The microfluidic-based fluorescence enhancement of delayed fluorescence silica-embedded carbon dots for the detection and quantification of unamplified nucleic acid in clinical samples

Delayed fluorescence silica-embedded carbon dots (DF@N-CDs/SiO₂) represent an innovative and highly effective platform for nucleic acid detection. These dots exhibit remarkable fluorescence characteristics that enable rapid and sensitive diagnostics. An example of their application is in the detection of the hepatitis C virus (HCV), which can cause asymptomatic chronic infections with serious clinical consequences. The timely and sensitive detection of HCV RNA is crucial for infection control and monitoring treatment response. While current technologies, such as PCR and isothermal amplification-based strategies, are specific, they are often expensive, labor-intensive, and time-consuming, which limits their use in field settings and smaller laboratories.

This study introduces a novel technology that utilizes the Crosslinked Enhanced Emission (CEE) phenomenon. In this context, fluorescent amino-functionalized silica-coated nitrogen-doped carbon dots (N-CDs/SiO₂/NH₂) exhibit substantial fluorescence enhancement upon interaction with unamplified Hepatitis C Virus (HCV) RNA extracted magnetically from clinical samples. This method has been effectively integrated into a 3D-printed microfluidic chip and a standard 96-well plate format, achieving detection limits of 500 IU/mL and 1000 IU/mL, respectively. The chip-based assay provides results in less than 20 minutes, significantly decreasing the processing time in comparison to traditional amplification-based techniques. Analyzing 141 patient samples yielded high sensitivity (96.47%) and specificity (98.79%), underscoring the platform’s efficacy in clinical diagnostics.

This investigation marks the initial application of N-CDs/SiO2 as fluorescent probes for nucleic acid detection, providing a versatile and cost-effective solution that can be readily integrated into existing laboratory environments. Furthermore, it enhances both the speed and accuracy of HCV RNA detection.