructed easily and possess excellent regeneration,which providing an important reference method for the efficient and sensitive determination of thrombin.（2）A functionalized nanocomposites-based electrochemiluminescence（ECL）aptamer sensor for the detection of thrombin was constructed.The Ru（bpy）₃²⁺/β-cyclodextrin-Au nanoparticles/nanographene([Ru（bpy）₃]²⁺/β-CD-AuNPs/NGP)composite nanomaterials were used to modify the glassy carbon electrode（GCE）surface,and the thrombin aptamers（TBA1 and TBA2 with a 1:1 molar ratio）were labeled with ferrocene Fc acting as the probes,were attached to the modified electrode surface via the host–guest recognition betweenβ-CD and Fc.In the absence of thrombin,the quenching of Fc to[Ru（bpy）₃]²⁺was maintained,and“signal-off”signal of ECL was observed.However,in case of the specific combination of the aptamer probes and thrombin,the configuration of probes changed and escaped from the electrode surface,which result the quenching disappeared,and the ECL signal was changed from“off”to“on”.The detection results showed that the thrombin concentration had a consideration linear relationship to the response signal between 4.0×10^-13 mol/L¹.0×10^-9mol/L,and the detection limit of thrombin was 2.3×10^-13 mol/L（S/N=3）.The application of nanocomposites in this sensor amplified the effect of“signal-on”,and can be used for sensitive detection of thrombin signal amplification.（3）In homogeneous system,an unmodified sensing analysis method was established to detect thrombin.The aptamer probe with the dual-hairpin structure was labeled with methylene blue（MB）at its 3’terminal and thiol（SH）at its 5’terminal respectively,the closed loop structure of probe impeded the connection between thiol and the surface of the electrode.However,the loop structure of dual-hairpin probe opened once treatment with the target protein of thrombin,consequently,the thiol could attach to the electrode surface rapidly to form Au-S self-assembly with the action of potential-assisted,and then the electrochemical signal of MB could be recorded for measurement.The detection results indicated that the thrombin concentration had a consideration linear relationship to the response signal between8.4×10^-12 mol/L⁴.2×10^-9 mol/L,and as low as 3.5×10^-12 mol/L thrombin has been detected（S/N=3）.The designed homogeneous strategy can be used to detect thrombin with high specificity and sensitivity.