Investigation of RCA Aptamer-Based Microfluidic System for E. Coli O157:H7 Detection

Abstract

Aptamers have been widely used as capturing agents to improve the sensitivity and specificity of microfluidic detection systems. This study uses rolling circle amplification (RCA) to produce repeating capturing aptamers or capturing RCA (cRCA) aptamers. The in situ cRCA reaction is carried out on the inner surfaces of microchannel to generate a long single-stranded DNA molecule containing tandem repeating cRCA aptamers that are specific for E. coli O157:H7 cells recognition. While optimizing the capturing performance of the cRCA aptamer-based microfluidic detection system, we find that the best capturing performance is achieved when the in situ cRCA reaction is carried out for 2 hours in order to modify the microchannel. In addition, the cRCA products are characterized using water contact angle measurement, fluorescence test, real-time quantitative PCR analysis, and atomic force microscopy (AFM). Furthermore, in order to test the sensitivity of the optimized detection system, the cRCA aptamer modified microchannel is used to detect iced tea and bottled water samples doped with E. coli O157:H7 cells. Our results show that the detection sensitivity and the capturing performance of this optimized cRCA aptamer detection system are about 10 times that of the conventional unit aptamer modified microfluidic detection system. In addition, our results also show that the cRCA aptamer approach had consistently higher target capturing efficiency at higher flow rates, suggesting its potential applications in sensitive detections under high throughput conditions.