Nanoengineered Calcium Receptors Coupled with Microscale Thermophoresis Enable Sensitive, Low-Volume Quantification of Calcium Ions in Complex Biological Fluids

Accurate quantification of calcium ions (Ca²⁺) in biological fluids is essential for elucidating cellular physiology and diagnosing calcium-associated disorders, yet conventional analytical tools often face challenges, including limited sensitivity, interference from complex matrices, and the need for large sample volumes. Here, we report nanoengineered calcium receptors, Sens4Ca and Sens2Ca, integrated with microscale thermophoresis (MST) to achieve sensitive, robust Ca²⁺ detection using minimal sample volumes. The calcium receptors are modular fusion proteins that combine calmodulin and calmodulin-binding peptides with rigid structural domains to tailor binding affinities and stoichiometries, enabling Ca²⁺ detection across nanomolar to micromolar concentrations. Unlike traditional fluorescence-based assays, our MST platform monitors shifts in thermophoretic mobility, mitigating matrix interference and enhancing assay precision. We validate this approach for accurate Ca²⁺ quantification in minimally processed biological and diagnostic samples, including tumorigenic cells, tears, blood, and urine, demonstrating broad applicability under diverse physiological conditions. This MST-based calcium sensing platform offers a low-volume, interference-resistant approach for high-performance Ca²⁺ quantification, with broad potential for different disciplines ranging from fundamental biological research to clinical diagnostics and environmental monitoring.