System and method for detecting and monitoring proteolysis of protein matrices

Abstract

Apparatus and methods are provided for detecting and monitoring proteolysis of protein matrices such as fibrin clots, extracellular matrix and collagen matrix. The apparatus and methods involve in general the measurement of diffusion-limited electrochemical currents generated by an electroactive species or elactomer in a synthetic protein matrix. The apparatus and methods are applied in various embodiments to detect and monitor the proteolytic activities of a sample, and more specifically fibrinolytic activities and collagenase activities of a sample. The apparatus and methods are utilized generally in the monitoring and diagnostics of cardiovascular, cerebralvascular, and oncology conditions.

1 .- 24 . (canceled) 
     
     
         25 . A method for detecting or monitoring a disease or disorder associated with fibrinolysis, comprising:
 (a) contacting a synthetic fibrin matrix with a biological sample from a subject, wherein:
 (i) the synthetic fibrin matrix comprises an elactomer and a small molecule electroactive species in contact with the synthetic fibrin matrix by diffusion within and across pores of the synthetic fibrin matrix, 
 (ii) the elactomer comprises a ferrocene-derivitized polymer, 
 (iii) the elactomer and a small molecule electroactive species are of an opposing oxidation state, and 
 (iv) the synthetic fibrin matrix is in contact with at least a portion of a working electrode or a counter electrode; 
   (b) applying a potential to the working electrode, thereby generating an electrochemical current through the working electrode;   (c) measuring the electrochemical current at a plurality of times to obtain a plurality of current measurements; and   (d) comparing at least two of the plurality of current measurements, wherein a difference between the at least two current measurements is indicative of fibrinolytic activity in the presence of the biological sample, thereby detecting or monitoring a disease or disorder associated with fibrinolysis.   
     
     
         26 . The method of  claim 25 , wherein the disease or disorder associated with fibrinolysis is deep vein thrombosis, pulmonary embolism, renal disease, hypertrophic keloid scars, coronary infarction, metastasis, inflammation, disseminated intravascular coagulation, atherosclerosis, rheumatoid arthritis, glomerulonephritis, systematic lupus erythematosus, autoimmune neuropathies, granulomatous disease, parasitic infection or allograft rejection. 
     
     
         27 . The method of  claim 25 , wherein the polymer is selected from the group consisting of polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP) and derivatives thereof. 
     
     
         28 . The method of  claim 25 , wherein the elactomer is a ferrocene-derivitized polyethylene glycol having the formula Fc-CO—NH—(CH 2 CH 2 O) n NH—CO-Fc and the small molecule electroactive species is ruthenium (III) hexamine (Ru(NH 3 ) 6   3+ ). 
     
     
         29 . The method of  claim 25 , wherein the relative concentrations of the elactomer and the small molecule electroactive species are determined such that essentially all of the current at the working electrode are generated by the elactomer, and not by the small molecule electroactive species. 
     
     
         30 . The method of  claim 25 , wherein the relative concentrations of the elactomer and the small molecule electroactive species are determined such that essentially all of the current at the working electrode are generated by the elactomer, and not by the small molecule electroactive species. 
     
     
         31 . The method of  claim 25 , wherein the biological sample is selected from blood, plasma, interstitial fluid, or other bodily fluid. 
     
     
         32 . The method of  claim 25 , where the counter electrode comprises a reference electrode and an auxiliary electrode. 
     
     
         33 . The method of  claim 25 , wherein the measuring is performed by chronoamperometry, potential step voltammetry, linear sweep voltammetry, cyclic voltammetry, square wave voltammetry, staircase voltammetry, anodic or cathodic stripping voltammetry, adsorptive stripping voltammetry, alternating current voltammetry, rotated electrode voltammetry, normal or differential pulse voltammetry, or chronocoulometry. 
     
     
         34 . The method of  claim 25 , wherein alternating currents or multiple direct current pulses are measured. 
     
     
         35 . The method of  claim 25 , wherein the measuring is based on electrochemical impedance spectroscopy, and wherein changes in a frequency domain are indicative of a level of the proteolytic activity in the sample. 
     
     
         36 . An apparatus for detecting or monitoring proteolysis of a protein matrix, comprising (i) a synthetic protein matrix, (ii) a working electrode, (iii) a counter electrode, (iv) an elactomer, and (v) a small molecule electroactive species;
 wherein the synthetic protein matrix is in contact with at least a portion of the working electrode or the counter electrode;   wherein the elactomer comprises a ferrocene-derivitized polymer wherein the polymer is a water-soluble macromolecule;   wherein the elactomer and the small molecule electroactive species are of an opposing oxidation state and are provided in contact with the synthetic protein matrix by diffusion within and across pores of the synthetic protein matrix;   wherein the elactomer is capable of generating diffusion-limited electrochemical currents at the working electrode and the small molecule electroactive species is capable of generating counter currents at the counter electrode; and   wherein the diffusion limited electrochemical currents are indicative of a level of proteolysis of the synthetic protein matrix.   
     
     
         37 . The apparatus of  claim 36 , wherein the water soluble macromolecule comprises a polypeptide. 
     
     
         38 . The apparatus of  claim 36 , wherein the water-soluble macromolecule comprises a protein. 
     
     
         39 . The apparatus of  claim 36 , wherein the synthetic protein matrix comprises a fibrin clot, gelatin, or a collagen matrix. 
     
     
         40 . The apparatus of  claim 36 , wherein the working electrode is larger than the counter electrode. 
     
     
         41 . The apparatus of  claim 36 , wherein the relative concentrations of the elactomer and the small molecule electroactive species are determined such that essentially all of the current at the working electrode are generated by the elactomer, and not by the small molecule electroactive species. 
     
     
         42 . The apparatus of  claim 36 , wherein the small molecule electroactive species is ruthenium (III) hexamine (Ru(NH 3 ) 6   3+ ). 
     
     
         43 . The apparatus of  claim 36 , further comprising a sample, wherein the sample is selected from the group consisting of blood, plasma, interstitial fluid, other bodily fluid, and a control with known proteolytic activity. 
     
     
         44 . The apparatus of  claim 36 , further comprising a data processing unit for analyzing diffusion limited currents using a predetermined formula or methodology, thereby producing a result indicative of proteolysis of the protein matrix.