Gold-nanoparticle-embedded hydrogel droplets with enhanced fluorescence for imaging and quantification of proteins in cells.

Conventional cellular protein detection techniques such as immunocytochemistry and flow cytometry require abundant cells, posing multiple challenges, including difficulty and cost for obtaining enough cells and the potential for clogging the instrument when using flow cytometry. Also, it is challenging to conduct cellular protein imaging and quantification simultaneously from a single experiment. We present a novel 3D platform, which integrates highly biocompatible cell-entrapped alginate hydrogel droplet array with gold-nanoparticle (AuNP)-based metal enhanced fluorescence (MEF), to achieve simultaneous imaging and quantification of proteins in intact cells in a sensitive manner. Compared to 2D immunocytochemistry, this 3D system allows for a higher cell loading capacity per unit area; together with the MEF-based signal enhancement from the embedded AuNPs, sensitive protein quantification was realized. Furthermore, compared to flow cytometry, this platform allows for protein imaging from individual cells. Taking the detection of EpCAM protein in ovarian cancer cells as a model, we optimized the AuNP size and concentration for optimal fluorescent signals. The 5 nm AuNPs at 6.54 × 10 particles/mL proved to be the most effective in signal enhancement, providing 2.4-fold higher signals compared to that without AuNPs and 6.4-fold higher signals than that of 2D immunocytochemistry. The number of cells required in our technology is 1-3 orders of magnitude smaller than that of conventional methods. This AuNP-embedded hydrogel platform combines the benefits of immunocytochemistry and flow cytometry, providing increased assay sensitivity while also allowing for qualitative analysis through imaging, suitable for protein determination in a variety of cells.