| Aptamers are synthetic oligonucleotides DNA or RNA, which are isolated from random-sequence nucleic acid bibraries by "in vitro selection". They can bind to molecular targets with high affinity and selectivity, such as proteins, small molecules, metal ion, DNA, even the whole cells. Owing to aptamer's relative ease of isolation and modification, good stability, the easier storage, the high binding affinity and simplicity of in vitro selection, it have attracted more and more attentions in the area of biochemical analysis. Thrombin aptamer has two kinds of nucleic acid sequence, which can bind to thrombin at different sites with binding affinity to form G-quartet structure. In this paper, thrombin used as a model protein to develop new thrombin detection methods using aptamers. The main work is summarized as follows:(1) We established a new method for electrochemical detection of thrombin combining Fe3O4/Au magnetic separation and CdTe-aptamer probe. Fe3O4/Au magnetic nanoparticles and L-cysteine modified CdTe nanoparticles were synthesized. Fe3O4/Au magnetic nanoparticles were used as DNA immobilization and separation material, while aptamer modified CdTe nanoparticles used as detection probe. Thiolated modified capture DNA which complementary with thrombin aptamer was anchored onto the surface of Au coated magnetic beads via sulfur-gold affinity. CdTe nanoparticles modified aptamer probe hybridized with capture DNA sequence modified on magnetic beads by hybridization reaction, and then the thrombin aptsensor was obtained. When thrombin added to the detection solution, CdTe nanoparticles modified aptamer was forced to dissociate from the magnetic beads. The thrombin was detected through current peak of Cd2+ in supernate of differential pulse anodic stripping voltammetry (DPV) after separation with an extermal magnetic field. This method is sensitive, no harmful reagents, requiring only a small sample volume and has a great potential for proteins detection.(2) A new thrombin sensor was development by using methylene blue (MB) as electrochemical probe and ZrO2/AuNPs as signal amplification and magnetic separation material. MB is used as the external electroactive indicator owing to its special interaction with DNA. In this paper, A thiolated capture DNA is immobilized on the surface of modified electrode and then hybridized with thrombin aptamer (29-mer). MB intercalates into the double-strands of nucleic acids and binds specifically to guanine bases, after which the aptasensors were obtained. In the presence of thrombin, thrombin interacted with its aptamer to form G-quadruplex, while MB desorbed from electrode surface, resulting in a decrease in current. The decrement of current is proportional to the amount of thrombin. This aptasensor has wide concentration range, low detection limit, high selectivity and good reproducibility and this aptasensor was able to sensitively detect thrombin.(3) A highly sensitive detection method was developed by using hybridization amplification and electrochemical impedance technique. Since one thrombin molecule has two active sites for its two kinds of aptamer, the electrode-aptamerâ… /thrombin/aptamerâ…¡-AuNPs sandwich structure was fabricated as the sensing platform. Here, we reported a simple and highly sensitive label-free impedimetric aptasensor for thrombin detection based on the hybridized signal amplification. This strategy could not only improve the detection sensitivity but also provide a simple model for the signal amplification of the impedimetric sensors. Meanwhile, this method exhibits good selectivity.
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