Intracellular metal enhanced fluorescence utilizing gold nanoparticles embedded in hydrogel droplets for sensitive protein detection in cells

Cellular protein detection is necessary for monitoring various biological pathways associated with complex diseases such as cancer. Detecting intracellular proteins is more challenging than cell surface protein detection because most antibodies cannot freely cross the cellular membrane. Cellular membrane permeabilization is thus required to facilitate the antibody diffusion and achieve adequate detection when combined with various conventional protein detection methods (e.g., flow cytometry and immunocytochemistry). However, these methods come with sensitivity challenges when a large number of cells are unavailable. Therefore, for early disease diagnostics and prognosis, novel methods are needed to achieve higher sensitivity to track disease-related proteins from small amounts of cells at early stages of disease progression. Here we applied a novel biosensing platform that co-entraps cells and gold nanoparticles in 3D hydrogel droplets to achieve sensitive fluorescence-based detection and quantification of intracellular proteins in intact cells. Importantly, intracellular metal enhanced fluorescence (iMEF) was achieved using gold nanoparticles via cell permeabilization. The intracellular goldnanoparticles, together with the high cell loading capacity of the hydrogel droplets, enhanced the fluorescence signals and in turn improved assay sensitivity compared to conventional methods. After assay optimization, we demonstrated the detection of Heat Shock Protein 70, a common cancer marker, with as little as 250 cells. This technology was also able to distinguish between cancerous and non-cancerous samples when mimicking a clinical fine needle aspiration procedure. Compared to conventional assays measuring the same protein, the assay sensitivity was improved by two to three orders of magnitude, allowing for highly sensitive intracellular protein detection from minimal amounts of cells.