System and method for performing G protein coupled receptor (GPCR) cell assays using waveguide-grating sensors

Abstract

The present invention includes a system and method that uses optical LID biosensors to monitor in real time agonist-induced GPCR signaling events within living cells. Particularly, the present invention includes a system and method for using an optical LID biosensor to screen compounds against a target GPCR within living cells based on the mass redistribution due to agonist-induced GPCR activation. In an extended embodiment, the present invention discloses different ways for self-referencing the optical LID biosensor to eliminate unwanted sensitivity to ambient temperature, pressure fluctuations, and other environmental changes. In yet another extended embodiment, the present invention discloses different ways for screening multiple GPCRs in a single type of cell or multiple GPCRs in multiple types of cells within a single medium solution. In still yet another extended embodiment, the present invention discloses different ways to confirm the physiological or pharmacological effect of a compound against a specific GPCR within living cells.

1 . A method for performing a living cell-based assay, said method comprising the step of: 
 using an optical label independent detection (LID) biosensor to monitor mass redistribution within living cells adherent on a surface of the optical LID biosensor.    
     
     
         2 . The method of  claim 1 , wherein said step of using enables one to monitor mass redistribution due to an agonist-induced G-protein coupled receptor (GPCR) activation within living cells by performing the following steps: 
 providing the optical LID biosensor;    placing the living cells in a cell medium to cover the optical LID biosensor so the living cells are able to attach to the surface of the optical LID biosensor;    applying a solution containing a compound into the cell medium located on the surface of the optical LID biosensor; and    interrogating the optical LID biosensor to obtain a time dependent optical response which indicates the mass redistribution within the living cells that enables one to monitor an agonist-induced GPCR translocation within the living cells.    
     
     
         3 . The method of  claim 2 , wherein prior to the step of applying the solution containing the compound into the cell medium located on the surface of the optical LID biosensor the following step is performed: 
 applying a buffer solution at least once into the cell medium located on the surface of the optical LID biosensor solution.    
     
     
         4 . The method of  claim 1 , wherein said step of using enables one to screen an agonist against a target G-protein coupled receptor (GPCR) based on mass redistribution within living cells by performing the following steps: 
 providing the optical LID biosensor;    placing the living cells in a cell medium to cover the optical LID biosensor so the living cells are able to attach to the surface of the optical LID biosensor;    applying a solution containing an antagonist with a known affinity at a certain concentration into the cell medium to stabilize the optical LID biosensor;    applying a solution containing a compound into the cell medium located on the surface of the optical LID biosensor, wherein a concentration of the compound is sufficiently high to compete off the antagonist; and    interrogating the optical LID biosensor to obtain a time dependent optical response which indicates the mass redistribution within the living cells that enables one to screen an agonist against a target GPCR based on mass redistribution within the living cells.    
     
     
         5 . The method of  claim 1 , wherein said step of using enables one to screen an antagonist against a target G-protein coupled receptor (GPCR) based on mass redistribution within living cells by performing the following steps: 
 providing the optical LID biosensor;    placing the living cells in a cell medium to cover the optical LID biosensor so the living cells are able to attach to the surface of the optical LID biosensor;    applying a solution containing an agonist with a known affinity at a certain concentration into the cell medium located on the surface of the optical LID biosensor;    applying, before translocation happens due to the presence of the agonist, a solution containing a compound into the cell medium located on the surface of the optical LID biosensor; and    interrogating the optical LID biosensor to obtain a time dependent optical response which indicates the mass redistribution within the living cells that enables one to screen an antagonist against a target GPCR based on mass redistribution within living cells.    
     
     
         6 . The method of  claim 1 , wherein said optical LID biosensor is formed into a self-referencing optical LID biosensor prior to performing the step of using by: 
 blocking a portion of the surface of the optical LID biosensor using a stamp that prevents attachment of the living cells;    placing the living cells in a cell medium to cover an unblocked portion of the surface of the optical LID biosensor so the living cells are able to attach to the unblocked portion of the surface of the optical LID biosensor; and    removing the stamp from the surface of the optical LID biosensor.    
     
     
         7 . The method of  claim 1 , wherein said optical LID biosensor is formed into a self-referencing optical LID biosensor prior to performing the step of using by: 
 blocking a portion of the surface of the optical LID biosensor using a stamp that prevents attachment of the living cells;    placing the living cells in a cell medium to cover an unblocked portion of the surface of the optical LID biosensor so the living cells are able to attach to the unblocked portion of the surface of the optical LID biosensor;    removing the stamp from the surface of the optical LID biosensor, and    and placing a second type of cells in the same cell medium to cover the blocked portion of the surface of the optical LID biosensor so the second type of cells are able to attach to the blocked portion of the surface of the optical LID biosensor.    
     
     
         8 . The method of  claim 1 , wherein said step of using optical LID sensor enables one to monitor the mass redistribution due to an agonist-induced G-protein coupled receptor (GPCR) activation within multiple types of the living cells by performing the following steps: 
 providing a chamber containing an array of the optical LID biosensors;    placing a first type of the living cells in a cell medium to cover at least one of the optical LID biosensors so the first type of the living cells is able to attach to the surface of the at least one optical LID biosensor;    placing a second type of the living cells in a cell medium to cover at least one of the remaining uncovered optical LID biosensors so the second type of the living cells is able to attach to the surface of the at least one remaining uncovered optical LID biosensor;    applying a solution containing a compound into the cell mediums located on the surfaces of the optical LID biosensors; and    interrogating the optical LID biosensors to obtain time dependent optical responses which indicates the mass redistributions within the living cells on each of the optical LID biosensors that enables one to monitor the agonist-induced GPCR activation within multiple types of the living cells.    
     
     
         9 . The method of  claim 1 , wherein said step of using optical LID sensor enables one to monitor mass redistribution due to an agonist-induced G-protein coupled receptor (GPCR) activation within multiple types of the living cells by performing the following steps: 
 providing the optical LID biosensor;    blocking a portion of the surface of the optical LID biosensor using a stamp that prevents attachment of the living cells to that portion of the optical LID biosensor;    placing a first type of the living cells in a cell medium to cover an unblocked portion of the surface of the optical LID biosensor so the first type of the living cells are able to attach to the unblocked portion of the surface of the optical LID biosensor;    removing the stamp from the surface of the optical LID biosensor;    placing a second type of the living cells in a cell medium to cover the optical LID biosensor so the second type of the living cells are able to attach to the uncovered portion of the surface on the optical LID biosensor;    applying a solution containing a compound into the cell medium located on the surface of the optical LID biosensor; and    interrogating the optical LID biosensors to obtain time dependent optical responses which indicates the mass redistributions within the two types of living cells on the optical LID biosensor that enables one to monitor the agonist-induced GPCR activation within multiple types of the living cells.    
     
     
         10 . The method of  claim 1 , wherein said step of using optical LID sensor enables one to screen agonists against multiple G-protein coupled receptors (GPCRs) within a single type of living cells by performing the following steps: 
 providing the optical LID biosensor;    placing the living cells in a cell medium to cover the optical LID biosensor so the living cells are able to attach to the surface of the optical LID biosensor;    applying a solution containing a cocktail solution of antagonists;    applying a solution containing a compound into the cell medium located on the surface of the optical LID biosensor; and    interrogating the optical LID biosensor to obtain a time dependent optical response which indicates the mass redistribution within the living cells that enables one to screen agonists against multiple GPCRs within the living cells.    
     
     
         11 . A system, comprising: 
 an interrogation system; and    an optical label independent detection (LID) biosensor, wherein said interrogation system emits an optical beam to said optical LID biosensor and receives an optical beam from said optical LID biosensor which enables said interrogation system to monitor mass redistribution within living cells located on a surface of the optical LID biosensor.    
     
     
         12 . The system of  claim 11 , wherein said interrogation system is further capable of monitoring an agonist-induced G protein coupled receptor (GPCR) activation within the living cells after the following steps are performed: 
 providing the optical LID biosensor;    placing the living cells in a cell medium to cover the optical LID biosensor so the living cells are able to attach to the surface of the optical LID biosensor;    applying a solution containing a compound into the cell medium located on the surface of the optical LID biosensor; and    interrogating the optical LID biosensor to obtain a time dependent optical response which indicates the mass redistribution within the living cells that enables one to monitor the mass redistribution due to an agonist-induced GPCR activation within the living cells.    
     
     
         13 . The system of  claim 12 , wherein prior to the step of applying the solution containing the compound into the cell medium located on the surface of the optical LID biosensor the following step is performed: 
 applying a buffer solution at least once into the cell medium located on the surface of the optical LID biosensor solution.    
     
     
         14 . The system of  claim 11 , wherein said interrogation system is further capable of screening an agonist against a target G-protein coupled receptor (GPCR) based on mass redistribution after the following steps are performed: 
 providing the optical LID biosensor;    placing the living cells in a cell medium to cover the optical LID biosensor so the living cells are able to attach to the surface of the optical LID biosensor;    applying a solution containing an antagonist with a known affinity at a certain concentration into the cell medium to stabilize the optical LID biosensor;    applying a solution containing a compound into the cell medium located on the surface of the optical LID biosensor, wherein a concentration of the compound is sufficiently high to compete off the antagonist; and    interrogating the optical LID biosensor to obtain a time dependent optical response which indicates the mass redistribution within the living cells that enables one to screen an agonist against a target GPCR based on mass redistribution within the living cells.    
     
     
         15 . The system of  claim 11 , wherein said interrogation system is further capable of screening an antagonist against a target G-protein coupled receptor (GPCR) based on mass redistribution after the following steps are performed: 
 providing the optical LID biosensor;    placing the living cells in a cell medium to cover the optical LID biosensor so the living cells are able to attach to the surface of the optical LID biosensor;    applying a solution containing an agonist with a known affinity at a certain concentration into the cell medium located on the surface of the optical LID biosensor;    applying, before translocation happens due to the presence of the agonist, a solution containing a compound into the cell medium located on the surface of the optical LID biosensor; and    interrogating the optical LID biosensor to obtain a time dependent optical response which indicates the mass redistribution within the living cells that enables one to screen an antagonist against a target GPCR based on mass redistribution within living cells.    
     
     
         16 . The system of  claim 11 , wherein said optical LID biosensor is formed into a self-referencing optical LID biosensor by: 
 blocking a portion of the surface of the optical LID biosensor using a stamp that prevents attachment of the living cells;    placing the living cells in a cell medium to cover an unblocked portion of the surface of the optical LID biosensor so the living cells are able to attach to the unblocked portion of the surface of the optical LID biosensor; and    removing the stamp from the surface of the optical LID biosensor.    
     
     
         17 . The system of  claim 11 , wherein said optical LID biosensor is formed into a self-referencing optical LID biosensor prior to performing the step of using by: 
 blocking a portion of the surface of the optical LID biosensor using a stamp that prevents attachment of the living cells;    placing the living cells in a cell medium to cover an unblocked portion of the surface of the optical LID biosensor so the living cells are able to attach to the unblock portion of the surface of the optical LID biosensor;    removing the stamp from the surface of the optical LID biosensor, and    and placing a second type of cells in the same cell medium to cover the blocked portion of the surface of the optical LID biosensor so the second type of cells are able to attach to the blocked portion of the surface of the optical LID biosensor.    
     
     
         18 . The system of  claim 11 , wherein said interrogation system is further capable of monitoring the mass redistribution due to an agonist-induced G-protein coupled receptor (GPCR) activation within multiple types of the living cells after the following steps are performed: 
 providing a chamber containing an array of the optical LID biosensors;    placing a first type of the living cells in a cell medium to cover at least one of the optical LID biosensors so the first type of the living cells is able to attach to the surface of the at least one optical LID biosensor;    placing a second type of the living cells in a cell medium to cover at least one of the remaining uncovered optical LID biosensors so the second type of the living cells is able to attach to the surface of the at least one remaining uncovered optical LID biosensor;    applying a solution containing a compound into the cell mediums located on the surfaces of the optical LID biosensors; and    interrogating the optical LID biosensors to obtain time dependent optical responses which indicates the mass redistributions within the living cells on each of the optical LID biosensors that enables one to monitor the agonist-induced GPCR translocation within multiple types of the living cells.    
     
     
         19 . The system of  claim 11 , wherein said interrogation system is further capable of monitoring the mass redistribution due to an agonist-induced G-protein coupled receptor (GPCR) activation within multiple types of the living cells after the following steps are performed: 
 providing the optical LID biosensor;    blocking a portion of the surface of the optical LID biosensor using a stamp that prevents attachment of the living cells to that portion of the optical LID biosensor;    placing a first type of the living cells in a cell medium to cover an unblocked portion of the surface of the optical LID biosensor so the first type of the living cells are able to attach to the unblock portion of the surface of the optical LID biosensor;    removing the stamp from the surface of the optical LID biosensor;    placing a second type of the living cells in a cell medium to cover the optical LID biosensor so the second type of the living cells are able to attach to the uncovered portion of the surface on the optical LID biosensor;    applying a solution containing a compound into the cell medium located on the surface of the optical LID biosensor; and    interrogating the optical LID biosensors to obtain time dependent optical responses which indicates the mass redistributions within the two types of living cells on the optical LID biosensor that enables one to monitor the agonist-induced GPCR translocations within multiple types of the living cells.    
     
     
         20 . The system of  claim 11 , wherein said interrogation system is further capable of screening agonists against multiple G-protein coupled receptors (GPCRs) within a single type of living cells after the following steps are performed: 
 providing the optical LID biosensor;    placing the living cells in a cell medium to cover the optical LID biosensor so the living cells are able to attach to the surface of the optical LID biosensor;    applying a solution containing a cocktail solution of antagonists;    applying a solution containing a compound into the cell medium located on the surface of the optical LID biosensor; and    interrogating the optical LID biosensor to obtain a time dependent optical response which indicates the mass redistribution within the living cells that enables one to screen agonists against multiple GPCRs within the living cells.