Protease detection assay

Abstract

The invention provides methods and compounds for detecting protease activity in a sample solution comprising contacting the sample solution with a protease substrate labeled with an electrochemically active marker, providing conditions under which any protease which may be present in the sample may degrade the protease substrate and electrochemically determining information relating to the electrochemically active marker.

1 - 18 . (canceled) 
     
     
         19 . A method for detecting protease activity in a sample solution comprising the steps of:
 i) contacting the sample solution with a protease substrate labeled with an electrochemically active marker, wherein the electrochemically active marker is a metallocene moiety,   ii) providing conditions under which a protease present in the sample solution may degrade the protease substrate, wherein the protease is capable of recognizing the protease substrate, and   iii) electrochemically determining information relating to the electrochemically active marker, thereby detecting the protease activity in the sample.   
     
     
         20 . A method as claimed in  claim 19  wherein the information relating to the electrochemically active marker is determined using voltammetry. 
     
     
         21 . A method as claimed in  claim 20  wherein the information relating to the electrochemically active marker is determined using differential pulse voltammetry. 
     
     
         22 . A method as claimed in  claim 19  wherein the information relating to the electrochemically active marker is determined using an amperometric technique. 
     
     
         23 . A method as claimed in  claim 19  wherein the information relating to the electrochemically active marker is determined using a technique that utilizes one or more electrodes that are functionally surrounded by a selectively permeable membrane. 
     
     
         24 . (canceled) 
     
     
         25 . A method as claimed in  claim 19  wherein the electrochemically active marker is a ferrocene moiety. 
     
     
         26 . A method as claimed in  claim 19  wherein the electrochemically active marker is attached to the protease substrate through a linker. 
     
     
         27 . A method as claimed in  claim 19  wherein each protease substrate molecule is, on average, labeled with more than one electrochemically active marker molecule. 
     
     
         28 . A method as claimed in  claim 19  wherein the protease substrate labeled with an electrochemically active marker is a single amino acid labeled with an electrochemically active marker. 
     
     
         29 . A method for detecting a disease in a subject the method comprising the method of  claim 19  further comprising a step of comparing said protease activity with a level of protease activity that is diagnostic of a disease in a subject, thereby detecting a disease in a subject. 
     
     
         30 . A method for detecting a pathogen the method comprising the method of  claim 19  further comprising a step of comparing said protease activity with a level of protease activity that is diagnostic of the presence of a pathogen, thereby detecting a pathogen. 
     
     
         31 . A method for screening for a protease inhibitor the method comprising the method of  claim 19  further comprising a step of contacting the sample solution with a putative protease inhibitor. 
     
     
         32 . An assay kit comprising a protease substrate labeled with an electrochemically active marker, wherein the electrochemically active marker is a metallocene moiety. 
     
     
         33 . An apparatus for detecting protease activity in a sample solution, the apparatus comprising a means for contacting the sample solution with a protease substrate labeled with an electrochemically active marker, a means for providing conditions under which a protease present in the sample solution may degrade the protease substrate, and a means for electrochemically determining information relating to the electrochemically active marker, wherein the means for electrochemically determining information relating to the electrochemically active marker is selected from the group consisting of a voltammeter, an amperometer, and one or more electrodes, wherein the electrochemically active marker is a metallocene moiety, wherein the electrochemically active marker is attached to the protease substrate through a linker, wherein the protease is capable of recognizing the protease substrate, and wherein the protease substrate labeled with an electrochemically active marker is at least a single amino acid labeled with an electrochemically active marker. 
     
     
         34 . A compound of formula IV,
   Mc-NR′—C(═O)—X—(Ar) n -(L) m -R   IV
   
       wherein
 Mc is a metallocenyl group in which each ring may independently be substituted or unsubstituted, 
 the metallocenyl group comprises a metal ion M selected from the group consisting of iron, chromium, cobalt, osmium, ruthenium, nickel, and titanium, 
 R′ is H or lower alkyl, 
 X is NR′ or O, 
 Ar is a substituted or unsubstituted aryl group, 
 n is 0 or 1, 
 L is a linker group, 
 m is 0 or 1, and 
 R is a protein, a peptide, or an amino acid residue. 
 
     
     
         35 . A compound comprising a metallocenyl group attached to a carboxyl group of molecule, the molecule selected from the group consisting of an amino acid residue, a peptide, and a protein. 
     
     
         36 . (canceled) 
     
     
         37 . A compound as claimed in  claim 35  having formula VI,
   (Mc) m -(CH 2 ) n —X-(L) p -R   VI
 
 
       wherein
 Mc is a metallocenyl group in which ring may be independently be substituted or unsubstituted, 
 the metallocenyl group comprises a metal ion M selected from the group consisting of iron, chromium, cobalt, osmium, ruthenium, nickel, and titanium. 
 m is 1, 2, 3 or 4 
 n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 
 X is NR′ or O 
 R′ is H or lower alkyl, 
 L is a linker group, 
 p is 0 or 1, and 
 R is a protein, a peptide or an amino acid residue. 
 
     
     
         38 . A compound as claimed in  claim 37  having formula V,
   Mc-(CH 2 ) n —X—R   V
 
 
       wherein
 Mc is a metallocenyl group in which ring may be independently be substituted or unsubstituted, 
 the metallocenyl group comprises a metal ion M selected from the group consisting of iron, chromium, cobalt, osmium, ruthenium, nickel, and titanium, 
 n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, 
 X is NR′ or 0, 
 R′ is H or lower alkyl, and 
 R is a protein, a peptide, or an amino acid residue.