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A Comparative Study of Impedance versus Optical Label-Free Systems Relative to Labelled Assays in a Predominantly Gi Coupled GPCR (C5aR) Signalling.

Halai R, Croker DE, Suen JY, Fairlie DP, Cooper MA - Biosensors (Basel) (2012)

Bottom Line: Here, we compare four agonists (native agonists, a peptide full agonist and a peptide partial agonist) that stimulate the human inflammatory GPCR C5aR.The receptor was challenged when present in human monocyte-derived macrophages (HMDM) versus stably transfected human C5aR-CHO cells.However, label-free read outs gave consistently lower potency values in both native and transfected cells.

View Article: PubMed Central - PubMed

Affiliation: Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia. r.halai@uq.edu.au.

ABSTRACT
Profiling ligand function on G-protein coupled receptors (GPCRs) typically involves using transfected cells over-expressing a target of interest, a labelled ligand, and intracellular secondary messenger reporters. In contrast, label-free assays are sensitive enough to allow detection in native cells, which may provide a more physiologically relevant readout. Here, we compare four agonists (native agonists, a peptide full agonist and a peptide partial agonist) that stimulate the human inflammatory GPCR C5aR. The receptor was challenged when present in human monocyte-derived macrophages (HMDM) versus stably transfected human C5aR-CHO cells. Receptor activation was compared on label-free optical and impedance biosensors and contrasted with results from two traditional reporter assays. The rank order of potencies observed across label-free and pathway specific assays was similar. However, label-free read outs gave consistently lower potency values in both native and transfected cells. Relative to pathway-specific assays, these technologies measure whole-cell responses that may encompass multiple signalling events, including down-regulatory events, which may explain the potency discrepancies observed. These observations have important implications for screening compound libraries against GPCR targets and for selecting drug candidates for in vivo assays.

No MeSH data available.


Overview of optical resonant waveguide and cell impedance label-free platforms. (a) Cross-section of the resonant waveguide grating optical biosensors in each well of a 384-well plate. A coating with a high index of refraction on the sensor surface reflects only a narrow band of wavelengths when illuminated with an optical beam. The incident angle of the reflected beam is sensitive to mass redistribution within the cell up to ~150 nm from the surface [17]; (b) Cells are plated onto gold microelectrode arrays, which when stimulated with a low voltage; generate an electric field sensitive to changes in the properties of a cell. Impedance measurement in Cell Index (CI) is zero when cells are not present. The impedance increases as cells attach and spread across the electrodes [19].
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biosensors-02-00273-f001: Overview of optical resonant waveguide and cell impedance label-free platforms. (a) Cross-section of the resonant waveguide grating optical biosensors in each well of a 384-well plate. A coating with a high index of refraction on the sensor surface reflects only a narrow band of wavelengths when illuminated with an optical beam. The incident angle of the reflected beam is sensitive to mass redistribution within the cell up to ~150 nm from the surface [17]; (b) Cells are plated onto gold microelectrode arrays, which when stimulated with a low voltage; generate an electric field sensitive to changes in the properties of a cell. Impedance measurement in Cell Index (CI) is zero when cells are not present. The impedance increases as cells attach and spread across the electrodes [19].

Mentions: The physical basis and general capabilities of commercially available label-free platforms have been reviewed previously [7,8,9,10,11]. Two generally adopted label-free detection platforms based on optical and impedance transduction are highlighted in Figure 1 [12]. However, there are other label-free transductions modes; acoustic biosensors such as the Quartz Crystal Microbalance in which acoustic frequency shifts are influenced by mass and viscoelastic property changes of receptor-analyte interactions, Isothermal Titration Calorimetry, which measures changes in heat as a result of binding complex formation, as well as many others [13]. Furthermore, there are also various forms of optical biosensors, such as, surface plasmon resonance (SPR) or resonant waveguide grating (RWG). This study explores the Corning EPIC® system which is an optical biosensor based on resonant waveguide grating (RWG) [14,15]. Alternatively referred to as a “photonic crystal”, it is comprised of a periodic arrangement of dielectric material in two or three dimensions. If the periodicity and symmetry of the crystal and dielectric constants of the materials are chosen appropriately, the photonic crystal will selectively couple energy at specific wavelengths (Figure 1). When embossed at the bottom of a 96- or 384-well plate, the crystal structure geometry can be designed to concentrate light into extremely small volumes, so that the sensor is sensitive to “mass changes” in cells close (~150–200 nm) to the base of the well plate [16,17].


A Comparative Study of Impedance versus Optical Label-Free Systems Relative to Labelled Assays in a Predominantly Gi Coupled GPCR (C5aR) Signalling.

Halai R, Croker DE, Suen JY, Fairlie DP, Cooper MA - Biosensors (Basel) (2012)

Overview of optical resonant waveguide and cell impedance label-free platforms. (a) Cross-section of the resonant waveguide grating optical biosensors in each well of a 384-well plate. A coating with a high index of refraction on the sensor surface reflects only a narrow band of wavelengths when illuminated with an optical beam. The incident angle of the reflected beam is sensitive to mass redistribution within the cell up to ~150 nm from the surface [17]; (b) Cells are plated onto gold microelectrode arrays, which when stimulated with a low voltage; generate an electric field sensitive to changes in the properties of a cell. Impedance measurement in Cell Index (CI) is zero when cells are not present. The impedance increases as cells attach and spread across the electrodes [19].
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4263554&req=5

biosensors-02-00273-f001: Overview of optical resonant waveguide and cell impedance label-free platforms. (a) Cross-section of the resonant waveguide grating optical biosensors in each well of a 384-well plate. A coating with a high index of refraction on the sensor surface reflects only a narrow band of wavelengths when illuminated with an optical beam. The incident angle of the reflected beam is sensitive to mass redistribution within the cell up to ~150 nm from the surface [17]; (b) Cells are plated onto gold microelectrode arrays, which when stimulated with a low voltage; generate an electric field sensitive to changes in the properties of a cell. Impedance measurement in Cell Index (CI) is zero when cells are not present. The impedance increases as cells attach and spread across the electrodes [19].
Mentions: The physical basis and general capabilities of commercially available label-free platforms have been reviewed previously [7,8,9,10,11]. Two generally adopted label-free detection platforms based on optical and impedance transduction are highlighted in Figure 1 [12]. However, there are other label-free transductions modes; acoustic biosensors such as the Quartz Crystal Microbalance in which acoustic frequency shifts are influenced by mass and viscoelastic property changes of receptor-analyte interactions, Isothermal Titration Calorimetry, which measures changes in heat as a result of binding complex formation, as well as many others [13]. Furthermore, there are also various forms of optical biosensors, such as, surface plasmon resonance (SPR) or resonant waveguide grating (RWG). This study explores the Corning EPIC® system which is an optical biosensor based on resonant waveguide grating (RWG) [14,15]. Alternatively referred to as a “photonic crystal”, it is comprised of a periodic arrangement of dielectric material in two or three dimensions. If the periodicity and symmetry of the crystal and dielectric constants of the materials are chosen appropriately, the photonic crystal will selectively couple energy at specific wavelengths (Figure 1). When embossed at the bottom of a 96- or 384-well plate, the crystal structure geometry can be designed to concentrate light into extremely small volumes, so that the sensor is sensitive to “mass changes” in cells close (~150–200 nm) to the base of the well plate [16,17].

Bottom Line: Here, we compare four agonists (native agonists, a peptide full agonist and a peptide partial agonist) that stimulate the human inflammatory GPCR C5aR.The receptor was challenged when present in human monocyte-derived macrophages (HMDM) versus stably transfected human C5aR-CHO cells.However, label-free read outs gave consistently lower potency values in both native and transfected cells.

View Article: PubMed Central - PubMed

Affiliation: Institute for Molecular Bioscience, The University of Queensland, Brisbane, QLD 4072, Australia. r.halai@uq.edu.au.

ABSTRACT
Profiling ligand function on G-protein coupled receptors (GPCRs) typically involves using transfected cells over-expressing a target of interest, a labelled ligand, and intracellular secondary messenger reporters. In contrast, label-free assays are sensitive enough to allow detection in native cells, which may provide a more physiologically relevant readout. Here, we compare four agonists (native agonists, a peptide full agonist and a peptide partial agonist) that stimulate the human inflammatory GPCR C5aR. The receptor was challenged when present in human monocyte-derived macrophages (HMDM) versus stably transfected human C5aR-CHO cells. Receptor activation was compared on label-free optical and impedance biosensors and contrasted with results from two traditional reporter assays. The rank order of potencies observed across label-free and pathway specific assays was similar. However, label-free read outs gave consistently lower potency values in both native and transfected cells. Relative to pathway-specific assays, these technologies measure whole-cell responses that may encompass multiple signalling events, including down-regulatory events, which may explain the potency discrepancies observed. These observations have important implications for screening compound libraries against GPCR targets and for selecting drug candidates for in vivo assays.

No MeSH data available.