Method for the detection of enzymatic activity with magnetically functionalized substrates

Abstract

The present invention provides methods for detecting an enzymatic activity, the method including combining at least one magnetic particle to an enzyme substrate to form a magnetically modified substrate, reacting the magnetically modified substrate with at least one enzyme; and detecting a change in a magnetic property of the magnetically modified substrate or its cleavage products, thereby detecting an activity of said at least one enzyme, wherein the method may be applied to a human subject to detect a disease selected from the group consisting of rheumatitis, arthritis, an injury, Dupuytren's disease, Peyronie's disease, a collagen related disease, steatosis, fibrosis, cirrhosis, metastasis, tissue regeneration, cancer, coronary disease, a liver disease, a metabolic condition, an infection and an inflammatory disease.

1 . A method for detecting an enzymatic activity, the method comprising:
 a) combining at least one magnetic particle to an enzyme substrate to form a magnetically modified substrate;   b) reacting the magnetically modified substrate with at least one enzyme; and   c) detecting a change in a magnetic property of at least one of said magnetically modified substrate and a cleavage product thereof, wherein said at least one of said magnetically modified substrate and said cleavage product thereof, comprise at least one magnetic particle,   
       thereby detecting an activity of said at least one enzyme. 
     
     
         2 . A method according to  claim 1 , wherein the change in said magnetic property is at least a partial change from superparamagnetic to paramagnetic. 
     
     
         3 . A method according to  claim 1 , wherein said detecting step comprises detecting at least one relaxation time of said magnetically modified substrate. 
     
     
         4 . A method according to  claim 3 , wherein said least one relaxation time comprises a first relaxation time prior to the reacting step and a second relaxation time after said reacting step. 
     
     
         5 . A method according to  claim 1 , wherein said detecting step is performed by at least one of magnetic resonance, magnetic resonance imaging (MRI), electronic paramagnetic resonance (EPR), magnetometry, Mössbauer spectrometry, electromagnetic inductance, atomic force microscopy, magnetorelaxometry and a combination thereof. 
     
     
         6 . A method according to  claim 5 , wherein said detecting step comprises the use of at least one of a fine probe and a contrast agent in an imaging process. 
     
     
         7 . A method according to  claim 6 , wherein said imaging process comprises detecting at least one of a signal intensity, T1, T2, diffusion parameter and a response to a signal saturation. 
     
     
         8 . A method according to  claim 6 , wherein said imaging process comprises detecting at least one change in a signal intensity, T1, T2, diffusion and a response to a signal saturation measured a) before the reacting step and b) after the reacting step. 
     
     
         9 . A method according to  claim 1 , wherein said change is indicative of at least one of:
 a. aggregation of said at least one magnetic particle;   b. aggregation of said magnetically modified substrate or its cleavage products;   c. a change in particle size of said at least one magnetic particle; and   d. a change in particle size of at least one of said magnetically modified substrate and a cleavage product thereof, wherein said at least one of said magnetically modified substrate and said cleavage product thereof comprise at least one magnetic particle.   
     
     
         10 . A method according to  claim 1 , wherein said enzyme substrate comprises at least one of: a protein, a polypeptide, a modified protein, a modified polypeptide, a glycoprotein, a lipoprotein, collagen and gelatin. 
     
     
         11 . A method according to  claim 1 , wherein said at least one enzyme comprises at least one of: a metal-dependent enzyme, a protease, a lipase, a lipoprotein lipase, a hormone sensitive lipase, a metalloprotease, a matrix metallo-proteinases (MMP), a collagenase and a gelatinase. 
     
     
         12 . A method according to  claim 1 , wherein said reacting step is performed in vivo. 
     
     
         13 . A method according to  claim 12 , wherein said reacting step is performed in a human subject in order to detect a disease or disorder associated with said activity of said at least one enzyme. 
     
     
         14 . A method according to  claim 13 , wherein said disease or disorder is selected from the group consisting of rheumatitis, arthritis, an injury, connective tissue condition related disease, Dupuytren's disease, Peyronie's disease, a collagen related disease, steatosis, fibrosis, cirrhosis, metastasis, tissue regeneration, cancer, coronary disease, a liver disease, a metabolic condition, an infection and an inflammatory disease. 
     
     
         15 . A method for detecting a disease in a human subject, the method comprising:
 a) combining at least one magnetic particle to a reactant to form a magnetically modified reactant;   b) reacting the magnetically modified reactant with at least one enzyme in said human subject; and   c) detecting a change in a magnetic property of the magnetically modified reactant thereby detecting said disease in said human subject.   
     
     
         16 . A method according to  claim 15 , wherein said reactant comprises at least one of: a protein, a polypeptide, a modified protein, a modified polypeptide, a glycoprotein, a lipoprotein, collagen and gelatin. 
     
     
         17 . A method according to  claim 15 , wherein said at least one enzyme comprises at least one of: a metal-dependent enzyme, a protease, a lipase, a lipoprotein lipase, a hormone sensitive lipase, a metalloprotease, a matrix metallo-proteinases (MMP), a collagenase and a gelatinase. 
     
     
         18 . A method according to  claim 15 , wherein said disease is selected from the group consisting of rheumatitis, arthritis, an injury, a connective tissue condition related disease, Dupuytren's disease, Peyronie's disease, a collagen related disease, steatosis, fibrosis, cirrhosis, metastasis, tissue regeneration, cancer, coronary disease, a liver disease, a metabolic condition, an infection and an inflammatory disease. 
     
     
         19 . A method according to  claim 1 , wherein said magnetic particle is combined with at least one of an enzyme substrate and a reactant in vivo. 
     
     
         20 . A method according to  claim 19 , wherein said change is indicative of at least one of:
 a. aggregation of said at least one magnetic particle;   b. aggregation of said magnetically modified substrate or its cleavage products;   c. a change in particle size of said at least one magnetic particle; and   d. a change in particle size of at least one of said magnetically modified substrate and a cleavage product thereof, wherein said at least one of said magnetically modified substrate and said cleavage product thereof comprise at least one magnetic particle.