The Effect of Calcium Buffering and Calcium Sensor Type on the Sensitivity of an Array-Based Bitter Receptor Screening Assay.

The genetically encoded calcium sensor protein Cameleon YC3.6 has previously been applied for functional G protein-coupled receptor screening using receptor cell arrays. However, different types of sensors are available, with a wide range in [Ca2+] sensitivity, Hill coefficients, calcium binding domains, and fluorophores, which could potentially improve the performance of the assay. Here, we compared the responses of 3 structurally different calcium sensor proteins (Cameleon YC3.6, Nano140, and Twitch2B) simultaneously, on a single chip, at different cytosolic expression levels and in combination with 2 different bitter receptors, TAS2R8 and TAS2R14. Sensor concentrations were modified by varying the amount of calcium sensor DNA that was printed on the DNA arrays prior to reverse transfection. We found that ~2-fold lower concentrations of calcium sensor protein, by transfecting 4 times less sensor-coding DNA, resulted in more sensitive bitter responses. The best results were obtained with Twitch2B, where, relative to YC3.6 at the default DNA concentration, a 4-fold lower DNA concentration increased sensitivity 60-fold and signal strength 5- to 10-fold. Next, we compared the performance of YC3.6 and Twitch2B against an array with 11 different bitter taste receptors. We observed a 2- to 8-fold increase in sensitivity using Twitch2B compared with YC3.6. The bitter receptor arrays contained 300 spots and could be exposed to a series of 18 injections within 1 h resulting in 5400 measurements. These optimized sensor conditions provide a basis for enhancing receptomics calcium assays for receptors with poor Ca2+ signaling and will benefit future high-throughput receptomics experiments.

Statistical models discriminating between complex samples measured with microfluidic receptor-cell arrays.

Data analysis for flow-based in-vitro receptomics array, like a tongue-on-a-chip, is complicated by the relatively large variability within and between arrays, transfected DNA types, spots, and cells within spots. Simply averaging responses of spots of the same type would lead to high variances and low statistical power. This paper presents an approach based on linear mixed models, allowing a quantitative and robust comparison of complex samples and indicating which receptors are responsible for any differences. These models are easily extended to take into account additional effects such as the build-up of cell stress and to combine data from replicated experiments. The increased analytical power this brings to receptomics research is discussed.

Calcium Imaging of GPCR Activation Using Arrays of Reverse Transfected HEK293 Cells in a Microfluidic System.

Reverse-transfected cell arrays in microfluidic systems have great potential to perform large-scale parallel screening of G protein-coupled receptor (GPCR) activation. Here, we report the preparation of a novel platform using reverse transfection of HEK293 cells, imaging by stereo-fluorescence microscopy in a flowcell format, real-time monitoring of cytosolic calcium ion fluctuations using the fluorescent protein Cameleon and analysis of GPCR responses to sequential sample exposures. To determine the relationship between DNA concentration and gene expression, we analyzed cell arrays made with variable concentrations of plasmid DNA encoding fluorescent proteins and the Neurokinin 1 (NK1) receptor. We observed pronounced effects on gene expression of both the specific and total DNA concentration. Reverse transfected spots with NK1 plasmid DNA at 1% of total DNA still resulted in detectable NK1 activation when exposed to its ligand. By varying the GPCR DNA concentration in reverse transfection, the sensitivity and robustness of the receptor response for sequential sample exposures was optimized. An injection series is shown for an array containing the NK1 receptor, bitter receptor TAS2R8 and controls. Both receptors were exposed 14 times to alternating samples of two ligands. Specific responses remained reproducible. This platform introduces new opportunities for high throughput screening of GPCR libraries.

A generic microfluidic biosensor of G protein-coupled receptor activation-monitoring cytoplasmic [Ca(2+)] changes in human HEK293 cells.

Cell lines expressing recombinant G-protein coupled receptors (GPCRs) are activated by specific ligands resulting in transient [Ca(2+)] rises that return to basal levels in 30-60s. Yellow Cameleon 3.6 (YC3.6) is a genetically encoded calcium indicator which can be co-expressed to monitor these cytosolic [Ca(2+)] 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 [Ca(2+)] 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 [Ca(2+)] 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 ~50µl and a flow cell surface of 170mm(2). Cells were activated by brief exposures to specific and non-specific analytes using an injection valve with a flexible sample volume (tested range 5-100µl) at a flow speed of 100µl/min. A flow cell surface of 0.2mm(2) with 50 cells was imaged every 2-4s to obtain signal kinetics. The lower limit of detection was 30pM Substance P (SP, 2pg/50µl), and reproducible responses to repeated injections every 3min were obtained at 1nM 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.

A broad set of different llama antibodies specific for a 16 kDa heat shock protein of Mycobacterium tuberculosis.

BACKGROUND: Recombinant antibodies are powerful tools in engineering of novel diagnostics. Due to the small size and stable nature of llama antibody domains selected antibodies can serve as a detection reagent in multiplexed and sensitive assays for M. tuberculosis. METHODOLOGY/PRINCIPAL FINDINGS: Antibodies for Mycobacterium tuberculosis (M. tb) recognition were raised in Alpaca, and, by phage display, recombinant variable domains of heavy-chain antibodies (VHH) binding to M. tuberculosis antigens were isolated. Two phage display selection strategies were followed: one direct selection using semi-purified protein antigen, and a depletion strategy with lysates, aiming to avoid cross-reaction to other mycobacteria. Both panning methods selected a set of binders with widely differing complementarity determining regions. Selected recombinant VHHs were produced in E. coli and shown to bind immobilized lysate in direct Enzymelinked Immunosorbent Assay (ELISA) tests and soluble antigen by surface plasmon resonance (SPR) analysis. All tested VHHs were specific for tuberculosis-causing mycobacteria (M. tuberculosis, M. bovis) and exclusively recognized an immunodominant 16 kDa heat shock protein (hsp). The highest affinity VHH had a dissociation constant (KD) of 4 × 10(-10) M. CONCLUSIONS/SIGNIFICANCE: A broad set of different llama antibodies specific for 16 kDa heat shock protein of M. tuberculosis is available. This protein is highly stable and abundant in M. tuberculosis. The VHH that detect this protein are applied in a robust SPR sensor for identification of tuberculosis-causing mycobacteria.