Microfluidic-based fluorescence enhancement of silica-embedded carbon dots for direct detection and quantification of unamplified HCV RNA in clinical samples

Background: Hepatitis C Virus (HCV) is an asymptomatic chronic infection with serious clinical consequences. Timely and sensitive detection of HCV RNA is critical for infection control, treatment response follow up. While current technologies, such as PCR or isothermal amplification-based strategies, are specific, they are expensive, labor intensive, time consuming limiting their use in field and small laboratories. Results: We introduce a novel technology for detecting nucleic acids, as exemplified by HCV RNA, in clinical specimens. This approach utilizes the crosslinked Enhanced Emission (CEE) phenomena upon mixing fluorescent amino functionalized silica-coated nitrogen-doped carbon dots (N-CDs/SiO₂/NH₂) with magnetic extracted unamplified HCV RNA showed a significant and immediate fluorescence enhancement. This method was integrated into a semi-automated 3D printed microfluidic chip, wherein the unamplified RNA was mixed with the N-CDs/SiO₂/NH₂. The assay was also employed on the conventional 96-well plate format. This assay offers high sensitivity with detection limit of 500 IU/ml and 1000 IU/ml for the chip and well plate respectively. The sample-to-result time was <20 min on the chip and is simpler than the amplification-based techniques. Analyzing 141 patient samples yielded sensitivity and specificity of 96.47 % and 98.79 % respectively. Significance: This application of N-CDs/SiO2 as nucleic acid fluorescent probes for the first time, offers a versatile, cost-effective, and universal platform for nucleic acids detection. The developed system can be employed in conventional microwell plate-based detection with minimal modifications to current laboratory setups. Additionally, it was integrated into a 3D-printed microfluidic chip enabling enhancement in specificity, sensitivity and accuracy.