Biosensors and methods of using the same

Abstract

The present invention relates to a method for detection of receptor antagonists comprising the following steps: (I) a sample containing the receptor antagonist is fractionated by use of a liquid-based separation means, preferably capillary electrophoresis, (II) a fraction containing the receptor antagonist or modulator is fed directly to a biosensor (9) which is activated by an appropriate receptor agonist and, as a result of this activation, is generating a measurable response, said agonist being fed to the biosensor through the liquid-based separation means together with the antagonist or modulator, said activation of the biosensor ( 9 ) being pulsed by delivery of the receptor agonist to the biosensor for short period of times, said periods being separated by other periods when no agonist is delivered to the biosensor, and (III) the change of the response resulting from deactivation of the receptor agonist-activated biosensor by the receptor antagonist or modulator is measured, preferably by means of a patch clamp electrode ( 8 ). It is further possible to resensitize the biosensor desensitized as above by use of pulsed superfusion of the biosensor. The present invention also relates to an apparatus usable for practicing the above mentioned method.

1 . A method for detection of a receptor antagonist or a receptor modulator, characterised in that it comprises the following steps: 
 (I) a sample containing the receptor antagonist or modulator is fractionated by use of a liquid-based separation means,    (II) a fraction containing the receptor antagonist or modulator is fed directly to a biosensor ( 9 ) which is activated by an appropriate receptor agonist and, as a result of this activation, is generating a measurable response, said agonist being fed to the biosensor through the liquid-based separation means together with the antagonist or modulator, said activation of the biosensor ( 9 ) being pulsed by delivery of the receptor agonist to the biosensor for short period of times, said periods being separated by other periods when no agonist is delivered to the biosensor, and    (III) the change of the response resulting from deactivation of the receptor agonist-activated biosensor ( 9 ) by the receptor antagonist or modulator is measured.    
     
     
         2 . A method according to  claim 1 , wherein the response generated from the biosensor ( 9 ) is measured during the periods when no agonist is delivered to the biosensor.  
     
     
         3 . A method according to  claim 1  or  2 , wherein the desensitised biosensor is resensitised by superfusion of the biosensor.  
     
     
         4 . A method according to  claim 3 , wherein the superfusion is performed by use of a column, placed in parallel to the liquid-based separation means.  
     
     
         5 . A method according to  claim 3 , wherein the superfusion is performed by use of a glass pipette placed with its outlet adjacent to the outlet of the liquid-based separation means.  
     
     
         6 . A method according to any one of the claims  1 - 5 , wherein the fractionation in step (I) is achieved by a microcolumn separation technique.  
     
     
         7 . A method according to  claim 6 , wherein the microcolumn separation technique is capillary electrophoresis.  
     
     
         8 . A method according to any one of claims  1 - 7 , wherein the response generated by the biosensor ( 9 ) is an electrical current.  
     
     
         9 . A method according to  claim 8 , wherein the electrical current is measured by the use of a patch clamp technique or a two-electrode voltage clamp technique.  
     
     
         10 . A method according to any one of claims  1 - 7 , wherein the response generated by the biosensor ( 9 ) is fluorescence.  
     
     
         11 . A method according to any one of the claims  1 - 10 , wherein the biosensor ( 9 ) is constituted by at least one cell or part of a cell comprising at least one receptor for the agonist used and for the antagonist to be detected.  
     
     
         12 . A method according to any one of the claims  1 - 11 , wherein the antagonist to be detected is a substance affecting ligand-gated ion channels.  
     
     
         13 . An apparatus for detection of a receptor antagonist or a receptor modulator comprising a capillary electrophoresis separation capillary ( 1 ) containing an electrolyte supplemented with an appropriate receptor agonist, the sample inlet part of which is connected to a high-voltage power supply ( 2 ) through a buffer vial ( 3 ) containing a buffer supplemented with an appropriate receptor agonist and the grounded outlet part ( 4 ) of which ends close to a patch-clamped biosensor ( 9 ) that is activated by the receptor agonist and deactivated by the fractionated antagonist; the apparatus further comprising a patch clamp electrode ( 8 ) and means to record currents detected by the patch clamp electrode.  
     
     
         14 . An apparatus according to  claim 13  wherein the capillary electrophoresis capillary ( 1 ) is a fused silica capillary.  
     
     
         15 . An apparatus according to  claim 13  or  14  further comprising at least one micropositioner ( 10 ) connected to the capillary and/or the patch clamp electrode.  
     
     
         16 . An apparatus according to any one of the claims  13 - 15 , wherein the patch-clamped biosensor ( 9 ) is placed in a cell bath ( 6 ).  
     
     
         17 . An apparatus according to  claim 16 , wherein the electrolyte, the buffer and the cell bath are constituted of the same medium, with the only difference that the electrolyte and the buffer but not the cell bath are supplemented with an appropriate agonist.  
     
     
         18 . An apparatus according to any one of the claims  13 - 17  further comprising a second capillary ( 14 ) for delivery of a buffer not comprising any agonist, said second capillary ( 14 ) being connected to a superfusion system ( 16 ), said apparatus also comprising means ( 10 ) for switching the position of the capillaries so that the patch-clamped biosensor ( 9 ) alternatively is placed in front of the outlet of the first capillary ( 1 ) and in front of the outlet of the second capillary ( 14 ).  
     
     
         19 . An apparatus according to any one of the claims  13 - 17  further comprising means ( 17 ) for superfusing the patch-clamped biosensor ( 9 ) with a buffer not comprising any agonist.  
     
     
         20 . An apparatus according to  claim 19  further comprising means for pulsing the flow of the buffer delivered from the superfusion means.  
     
     
         21 . A method according to  claim 20 , wherein said means ( 17 ) is a micropipette.  
     
     
         22 . An apparatus according to any one of the claims  13 - 21 , wherein the biosensor ( 9 ) is constituted of at least one cell or part of a cell comprising receptors for the antagonist or modulator to be detected.