Many biological events such as mutations or aberrant post-translational modifications can alter the conformation and/or folding stability of proteins and their subsequent biological function, which may trigger the onset of diseases like cancer. Evaluating protein folding is hence crucial for the diagnosis of these diseases. Yet, it is still challenging to detect changes in protein folding, especially if they are subtle, in a simple and highly sensitive manner with the current assays. Herein, we report a new colloidal-based interfacial biosensing approach for qualitative and quantitative profiling of various types of changes in protein folding; from denaturation to variant conformations in native proteins, such as protein activation via mutations or phosphorylation. The approach is based on the direct interfacial interaction of proteins freely available in solution with added tannic-acid-capped gold nanoparticles, to interrogate their folding status in their solubilized form. We found that under the optimized conditions, proteins can modulate colloids solvation according to their folding or conformational status, which can be visualized in a single step, by the naked eye, with minimal protein input requirements (limit of detection of 1 ng/μL). Protein folding detection was achieved regardless of protein topology and size without using conformation-specific antibodies and mutational analysis, which are the most common assays for sensing malfunctioning proteins. The approach showed excellent sensitivity, superior to circular dichroism, for the detection of the very subtle conformational changes induced by activating mutations and phosphorylation in epidermal growth factor receptor (EGFR) and extracellular signal-regulated kinase (ERK) proteins. This enabled their detection even in complex samples derived from lung cancer cells, which contained up to 95% excess of their wild-type forms. A broader clinical translation was shown via monitoring the action of conformation-restoring drugs, such as tyrosine kinase inhibitors, on EGFR conformation and its downstream protein network, using the ERK protein as a surrogate.
Keywords: colorimetric response; gold nanoparticles; protein conformation; protein folding; tannic acid.