A portable microchip for ultrasensitive and high-throughput assay of thrombin by rolling circle amplification and hemin/G-quadruplex system.
In this work, a convenient and high-throughput colorimetric assay was developed on an aptamer-modified microchip for ultrasensitive detection of thrombin using rolling circle amplification and G-quadruplex DNAzyme. This system consisted of an aptamer-modified microchip and a secondary aptamer. The secondary aptamer contained a thrombin aptamer and a primer with a G-quadruplex circular template. RCA technology was used to improve the sensitivity by producing the multiple G-quadruplex units. To generate colorimetric signal, G-quadruplex DNAzyme was used to catalyze the H2O2-mediated oxidation of 2,2'-azinobis (3-ethylbenzothiozoline)-6-sulfonic acid. At the optimal conditions, the linear range for thrombin was 0.100-50.000 pg/mL, and the limit of detection was down to 0.083 pg/mL. Moreover, the developed method was successfully applied to detect thrombin from human plasma and serum, indicating that this approach has great potential in clinical diagnosis and medical investigation.
State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing 100029, China; Department of Chemistry, Beijing Key Laboratory of Microanalytical Methods and Instrumentation, Tsinghua University, Beijing 100084, China.
Xuexia
X
Lin
Department of Chemistry, Beijing Key Laboratory of Microanalytical Methods and Instrumentation, Tsinghua University, Beijing 100084, China.
Qiushui
Q
Chen
Department of Chemistry, Beijing Key Laboratory of Microanalytical Methods and Instrumentation, Tsinghua University, Beijing 100084, China.
Wu
W
Liu
Department of Chemistry, Beijing Key Laboratory of Microanalytical Methods and Instrumentation, Tsinghua University, Beijing 100084, China.
Haifang
H
Li
Department of Chemistry, Beijing Key Laboratory of Microanalytical Methods and Instrumentation, Tsinghua University, Beijing 100084, China. Electronic address: jmlin@mail.tsinghua.edu.cn.
Jin-Ming
JM
Lin
D052157:Aptamers, Nucleotide / Q000737:chemistry / Q000378:metabolism*; D001483:Base Sequence; D052160:Benzothiazoles / Q000378:metabolism; D015374:Biosensing Techniques / Q000295:instrumentation; D003124:Colorimetry / Q000295:instrumentation; D021881:DNA, Catalytic / Q000737:chemistry / Q000378:metabolism*; D004867:Equipment Design; D054856:G-Quadruplexes; D006427:Hemin / Q000378:metabolism*; D057166:High-Throughput Screening Assays / Q000295:instrumentation; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation*; D008969:Molecular Sequence Data; D021141:Nucleic Acid Amplification Techniques / Q000295:instrumentation; D010084:Oxidation-Reduction; D013451:Sulfonic Acids / Q000378:metabolism; D013917:Thrombin / Q000032:analysis* / Q000378:metabolism
D052157:Aptamers, Nucleotide; D052160:Benzothiazoles; D021881:DNA, Catalytic; D013451:Sulfonic Acids; C081676:thrombin aptamer; C002502:2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid; D006427:Hemin; D006861:Hydrogen Peroxide; D013917:Thrombin
Colorimetric assay; G-quadruplex; Microchip; Rolling circle amplification