A portable microchip for ultrasensitive and high-throughput assay of thrombin by rolling circle amplification and hemin/G-quadruplex system

•The ultrasensitivity of thrombin detection was based on rolling circle amplification and G-quadruplex DNAzyme catalysis.•The colorimetric functional nucleic acid assay was applied onto a microfluidic chip by G-quadruplex/hemin with ABTS and peroxide.•The multichannels microchips can be used as a promising point-of-care device with low cost and simple operation for disease diagnosis.•The proposal method can be well used to analyze thrombin in human plasma.

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.

Graphical abstractA simple and versatile microchip for thrombin detection was developed based on rolling circle amplification and ABTS–H2O2/hemin/G-quadruplex system.Figure optionsDownload full-size imageDownload as PowerPoint slide