Label-free methods using a resonant waveguide grating biosensor to determine GPCR signaling pathways
First Claim
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1. A label-free method for GPCR signaling pathway determination, the method comprising:
- immobilizing live cells on a resonant waveguide grating (RWG) biosensor;
contacting the immobilized live cells with a substance that elevates intracellular cAMP and activates protein kinase A in the live cells;
contacting the substance-contacted live cells with a GPCR ligand;
detecting and comparing the GPCR ligand-induced biosensor response of the live cells in the presence and absence of the substance; and
determining the signalling pathways of the GPCR activated by the ligand, wherein the substance-induced inhibition of the GPCR ligand-induced response indicates that the GPCR signals via Gα
s pathway, the substance-induced partial suppression of the GPCR ligand-induced response indicates that the GPCR signals via a Gα
q pathway, and the substance-induced potentiation of the GPCR ligand-induced response indicates that the GPCR signals via a Gα
i pathway, wherein the response is a dynamic mass redistribution (DMR) signal measured by the resonant waveguide biosensor, and the substance is a probe molecule to differentiate all classes of signalling mediated through the GPCR.
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Abstract
A system and method for GPCR signaling pathway analysis and elucidation using a biosensor, a live-cell, and a pathway active compound, as defined herein.
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Citations
20 Claims
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1. A label-free method for GPCR signaling pathway determination, the method comprising:
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immobilizing live cells on a resonant waveguide grating (RWG) biosensor; contacting the immobilized live cells with a substance that elevates intracellular cAMP and activates protein kinase A in the live cells; contacting the substance-contacted live cells with a GPCR ligand; detecting and comparing the GPCR ligand-induced biosensor response of the live cells in the presence and absence of the substance; and determining the signalling pathways of the GPCR activated by the ligand, wherein the substance-induced inhibition of the GPCR ligand-induced response indicates that the GPCR signals via Gα
s pathway, the substance-induced partial suppression of the GPCR ligand-induced response indicates that the GPCR signals via a Gα
q pathway, and the substance-induced potentiation of the GPCR ligand-induced response indicates that the GPCR signals via a Gα
i pathway, wherein the response is a dynamic mass redistribution (DMR) signal measured by the resonant waveguide biosensor, and the substance is a probe molecule to differentiate all classes of signalling mediated through the GPCR. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)
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14. A method for GPCR signaling pathway determination, the method comprising:
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immobilizing live cells on a resonant waveguide grating (RWG) biosensor; contacting the immobilized live cells with a GPCR ligand; contacting the GPCR ligand-contacted live cells with a substance that elevates intracellular cAMP and activates protein kinase A in the live cells; detecting and comparing the substance-induced biosensor response of the live cells in the presence and absence of the GPCR ligand; and determining the signal pathway of the GPCR induced by the ligand, wherein the alteration of the substance-induced response by the ligand indicates the signalling pathway being activated by the ligand, the substance-induced response is a dynamic mass redistribution response, and the substance is a probe molecule to differentiate all classes of signalling mediated through the GPCR. - View Dependent Claims (15, 16)
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17. A method for GPCR signaling pathway determination, the method comprising:
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immobilizing live cells on a resonant waveguide grating (RWG) biosensor; contacting the immobilized live-cells with a substance that suppresses intracellular phospholipase C (PLC) activity in the live cells; contacting the PLC-suppressed live cells with a GPCR ligand; detecting and comparing the GPCR ligand-induced biosensor response of the live cells in the presence and absence of the substance; and determining the GPCR signal pathway of the receptor induced by the ligand, wherein the inhibition of the GPCR ligand-induced response indicates that the GPCR signals via Gα
q pathway, the substance-induced partial suppression of the GPCR ligand-induced response indicates that the GPCR signals via a Gα
i pathway, the substance-induced little change of the GPCR ligand-induced response indicates that the GPCR signals via a Gα
s pathway, and the substance is a probe molecule to differentiate all classes of signalling mediated through the GPCR. - View Dependent Claims (18, 19, 20)
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Specification