Directed evolution biosensors

Abstract

The invention exploits the evolutionary principles responsible for the development of the broad spectrum general odorant detector system, to create a G-protein coupled receptor (GPCR) based system capable of detecting and discriminating between thousands of chemicals. The means is to subject a defined set of receptors such as G-protein coupled receptors, tyrosine kinase receptors, and/or ion channels, to the types of evolutionary forces that have created the array of approximately 1,000 natural receptors used in general olfaction by higher animals. This goal is accomplished by ‘directed evolution-in-a-test-tube’ by imposing very high rates of mutation and extremely strict selection criteria to create a sensor. The novel sensor is selected using a sensitive melanophore-based functional bioassay. Stimulation of the sensor upon interaction with chemical signatures derived from ordinances will result in a calcium ion flux rapidly detectable as a fluorescent signal.

I claim:  
     
         1 . A detector for identifying the presence of a ligand which binds to a cell surface receptor comprising: 
 a container housing cells, the cells expressing a receptor which binds the ligand and producing a detectable intracellular signal when the ligand binds the receptor,    an inlet port in the container for introducing a sample containing the ligand into the container, and a sensor attached to the container for detecting the intracellular signal.    
     
     
         2 . The detector of  claim 1 , wherein the receptor is an exogenous cell surface receptor.  
     
     
         3 . The detector of  claim 1 , wherein the receptor is an exogenous cell surface receptor mutated to have altered binding to its natural ligand.  
     
     
         4 . The detector of  claim 1 , wherein the signal detected is compared to a signal generated by a cell expressing a non-mutated receptor of the same type as the mutated receptor, thereby permitting the identification of a mutated receptor with an altered binding specificity for the ligand versus the non-mutated receptor.  
     
     
         5 . The detector of  claim 1 , wherein the signal is compared to a cell expressing a different mutated receptor which binds the ligand.  
     
     
         6 . The detector of  claim 1 , wherein the ligand is not the natural ligand for the receptor.  
     
     
         7 . The detector of  claim 1 , wherein the signal can be compared to a cell expressing a different mutated receptor which binds the ligand.  
     
     
         8 . The detector according to any one of claims  1 - 7 , wherein the signals generated are second messenger signals.  
     
     
         9 . The detector of  claim 8 , wherein the second messenger signals result in pigment dispersion or aggregation.  
     
     
         10 . The detector of  claim 1 , wherein the cells are melaniferous.  
     
     
         11 . The detector of  claim 1 , wherein the cells are lower animal pigment cells.  
     
     
         12 . The detector of  claim 1 , wherein the second messenger signals cause alterations in calcium levels in the cell and the signal is calcium mediated fluorescence.  
     
     
         13 . The detector of  claim 1 , wherein the cells are an array of cells expressing an array of mutated receptors.  
     
     
         14 . The detector of  claim 1 , wherein the ligand is selected from the group consisting of chemical warfare agents, explosives, drugs, fragrances, impurities, environmental toxins and pollutants.