Electrochemical Aptasensor For Sensitive Detection Of Protein

Proteins run through the whole life process and they are the importantcontents of life science research. The concentration of some proteins is solow. The traditional method of quantitative protein, such as gelelectrophoresis, kjeldahl method, turbidimetric method, andradioimmunoassay，just can get low sensitivity. Therefore, ultrasensitiveassays for proteins play an important role in the prevention and diagnosis ofdisease. Electrochemical sensors have widely been used in the field ofclinical detection and environmental monitoring and provide a strongsupport for the development of the cause of human health due to their fastresponse, high sensitivity, low cost, and simple instrumentation. Aptamercould bind a range of ligand targets with affinity constants comparable totheir antibody counterparts. However, they could provide several greatadvantages in biological analysis and diagnostics, such as low cost, stability,and easy modification. Electrochemical aptasensors have received particularattention. In this paper, the interaction between the aptamer and thrombinwas investigated. A hairpin electrochemical aptasensor for sensitive andspecific detection of thrombin was developed with a gold nanoparticle platform. This dissertation includes the following three parts:1. Preparation of electrochemical aptamer sensorAn anti-thrombin aptamer was engineered into an aptamer hairpin byadding specific nuleotides. And a new aptamer hairpin electrochemicalsensor was developed for thrombin detection. Upon thrombin binding, theconformation of the hairpin could “switch on”, and the aptamer-thrombincomplex gave a single stranded tail sequence complementary to thesurface-tethered C-probe in the presence of the thrombin. The remainingC-probe could hybridize with the biotinylated D-probe and the thrombincould be detected. Electrochemical impedance spectroscopy (EIS) andsquare wave voltammetric (SWV) were used to monitor the fabrication ofthe aptasensor during stepwise modifications.The fabricated aptasensorcould provide both a wide linear dynamic range (3×10-2to3nM) and a lowdetection limit (0.01nM). Nonspecific proteins had almost negligble currentresponse. A hairpin electrochemical aptasensor for thrombin detection wassuccessfully developed and exhibited high sensitivity, good selectivity.2. A Hairpin electrochemical aptasensor for sensitive and specific detectionof thrombin based on AuNPs amplificationAn electrochemical aptasensor was developed for sensitive and specificdetection of thrombin by combining gold nanoparticles (AuNPs)amplification and hairpin aptamer used in part one. Streptavidin-alkalinephosphatase was used as reporter molecule. The proposed aptasensor successfully improved the stability and high affinity of the aptamer hairpinthrough homogenous recognition, which enhanced the sensitivity andselectivity of the sensors effectively. Additionally, AuNPs were employed toincrease the active area and conductivity of electrode, thus, improving thesensitivity of the aptasensor. Under the optimal conditions, the designedthrombin detection sensor obtained a lower detection limit of0.52pM inbuffer,6.9pM in blood serum.