Antibody-Driven Assembly of Plasmonic Core-Satellites to Increase the Sensitivity of a SERS Vertical Flow Immunoassay.

Here, we describe a SERS-based vertical flow assay as a platform technology suitable for point-of-care (POC) diagnostic testing. A capture substrate is constructed from filter paper embedded with spherical gold nanoparticles (AuNPs) and functionalized with an appropriate capture antibody. The capture substrate is loaded into a filtration device and connected to a syringe to rapidly and repeatedly pass the sample through the sensor for efficient antigen binding. The antigen is then labeled with a SERS-active detection probe. We show that only a few Raman reporter molecules, exclusively located adjacent to the plasmonic capture substrate, generate detectible signals. To maximize the signal from underutilized Raman reporter molecules, we employ a secondary signal enhancing probe that undergoes antibody-directed assembly to form plasmonic core-satellites. This facile enhancement step provides a 3.5-fold increase in the signal and a detection limit of 0.23 ng/mL (1.6 pM) for human IgG. This work highlights the potential to rationally design plasmonic architectures using widely available and reproducible spherical AuNPs to achieve large SERS enhancements for highly sensitive POC diagnostics.

SERS-based immunoassay on a plasmonic syringe filter for improved sampling and labeling efficiency of biomarkers.

Rapid, sensitive, and quantitative detection of biomarkers is needed for early diagnosis of disease and surveillance of infectious outbreaks. Here, we exploit a plasmonic syringe filter and surface-enhanced Raman spectroscopy (SERS) in the development of a rapid detection system, using human IgG as a model diagnostic biomarker. The novel assay design facilitates multiple passages of the sample and labeling solution through the detection zone enabling us to investigate and maximize sampling efficiency to the capture substrate. The vertical flow immunoassay process in this study involves the utilization of filter paper embedded with gold nanoparticles (AuNPs) to form a plasmonic substrate. Capture antibody (anti-human IgG) is then immobilized onto the prepared plasmonic paper and inserted into a vertical flow device (syringe filter holder). Sample solution is passed through the filter paper and the target antigen (human IgG) is selectively captured by the immobilized antibody to form an antibody-antigen complex. Next, functionalized AuNPs as extrinsic Raman labels (ERLs) are passed through the filter paper to label the captured biomarker molecules forming a layered structure. This sandwiched geometry enhances plasmonic coupling and SERS signal to provide highly sensitive detection of biomolecules. Systematic studies to investigate the impact of multiple infuse/withdraw cycles of the sample and labeling solutions reveal that antigen and ERL binding are maximized with 10 and 20 cycles, respectively. The optimized assay achieves a detection limit of ∼0.2 ng mL-1 for human IgG with a total assay time of less than 5 minutes, meeting the demands for rapid point of care diagnostics. Additionally, the optimized platform was implemented in the quantitative analysis of the SARS-CoV-2 nucleocapsid protein, the typical target in commercial, FDA-approved rapid antigen tests for COVID-19.