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.

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