Methods, microfluidic devices, and systems for detection of an active enzymatic agent

Abstract

Embodiments of the present invention provide methods, microfluidic devices, and systems for the detection of an active target agent in a fluid sample. A substrate molecule is used that contains a sequence which may cleave in the presence of an active target agent. A SNAP25 sequence is described, for example, that may be cleaved in the presence of Botulinum Neurotoxin. The substrate molecule includes a reporter moiety. The substrate molecule is exposed to the sample, and resulting reaction products separated using electrophoretic separation. The elution time of the reporter moiety may be utilized to identify the presence or absence of the active target agent.

What is claimed is: 
     
         1 . A method for detecting enzymatic activity of an active target agent in a sample, the method comprising:
 concentrating said sample at a membrane within a microfluidic device;   concentrating a substrate molecule larger than 25 kDa at the membrane within the microfluidic device, the substrate molecule comprising a reporter moiety and a protein sequence configured to cleave in the presence of the active target agent into reaction products, at least one reaction product including the reporter moiety and having a size larger than 25 kDa;   measuring an elution time for the reporter moiety to determine cleavage of said sequence, said cleavage at least in part identifying the presence of the active target agent in said sample.   
     
     
         2 . The method according to  claim 1  wherein the act of concentrating the substrate molecule comprises applying an electric field across the membrane of less than 80 V/cm. 
     
     
         3 . The method according to  claim 1  wherein the protein sequence comprises a SNAP25 sequence. 
     
     
         4 . The method according to  claim 3  wherein the active target agent comprises Botulinum Neurotoxin. 
     
     
         5 . The method according to  claim 1  wherein the protein sequence comprises a VAMP-2 sequence. 
     
     
         6 . The method according to  claim 5  wherein the active target agent comprises Tetanus Neurotoxin. 
     
     
         7 . The method according to  claim 1  further comprising incubating the substrate molecules and the active target agent in the vicinity of the membrane to generate reaction products. 
     
     
         8 . The method according to  claim 7  further comprising transporting the reaction products to a second location within the microfluidic device prior to the act of measuring the elution time. 
     
     
         9 . The method according to  claim 8  further comprising exposing the reaction products to a denaturing agent at the second location. 
     
     
         10 . The method according to  claim 1  further comprising:
 providing an indication of whether Botulinum Neurotoxin was present in the sample based, at least in part, on the elution time. 
 
     
     
         11 . The method according to  claim 7  wherein the substrate molecule comprises a SNAP25 substrate molecule and wherein the reaction products contain the intact SNAP25 substrate molecule including reporter moiety when the sample did not contain active Botulinum Neurotoxin. 
     
     
         12 . The method according to  claim 7  wherein the substrate molecule comprises a SNAP25 molecule and wherein the reaction products contain a cleaved reporter moiety and a second cleaved portion of the SNAP25 substrate molecule not including the reporter moiety when the sample did contain active Botulinum Neurotoxin. 
     
     
         13 . The method according to  claim 1  wherein the act of measuring the elution time comprises performing electrophoretic separation on the reaction products. 
     
     
         14 . A method for detecting enzymatic activity of an active target agent in a sample, the method comprising:
 concentrating said sample at a charged membrane within a microfluidic device;   concentrating a substrate molecule at the membrane within the microfluidic device, the substrate molecule comprising a reporter moiety and a protein sequence configured to cleave in the presence of the active target agent into reaction products, at least one reaction product including the reporter moiety and having a size equal to or less than 25 kDa, the charge of the charged membrane configured to exclude the reaction product having the size equal to or less than 25 kDa according to a pH of the reaction product; and   measuring an elution time for the reporter moiety to determine cleavage of said sequence, said cleavage at least in part identifying the presence of the active target agent in said sample.   
     
     
         15 . The method according to  claim 14  further comprising incubating the substrate molecules and the active target agent in the vicinity of the membrane to generate reaction products. 
     
     
         16 . The method according to  claim 15  further comprising transporting the reaction products to a second location within the microfluidic device prior to the act of measuring the elution time. 
     
     
         17 . The method according to  claim 16  further comprising exposing the reaction products to a denaturing agent at the second location.