Target-triggered catalytic hairpin assembly and TdT-catalyzed DNA polymerization for amplified electronic detection of thrombin in human serums.
Specific and sensitive detection of protein biomarkers is of great importance in biomedical and bioanalytical applications. In this work, a dual amplified signal enhancement approach based on the integration of catalytic hairpin assembly (CHA) and terminal deoxynucleotidyl transferase (TdT)-mediated in situ DNA polymerization has been developed for highly sensitive and label-free electrochemical detection of thrombin in human serums. The presence of the target thrombin leads to the unfolding and capture of a significant number of hairpin signal probes with free 3'-OH termini on the sensor electrode. Subsequently, TdT can catalyze the elongation of the signal probes and formation of many G-quadruplex sequence replicates with the presence of dGTP and dATP at a molar ratio of 6:4. These G-quadruplex sequences bind hemin and generate drastically amplified current response for sensitive detection of thrombin in a completely label-free fashion. The sensor shows a linear range of 0.5pM-10.0nM and a detection limit of 0.12pM for thrombin. Moreover, the developed sensor can selectively discriminate the target thrombin against other non-target proteins and can be employed to monitor thrombin in human serum samples.
Key Laboratory of Luminescent and Real-Time Analytical Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China.
Kai
K
Shi
Key Laboratory of Luminescent and Real-Time Analytical Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China.
Baoting
B
Dou
Key Laboratory of Luminescent and Real-Time Analytical Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China.
Jianmei
J
Yang
Key Laboratory of Luminescent and Real-Time Analytical Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China.
Ruo
R
Yuan
Key Laboratory of Luminescent and Real-Time Analytical Chemistry, Ministry of Education, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China. Electronic address: yunatswu@swu.edu.cn.
Yun
Y
Xiang
D052157:Aptamers, Nucleotide / Q000737:chemistry / Q000378:metabolism*; D015374:Biosensing Techniques / Q000379:methods*; D004253:DNA Nucleotidylexotransferase / Q000378:metabolism*; D055664:Electrochemical Techniques / Q000379:methods; D054856:G-Quadruplexes; D006427:Hemin / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D057230:Limit of Detection; D058105:Polymerization; D013917:Thrombin / Q000032:analysis* / Q000378:metabolism
D052157:Aptamers, Nucleotide; C081676:thrombin aptamer; D006427:Hemin; D004253:DNA Nucleotidylexotransferase; D013917:Thrombin
Catalytic hairpin assembly; Electrochemical biosensor; Signal amplification; Terminal deoxynucleotidyl transferase; Thrombin