Engineered Calmodulin Assembles into Core-Shell Particles Serving as Robust Fluorescent Calcium Sensors

Calcium is a strictly regulated physiological ion whose levels in blood and saliva act as biomarkers for a wide range of ailments, including renal, endocrine, and bone diseases. Current diagnostic approaches mainly rely on centralised laboratory assays that are accurate but slow and depend on infrastructural support. Thus, robust biosensors capable of rapid, selective calcium detection in complex biofluids are needed to enable real-time diagnostics with minimal invasive sampling. In this study, we engineered the soluble fluorescent sensor NCaMP7, a fusion of calmodulin and split neongreen fluorescent proteins, to self-assemble and bioconjugate to biopolymer particles (BPs) serving as robust and low-cost particulate calcium sensors. These particles were produced inside recombinant E. coli through a one-step, cost-effective, and scalable production process. Upon purification, the BPs retained their natural spherical structure, with the fusion proteins retaining their native conformation. Calcium-sensing functionality was achieved only when NCaMP7 was fused to the N-terminus of the biopolymer assembly protein domain (CAM-BP), enabling detection of free calcium in buffered media as well as complex biological matrices such as artificial saliva and plasma. These observations were further corroborated by fluorescence microscopy, which revealed a pronounced increase in fluorescence intensity following calcium addition, along with minimal signal decay over time, indicating low levels of photobleaching. The BP-based biosensor was also found to be moderately thermostable, retaining its calcium detection sensitivity for four weeks at 25 and 37 °C. Overall, these results position CAM-BPs as a robust, scalable, and cost-efficient platform for in vitro calcium sensing with broad application potential.