Gold-nanoparticle-embedded membrane (GEM) for highly sensitive multiplexed sandwich immunoassays

Abstract

For early disease diagnostics, highly sensitive biosensing methods are required to detect disease-related biomolecules in biofluids at low concentrations. Further, for complex diseases such as cancer, multiplexed detection is required to simultaneously measure multiple analytes in a single assay. Current technologies either lack sensitivity, multiplexing capabilities, or require complicated micro/nano-fabrication processes and specialized instrumentation, limiting their broader use in the biomedical research community as well as in clinical diagnostic applications. In this work, we have developed a gold-nanoparticle-embedded membrane (GEM) platform that consists of 3D nitrocellulose membranes preloaded with gold nanoparticles in microliter compartments for highly sensitive, multiplexed metal enhanced fluorescence (MEF)-based detection. After the development of the compartmentalized GEM and the optimization of size, shape, and concentration of the gold nanoparticles to achieve optimal MEF, we demonstrated its application for scalable multiplexed sandwich immunoassays in a microarray format for the quantification of three cancer-related proteins in blood plasma. In PBS buffer solution, the limit of detection for GM-CSF, TNF-α, and EGFR were improved by 287-folds, 949-folds, and 9.8-folds, respectively, when using the GEM compared to conventional ELISA. When spiked in pooled human plasma, low pg/mL sensitivity was also achieved for each protein, demonstrating its application for complex samples. Long-term storage was demonstrated for potential distribution of the GEM for wide availability among the biomedical and clinical community, facilitating technology dissemination. This technology offers simple, highly sensitive, and multiplexed detection capabilities with low reagent and sample consumption, suitable for accurate and early disease diagnostics.