Biosensor involving the use of optically sensitive moieties

Abstract

The present invention provides biosensors which include or are fabricated using optically sensitive moieties. The use of optically sensitive moieties provides advantages in the synthesis of the biosensors. Further the inclusion of optically sensitive moieties in the biosensor membrane provides an increase in the sensitivity of detection.

1 . A method of fabricating a biosensor in which there is at least one discrete test and at least one discrete control zone, the method comprising the following steps: 
 (i) providing a conductive substrate;    (ii) forming a membrane including membrane spanning lipids and ion channels comprising first and second half membrane spanning monomers, the membrane being attached to the conductive substrate in a manner such that a functioning reservoir exists between the membrane and the conductive substrate;    (iii) linking a ligand reactive with an analyte of interest to the ion-channel and linking a ligand reactive with an analyte of interest to the membrane spanning lipids via photocleavable linkers; and    (iv) exposing the membrane to a focused light source to cleave the photocleavable linkers thereby releasing the ligands from the ion-channel and membrane spanning lipid components in discrete areas of the membrane.    
     
     
         2 . A method as claimed in  claim 1  in which the method further includes the following step: 
 (v) binding control ligands to the ion channels and membrane spanning lipid components after the ligands have been removed in step (iv).  
 
     
     
         3 . A method as claimed in  claim 2  in which the membrane is rinsed between steps (iv) and (v).  
     
     
         4 . A method of fabricating a biosensor in which there is at least one discrete test and at least one discrete control zone, the method comprising the following steps: 
 (i) providing a conductive substrate;    (ii) forming a membrane including membrane spanning lipids and ion channels comprising first and second half membrane spanning monomers, the membrane being attached to the conductive substrate in amanner such that a functioning reservoir exists between the membrane and the conductive substrate;    (iii) providing on the ion channels and membrane spanning lipids a photoactivatable group which when illuminated will bind a receptor;    (iv) illuminating discrete areas of the membrane and linking a ligand reactive with an analyte of interest to the ion-channel and membrane spanning lipid components of the tethered membrane via photoactivated group to form test areas;    (v) removing unbound ligand; and    (vi) linking a control ligand to the remainder of the ion-channels and membrane spanning lipid components of the tethered membrane to form control areas.    
     
     
         5 . A method as claimed in  claim 4  in which the photoactivatable group is caged biotin.  
     
     
         6 . An improved biosensor, the biosensor comprising a membrane and an electrode having a conductive subsbate, the membrane including membrane spanning lipids and ion channels comprising first and second half membrane spanning monomers, the membrane being attached to the conductive substrate such that a functioning reservoir exists between the membrane and the conductive substrate, ligands specific for an analyte attached the ion channels and membrane spanning lipids, the improvement comprising providing on at least one of the first and second half membrane spanning monomers a photocleavable/switchable group which inhibits dimer formation.  
     
     
         7 . An improved biosensor, the biosensor comprising a membrane and an electrode having a conductive substrate, the membrane including membrane spanning lipids and ion channels comprsing first and second half membrane spanning monomers, the membrane being attached to the conductive substrate such that a functioning reservoir exists between the membrane and the conductive substrate, ligands specific for an analyte attached the ion channels and membrane spanning lipids, the improvement comprising the attachment of at least one of the ligands specific for an analyte attached the ion channels and membrane spanning lipids being attached by means of a photocleavable/switchable group.  
     
     
         8 . A method of detecting the presence of an analyte in a sample, the method including the steps of: 
 (i) adding a sample suspected to contain the analyte to the biosensor as claimed in  claim 6  or  claim 7;     (ii) determining the conductance or impedance of the membrane;    (iii) exposing the biosensor to irradiation to which the photocleavable/switchable group is sensitive;    (iv) determining the conductance or impedance of the membrane following irradiation;    (v) comparing the conductance or impedance determination in step (ii) with the determination in step (iv); and    (vi) optionally repeating steps (ii) to (v).    
     
     
         9 . A method of producing an improved biosensor, the method comprising the following steps: 
 (i) providing a conductive substrate;    (ii) forming a membrane including membrane spanning lipids and ion channels comprising first and second half membrane spanning monomers, the membrane being attached to the conductive substrate such that a functioning reservoir exists between the membrane and the conductive substrate;    (iii) providing on the half membrane spanning monomer remote from the conductive substrate a photoswitchable binder to streptavidin and providing biotin on the membrane spanning lipid;    (iv) adding streptavidin;    (v) triggering a light source and rinsing;    (vi) triogering the light source off and adding streptavidin;    (vii) optionally repeating steps (v) and (vi); and    (viii) adding ligands specfic to an analyte to the ion channels and membrane spanning lipids.    
     
     
         10 . A method as claimed in  claim 9  in which the photoswitchable binder to streptavidin is caged biotin, HABA or derivative thereof.  
     
     
         11 . An improved biosensor, the biosensor comprising a membrane and an electrode having a conductive substrate, the membrane including membrane spanning lipids and ion channels comprising first and second half membrane spanning monomers, the membrane being attached to the conductive substrate such that a functioning reservoir exists between the membrane and the conductive substrate, and ligands specific for an analyte attached to the ion channels and membrane spanning lipids, the improvement comprising providing on at least one of the first and second half membrane spanning monomers or membrane spanning lipids a photoswitchable group derived from a compound in accordance with Formula 1:  
       
         
           
           
               
               
           
         
         wherein R 1  represents 0 to about 3 groups where each is independently H or saturated or unsaturated, substituted or unsubstituted C 1 -C 10  hydrocarbon, preferably saturated or unsaturated, substituted or unsubstituted C 1-4  hydrocarbon; R 2  represents 0 to about 3 groups where each is independently hydrogen or satmuated or unsaturated, substituted or unsubstituted C 1-10  hydrocarbon, preferably saturated or unsaturated, substituted or unsubstituted C 1-4  hydrocarbon; Y represents H, saturated or unsaturated, substituted or unsubstituted C 1-4  hydrocarbon, preferably saturated or unsaturated, substituted or unsubstituted C 1-4  hydrocarbon, COR 6 , CONR 7 R 8 , COOR 14 , S(O) n R 15  where n is 0, 1 or 2, R 6 , R 7 , R 8 , R 14  and R 15  are each independently represent H, saturated or unsaturated, substituted or unsubstituted C 1-10  hydrocarbon, preferably saturated or unsaturated, substituted or unsubstituted C 1-4 , hydrocarbon or aryl; R 9  is —C(O)X where X represents H, saturated or unsaturated, substituted or unsubstituted C 1-10  hydrocarbon, preferably saturated or unsaturated, substituted or unsubstituted C 1-4  hydrocarbon, or OH, or OR 10  in which R 10  is alkyl, or NR 11 R 12  in which R 11  and R 12  are H, alkyl or taken together with N form a rincg, or arl or R 9  together with R 1  form a substituted or unsubstituted 5-6 member cyclic or heterocyclic ring; Z represents O or NR 13  R 13  is H, saturated or unsaturated, substituted or unsubstituted C 1-10  hydrocarbon, preferably saturated or unsaturated, substituted or unsubstituted C 1-4  hydrocarbon or aryl, the compound including a functional group at Y or R 9  such that the compound can be linked to the the at least one of the first and second half membrane spanning monomers.  
       
     
     
         12 . An improved biosensor as claimed in  claim 11  in which the photoswitchable linkers are derived are shown in Formula 2 below.  
       
         
           
           
               
               
           
         
         wherein R 1  represents 0 to about 3 groups where each is independently hydrogen or saturated or unsaturated, substituted or unsubstituted C 1-10  hydrocarbon, preferably saturated or unsaturated, substituted or uinsubstituted C 1-4  hydrocarbon; R, represents 0 to about 3 groups where each is independently hydrogen or saturated or unsaturated, substituted or unsubstituted C 1-10  hydrocarbon, preferably saturated or unsaturated, substituted or unsubstituted C 1-4  hydrocarbon; X represents H, saturated or unsaturated, substituted or unsubstituted C 1-10  hydrocarbon, preferably saturated or unsaturated, substituted or unsubstituted C 1-4  hydrocarbon, or aryl, or OH, or OR 10  in which R 10  is alkyl, or NR 11 R 12  in which R 11  and R 12  are H, alkyl or taken together with N form a ring,; Y represents H, or saturated or unsaturated, substituted or unsubstituted C 1-10  hydrocarbon, preferably saturated or unsaturated, substituted or unsubstituted C 1-4  hydrocarbon, COR 6 , CONR 7 R 8 , COOR 14 , S(O) n R 15  where n is 0, 1 or 2, R 6 , R 7 , R 8 , R 14  and R 15  are each independently represent H, saturated or unsaturated, substituted or unsubstituted C 1-10  hydrocarbon, preferably saturated or unsaturated, substituted or unsubstituted C 1-4  hydrocarbon or aryl; Z represents O or NR 13  R 13  is H, saturated or unsaturated, substituted or unsubstituted C 1-10  hydrocarbon, preferably saturated or unsaturated. substituted or unsubstituted C 1-4  hydrocarbon or aryl.  
       
     
     
         13 . A compound in accordance with Formula 1:  
       
         
           
           
               
               
           
         
         wherein R 1  represents 0 to about 3 groups where each is independently H or saturated or unsuaturated, substituted or usubstituted C 1-10  hydrocarbon, preferably saturated or unsaturated, substituted or unsubstituted C 1-4  hydrocarbon; R 2  represents 0 to about 3 groups where each is independently hydrogen or saturated or unsaturated, substituted or unsubstituted C 1-10  hydrocarbon, preferably saturated or unsaturated, substituted or unsubstituted C 1-4  hydrocarbon; Y represents H, saturated or unsaturated, substituted or unsubstituted C 1-10  hydrocarbon, preferably saturated or unsaturated, substituted or unsubstituted C 1-4  hydrocarbon, COR 6 , CONR 7 R 8 , COOR 14 , S(O) n R 15  where n is 0, 1, or 2, R 6 , R 7 , R 14  and R 15  are each independently represent H, saturated or unsaturated, substituted or unsubstitusted C 1-10  hydrocarbon, preferably, saturated or unsaturated, substituted or unsubstituted C 1-4  hydrocarbon or aryl; R 9  is —C(O)X where X represents H, saturated or unsaturated, substituted or unsubstituted C 1-10  hydrocarbon, preferably saturated or unsaturated, substituted or unsubstituted C 1-4  hydrocarbon, or OH, or OR 10  in which R 10  is alkyl, or NR 11 R 12  in which R 11  and R 12  are H, alkyl or taken together with N form a ring, or aryl or R 9  together with R 1  form a substituted or usubstituted 5-6 member cyclic or heterocyclic ring; Z represents O or NR 13  R 13  is H, saturated or unsaturated, substituted or unsubstituted C 1-10  hydrocarbon, preferably saturated or unsaturated, substituted or unsubstituted C 1-4  hydrocarbon or aryl, with the proviso that when R is —C(O)X and X is H, at least one of R 1 , R 2 , R 3  or Y is other than H.