(360d) Self-Assembling Biosensor Protein Coatings for the Detection of Calcium Toward the Early-Stage Diagnosis of Cancer

Biosensor proteins have attracted significant attention due to their utilities in various fields, such as healthcare, environmental monitoring, and biotechnology. These sensors are designed to interact with specific analytes, such as small molecules or ions, and transduce measurable signals. For example, genetically engineered calcium ion indicators (GECIs) visualize the dynamics of intracellular Ca²⁺ by quantifying the fluorescent intensity as a function of Ca²⁺ concentration. Because of their robustness and fast response rates, GECIs have been widely used in the detection of neuronal activity. Their use would also be promising for detecting Ca²⁺ in different biological systems for cardiology and cancer research. However, there is a critical limit of GECIs in the detection of Ca²⁺ outside cells, for example, in the extracellular matrix, as Ca²⁺ is present in the range of millimoles. Furthermore, quantifying Ca²⁺ using GECIs may require a high-cost microscope. To address these challenges, we investigated the self-assembly of protein coating materials, using recombinant proteins containing GECIs, that increase the density of GECIs and modulate the sensing range of Ca²⁺ concentrations.

In this study, we used a two-component system for protein coating assembly, which exploits the specific dimerization of coiled-coil motifs and the inverse phase transition elastin-like polypeptides (ELPs). Coiled-coil motifs are helical proteins that wrap each other into super-coiled helices, and ELPs are thermo-responsive proteins that generate microstructures via inverse-phase transition, showing lower critical solution temperature (LCST) behavior. Recombinant proteins of coiled coils fused with a fluorescent protein or ELP were produced and incubated above LCST to form a coating on substrates. Microscope images confirmed the formation of a microstructured protein coating on the glass surface and incorporation of the fluorescent protein. Repeating the steps of protein deposition, we were able to improve the stability and density of the self-assembled protein. After optimization of the number of repeated steps, we showed the protein coating formation on various substrates, including glass, polymers, and metal substrates. Then, we assembled protein coatings using a GECI fusion protein and verified the functionality. We quantified the fluorescence from protein coatings using a smartphone camera, and the results showed that the intensity of the GECI coating in the Ca²⁺-free state was restored after the addition of Ca²⁺. The intensity difference depended on the concentration of Ca²⁺, which was close to the range of calcium levels in the extracellular matrix (from 0.5 to 3 mM). This is distinct from the property of soluble GECI proteins. Next, comparing the fluorescence intensity, we were able to detect the difference between the Ca²⁺ concentration at the normal (1.3 mM) and hypercalcemia state (1.8 mM) in biologically relevant fluids. This result indicates that the early diagnosis of cancers that are associated with hypercalcemia could be enabled using this biosensor platform.

In conclusion, we demonstrate a facile, cost-effective method for the fabrication of fluorescent biosensor protein coatings. We were able to quantity of Ca²⁺ in the concentration range of the extracellular matrix and detect the calcium level changes in hypercalcemia. Therefore, this new platform, which is based on cost-effective protein coating materials and fluorescent imaging using a smartphone camera, holds great promise for point-of-care (POC) applications for the early detection of cancer-related hypercalcemia.