PCR-coupled Paper-based Surface-enhanced Raman Scattering (SERS) Sensor for Rapid and Sensitive Detection of Respiratory Bacterial DNA

Abstract

A paper-based surface-enhanced Raman scattering (SERS) substrate composed of silver-nanowires (AgNWs) was coupled with polymerase chain reaction (PCR) for rapid and sensitive determination of respiratory bacterial DNA. EvaGreen dye, which is a DNA intercalating molecule, was introduced to the low-cycled PCR product, and the difference in dye intercalation in the DNA structure was quantified by Raman spectroscopy to verify the presence of a target gene. Mycoplasma pneumoniae (M. Pne), was selected as a target for the sensing demonstration. At various gene concentrations and amplification cycles, detection capability of fluorescence-based real-time PCR (RT-PCR) and the PCR-coupled SERS method were compared to develop a DNA detection system capable of low-cycle amplification and sensitive DNA sensing. After 10 cycles, the PCR-coupled SERS method showed enhanced detection capability with a DNA detection limit of 3.12 pg/μL. Then, the SERS substrate was prepared as a rapid kit that included a test line and negative and positive control lines. Target DNA after 10 cycles of amplification was successfully discriminated compared to non-target DNA and it was statistically relevant. The developed system is expected to be used as a detection method for various bacteria and viruses, and also be integrated with diverse POC diagnostic systems.