Receptomics, design of a microfluidic receptor screening technology

Abstract

Chapter 1 Introduction Chapter 2 A generic microfluidic biosensor of GPCR-activation-monitoring cytoplasmic Ca 2+ flux in human HEK293 cells Chapter 3 Metabolomics meets functional assays: coupling LC-MS and microfluidic cell-based receptor-ligand analyses Chapter 4 Calcium imaging of GPCR activation using arrays of reverse transfected HEK293 cells in a microfluidic system Chapter 5 The effect of calcium buffering and calcium sensor type on the sensitivity of an array-based bitter receptor screening assay Chapter 6 Statistical models discriminating between complex samples measured with microfluidic receptor-cell arrays Chapter 7 General discussion References Summary Nederlandse samenvatting Abbreviations Acknow ledgements About the author List of publications Overview of completed training activities C H A P T E R 18 Chapter 1 | Introduction Figure 3, Receptor cell assay platforms (diagram adapted from Martins et al 2012). A, The multiwell plate format; cells are seeded on the surface of a multi well plate and (top-down) transfected with receptor coding plasmid DNA. Calcium dye loading, subsequent washing steps and test sample dosing are done using fluid dispensers. The test conditions are multiplexed by using multiple wells and multiple plates making this platform compatible with automation but at relatively high costs and low assay flexibility [90]. B, Receptor cell arrays are created within one well chamber by printing cells [97] or by printing receptor coding plasmid DNA into a micro well and by reverse transfection with cells [81, 85]. Arrays are co-transfected with fluorescent indicator proteins to locate the array. Array washing and sample dosing are done using fluid dispensers. This combines the HTS nature of multi-well plates with the possibility to screen multiple receptors and controls in a single well. C, In microfluidic platforms the cells are cultured or assembled into a microfluidic system [98, 99]. A system can be designed so that different channels address different chambers with (reverse transfected) cells analogous to A but with the option of repeated exposures [84] or a reverse transfected array can be enclosed in one large flowcell, sequentially addressing all spots at once with different samples analogous to B but with the option of repeated challenges and larger arrays [100]. Fluid dispenser Fluid dispenser Adherent cells Substrate Cover layer / flowcell with gasket Fluid Multiwell plate Adherent cell layer Adherent cells Substrate / well plate (A) Well plate HTS (B) Cell microarrays (C) Cell microfluidics Live cell assay platform Multiplexing Single flowcell cell array: Receptomics Multiple chambers Cell array in a well or on a slide Multiple well plates

Tongue-on-a-Chip: Parallel Recording of Sweet and Bitter Receptor Responses to Sequential Injections of Pure and Mixed Sweeteners

“…19 The slide or tongue-on-a-chip is then enclosed in a flow cell and connected to a microfluidic system, which ensures a constant flow of assay buffer across the receptor array and allows for sample loop injections into the existing flow, leading to precisely timed periods of ligand exposure (Figure 1). The strength of this specific tongue-on-achip application 20 is the similarity it holds to a human sensory experience of exposure windows to tastant solutions. It also facilitates the parallel assaying of an array of taste receptors versus a series of samples, each tested for a controlled period of time (typically 30−60 s) with ∼5 min intervals to allow sample wash-out and return of the cell calcium levels to baseline.…”