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

Roelse, Margriet; Ruijter, N.C.A. de; Vrouwe, E.X.; Jongsma, Maarten A.

doi:10.1016/j.bios.2013.03.065

Cited by 12 publications

(20 citation statements)

References 30 publications

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“…They are used in a wide range of analytical applications, including diagnosis and treatment of infectious diseases, biowarfare detection (A. H. Peruski et al, 2002;A. H. Peruski and L. F. Peruski, 2003), environmental monitoring (Palchetti and Mascini, 2008) (Rebe Raz and Haasnoot, 2011), drug discovery (Cooper and Cooper, 2002;Myszka and Rich, 2000), cell biology (H. Lee et al, 2008;Roelse et al, 2013), cancer research (Voura et al, 2004) (Wang, 2006) and point-of-care diagnostic testing (Wan et al, 2013) (Watkins et al, 2013). Here, we address a key challenge to developing label-free homogenous biosensors for sensitive biomolecular detection and kinetic analysis using microfluidic biosensing systems that normally require fluorescently labeled biomolecules and optical quantification.…”

Section: Introductionmentioning

confidence: 99%

Label-free biomolecular detection at electrically displaced liquid interfaces using interfacial electrokinetic transduction (IET)

Mavrogiannis¹,

Crivellari²,

Gagnon³

2016

Biosensors and Bioelectronics

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Biosensors require a biorecognition element that specifically binds to a target analyte, and a signal transducer, which converts this targeted binding event into a measurable signal. While current biosensing methods are capable of sensitively detecting a variety of target analytes in a laboratory setting, there are inherent difficulties in developing low-cost portable biosensors for point-of-care diagnostics using traditional optical, mass, or electroanalytical-based signal transducers. It is therefore important to develop new biosensing transducer elements for recognizing binding events at low cost and in portable environments. Here, we demonstrate a novel electrokinetic liquid biosensing method for the sensitive label-free detection of a model biomolecule against a background of serum protein. The biosensor is based on the motion of a microfluidic-generated electrical liquid interface when subjected to an external alternating current electrical field. We demonstrate that the electric field-induced motion of the interface can be used as a sensitive and specific transducer for the detection of avidin at femtomolar concentrations in solution. This new detection strategy does not require surface functionalization or fluorescent labels, and has the potential to serve as a sensitive low-cost method for portable biomarker detection.

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Section: Introductionmentioning

confidence: 99%

Label-free biomolecular detection at electrically displaced liquid interfaces using interfacial electrokinetic transduction (IET)

Mavrogiannis¹,

Crivellari²,

Gagnon³

2016

Biosensors and Bioelectronics

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“…These micro-well plate based high-throughput screening platforms of compound libraries are well established. Furthermore, microarray and microfluidic technologies may hold potential for miniaturization, automation, and biosensor integration [94][95][96] (Figure 3).…”

Section: Gpcr Screening Assaysmentioning

confidence: 99%

“…The TRPV1 receptor gene was obtained as a cDNA clone from GeneCopoeia (W1312) and cloned into pT2A-YC3.6 without a stop codon using the AscI and EcoRI sites. The expression vector containing T2A [132], [133] and YC3.6 in a single reading frame and the control construct mCherry-T2A-YC3.6 have been previously described [95]. Plasmids containing single CFP (cyan fluorescent protein) and YFP (yellow fluorescent protein), pcDNA3-CFP (Addgene plasmid 13030) and pcDNA3-YFP (Addgene plasmid 13033) were used for recording reference spectra.…”

Section: Plasmid Constructionmentioning

confidence: 99%

“…However, TRPV1 was first identified as a receptor for capsaicin; a pungent compound found in foods containing chili peppers. We used capsaicin as standard to test the general properties of the TRPV1 biosensor which uses the intracellular calcium ion sensor, YC3.6, to detect the cytosolic calcium changes that are induced by the activation of the TRPV1 ion channel [95,131]. We tested the TRPV1 biosensor using fruit pericarp extracts from three contrasting Capsicum varieties.…”

Section: Introductionmentioning

confidence: 99%

“…This set-up allowed routine monitoring of the ionic calcium responses of each individual spot to sequentially injected samples. As a proof-of-concept, we used the NK1 receptor which was previously characterized in this flowcell [95,96] and the bitter taste receptor hTAS2R8 [33]. The activation of these receptors by their respective ligands led to a transient increase in the cytoplasmic calcium ion concentration via a signal transduction pathway involving uncoupling of the G proteins, activation of phospholipase C, inositol trisphosphate (IP 3 ) production, and opening of an IP 3 -gated calcium channel of the endoplasmic reticulum [67,137].…”

Section: Introductionmentioning

confidence: 99%

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Receptomics, design of a microfluidic receptor screening technology

Roelse¹

Self Cite

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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

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