Calcium ions play an essential role in a wide range of physiological processes, including neurotransmission, hormone secretion, and cell proliferation, leaving a strong need for
accurate sensing of calcium concentrations both inside and outside cells. For instance, monitoring intracellular calcium dynamics can capture neuronal activity, while detecting
disruptions in extracellular calcium homeostasis can help identify pathological conditions such as cardiovascular disease or cancer. To measure calcium levels in cells, green
fluorescent protein has been engineered into genetically encoded calcium indicator proteins, such as GCaMP. The calcium sensor protein is incorporated into cells as plasmid
DNA, which enables its expression and localization in cells. However, simultaneous fluorescent imaging calcium signals from many cells often suffers from low signal-to-noise
ratio and artifactual correlations of neural activity. Moreover, GCaMP is primarily designed for intracellular calcium sensing, limiting its use for extracellular calcium detection. To
address these challenges, we developed modular, genetically programmable protein assemblies for enhanced intracellular calcium imaging as well as robust extracellular
calcium sensing, using GCaMP. Our strategy leverages leucine zippers, also known as coiled coils, which are α-helical motifs that form highly specific and stable dimers. By
recombining coiled coils with GCaMP, we created a modular protein assembly system for building supramolecular fluorescent biosensor materials. In neuronal cells, expression of
coiled-coil-fused GCaMP led to the formation of sensor protein materials localized to function specifically within the cell body, significantly enhancing the accuracy of intracellular
calcium imaging. Additionally, we designed and produced recombinant proteins that form self-assembled fluorescent biosensor protein films. These coatings exhibit the enhanced
signal intensity and are highly processable to fabricate biosensor devices that detect calcium ions in a low-cost portable setting, offering a promising point-of-care solution to
detect a disruption in extracellular calcium homeostasis such as hypercalcemia. In summary, our work demonstrates a modular protein assembly strategy to construct calcium-
sensing fluorescent protein materials for enhanced functionality and broad applicability with strong potential for extension to detect a range of biological target molecules.
