Enzyme detection device

Abstract

An enzyme detection device ( 1 ) for detecting the presence, in a sample, of an enzyme capable of modifying a provided substrate ( 10 ). The device ( 1 ) comprises a substrate which has a modification region ( 14 ) that is sensitive to modification by the enzyme from an unmodified state to a modified state. The device ( 1 ) further comprises a substrate recognition molecule ( 16 ) which binds the modification region ( 14 ) in either the modified or the unmodified state. The modification region 14 of the substrate is preferentially bound by the substrate recognition molecule ( 16 ) as compared with the enzyme when mixed. The device further comprises a detectable label ( 18 ) coupled to the substrate recognition molecule ( 17 ).

1 . An enzyme detection device for detecting the presence in a sample of an enzyme capable of modifying a provided substrate comprising:
 (i) a substrate comprising a modification region sensitive to modification by the enzyme from an unmodified state to a modified state;   (ii) a substrate recognition molecule capable of specifically binding the modification region in the unmodified state, the binding site of the substrate recognition molecule being such that the modification region of the substrate is preferentially bound by said substrate recognition molecule as compared with the enzyme when mixed; and   (iii) a detectable label couplable to the substrate recognition molecule.   
     
     
         2 . The enzyme detection device of  claim 1  wherein the affinity of the substrate recognition molecule for the substrate comprises a relatively low dissociation rate. 
     
     
         3 . The enzyme detection device of  claim 1  wherein the affinity of the substrate recognition molecule for the substrate comprises a relatively low dissociation rate and a relatively high association rate. 
     
     
         4 . The enzyme detection device of  claim 1  wherein the substrate recognition molecule has a lower dissociation rate and a higher association rate for the substrate than the enzyme for the substrate. 
     
     
         5 . An enzyme detection device according to  claim 1  wherein the substrate further comprises an attachment region and the enzyme detection device further comprises a solid support, the attachment region being attachable to the solid support. 
     
     
         6 . An enzyme detection device according to  claim 1  further comprising a chromatographic medium. 
     
     
         7 . An enzyme detection device according to  claim 6  wherein the substrate further comprises an attachment region attachable to the chromatographic medium. 
     
     
         8 . An enzyme detection device according to  claim 7  wherein the chromatographic medium comprises a first capture recognition molecule, immobilised on or in the chromatographic medium and capable of binding the attachment region of the substrate. 
     
     
         9 . An enzyme detection device according to  claim 8  wherein the chromatographic medium further comprises a second capture recognition molecule, immobilised on or in the chromatographic medium, and capable of binding the substrate recognition molecule, optionally in combination with a fragment of the substrate. 
     
     
         10 . An enzyme detection device according to  claim 8  or  9  wherein the first capture recognition molecule and the attachment region and/or the second capture recognition molecule and the substrate recognition molecule are each two halves of a binding pair wherein the binding pair is: an antigen and antibody or antigen-binding fragment specific therefor; biotin and avidin, streptavidin, neutravidin, or captavidin; protein A and G; a carbohydrate and a lectin; two complementary nucleotide sequences; an effector and a receptor molecule; a hormone and a hormone binding protein; an enzyme cofactor and an enzyme; an enzyme inhibitor and an enzyme; a cellulose binding domain and cellulose fibres; immobilised aminophenyl boronic acid and cis-diol bearing molecules; xyloglucan and cellulose fibres and analogues, derivatives and fragments thereof. 
     
     
         11 . An enzyme detection device according  claim 1  wherein the substrate recognition molecule is an antibody or antigen binding fragment thereof; a lectin; a nucleotide sequence; a receptor molecule; or a hormone binding protein capable of binding the modification region in either the modified or the unmodified state. 
     
     
         12 . An enzyme detection device according to  claim 1  wherein the substrate recognition molecule is avidin, streptavidin, neutravidin, or captavidin capable of binding unmodified biotin. 
     
     
         13 . An enzyme detection device according  claim 1  wherein the enzyme is a hydrolase, preferably a peptidase, lipase, nuclease, carbohydrase, phosphatase, sulphatase, neuraminidase, esterase, DNAase or RNAase. 
     
     
         14 . An enzyme detection device according  claim 1  wherein the enzyme is a kinase, a glycosyl transferase, an oxidase, a reductase or a transaminase. 
     
     
         15 . An enzyme detection device according  claim 1  further comprising a detectable label attached to the substrate recognition molecule. 
     
     
         16 . An enzyme detection device according to  claim 15  wherein the detectable label is covalently bound to the substrate recognition molecule. 
     
     
         17 . An enzyme detection device according to  claim 15  wherein the label is a fluorophore, a gold particle, a chromogen, a luminescent compound; a radioactive compound; a visible compound, a liposome or other vesicle containing signal producing substances, an electroactive species or a combination of an enzyme and its substrate. 
     
     
         18 . An enzyme detection device according to  claim 1  comprising first and second substrates, each comprising a modification region, the modification region of the first substrate being sensitive to modification by a first enzyme, the modification region of the second substrate being sensitive to modification by a second enzyme. 
     
     
         19 - 37 . (canceled)