In our lab we have previously established a reverse-transfected cell array technology in a microfluidic system to screen GPCR libraries against sequentially injected samples. Activated receptors induce a transient calcium increase that is visualized by a fluorescence-based ratiometric FRET calcium sensor using a fluorescence microscope. However, such specialized read-out equipment is costly and not easily miniaturized for use as a screening tool outside a biological lab. Furthermore, complex samples like coffee and milk are problematic to measure due to sample fluorescence and light reflection. To avoid the need for a fluorescence-excitation light source we switched to a luciferase-based calcium reporter system. Bioluminescent calcium sensors are available as ratiometric BRET-sensors like CalfluxVTN and intensity-based split luciferase sensors like GeNL Ca²⁺. The aim of this study was to evaluate these sensors for GPCRs activation in the context of a smartphone-based recording system.
Luciferase enzymes yield a constant glow of light at a stable concentration of substrate, but typically these substrates may be rapidly oxidized when mixed prior with different samples. To prevent oxidation we developed a substrate mixing setup where the substrate stock solution is merged immediately prior to entering the receptomics flowcell. We found that an iPhone12ProMax colour camera operated by a low light imaging app, mounted with a macro-lens and placed upon a light-tight box containing a microfluidic flowcell attached to a pump was all we needed to perform a BRET-analysis using the separate RGB signals of the colour camera. One to two second exposures resulted in high quality calcium signals from 300 receptor spots on a 1 square cm array. This demonstrated the potential of the development of robust BRET assays in small instruments outside the lab.

This work was supported by NWO project TO2 Luminose, a joint project between BU Bioscience and Insectsense BV.