A generic microfluidic biosensor of G protein-coupled receptor activation—monitoring cytoplasmic [Ca2+] changes in human HEK293 cells

•A biosensor for monitoring cytoplasmic [Ca2+] levels in mammalian cell lines is presented.•The biosensor is designed to allow monitoring of activation of specific membrane receptors.•A resealable microfluidic flow cell allows easy surface modification and cell deposition.•The biosensor can detect activating substances in sequential series of analytes every 3 min.•The read out is based on fluorescent image acquisition and analysis every 2–4 s.

Cell lines expressing recombinant G-protein coupled receptors (GPCRs) are activated by specific ligands resulting in transient [Ca2+] rises that return to basal levels in 30–60 s. Yellow Cameleon 3.6 (YC3.6) is a genetically encoded calcium indicator which can be co-expressed to monitor these cytosolic [Ca2+] changes in real-time using Förster (Fluorescence) resonance energy transfer (FRET). On this basis, we designed the prototype of a generic microfluidic biosensor of GPCR activation, imaging [Ca2+] changes in recombinant human HEK293 cells, which express a combination of a GPCR (Neurokinin 1) and YC3.6. An internal reference for non-specifically induced [Ca2+] changes were YC3.6 cells without GPCR but expressing a red fluorescent protein (mCherry) for identification. These cell lines were grown as a mixed population in a flow cell with a volume of ~50 µl and a flow cell surface of 170 mm2. Cells were activated by brief exposures to specific and non-specific analytes using an injection valve with a flexible sample volume (tested range 5–100 µl) at a flow speed of 100 µl/min. A flow cell surface of 0.2 mm2 with 50 cells was imaged every 2–4 s to obtain signal kinetics. The lower limit of detection was 30 pM Substance P (SP, 2 pg/50 µl), and reproducible responses to repeated injections every 3 min were obtained at 1 nM SP. This biosensor was designed for ~50 cells for statistical reasons, but at a lower limit of 1 receptor- and 1 reference-cell, specific ligand detection is still feasible.