Development of One-step Vertical Flow Immunoassay System for Onsite Diagnosis Assisted by Surface-enhanced Raman Scattering

Objectives: A study was conducted to address two major limitations of the vertical flow assay (VFA) used in point-of-care diagnostics: low sensitivity due to visual analysis, and the inconvenience of requiring multiple steps, such as washing.Methods: To overcome these issues, in this study, the VFA was integrated with surface-enhanced Raman scattering (SERS) for high sensitivity and applied a one-step analysis method using a premix.The developed one-step SERS-based VFA system is labeled by a Raman probe consisting of detection antibodies, Raman reporter (4-mercaptobenzoic acid [4-MBA]), and gold nanoparticles, and is based on a sandwich system composed of capture antibodies and analytes.To facilitate immediate SERS measurement, a customized cartridge was designed to eliminate the need for removal before analysis.Sensitivity was further improved by optimizing the sample dilution buffer, concentration of the capture antibody, and size of gold nanoparticles embedded in the Raman probe.Results: Using only 60 L of analyte, the system exhibited excellent linearity (R = 0.9988) up to 10 ng/mL, with a detection limit of 0.66 ng/mL for the C-reactive protein (CRP) antigen, outperforming traditional lateral flow and VFAs.Conclusion: This is superior to the lateral flow assay and VFA, which use CRP antigen as a biomarker.It is emphasized that the developed one-step SERS-based VFA system can be used as a potential point-of-care testing tool for diagnosing various biomarkers as well as CRP.