Microstructure apparatus and method for separating differently charged molecules using an applied electric field

Abstract

The field of the present invention relates generally to a microstructure apparatus which may be used in a high-throughput screening context to monitor the rate of reaction of an enzyme with its substrate in cases where the product of the reaction has an altered net charge. For example, the systems and methods disclosed herein may be used to detect the activity of phosphatase enzymes, proteases and kinases on charged peptide substrates. The microstructure devices of the present invention comprise a plurality of microstructures, wherein each microstructure comprises a capture matrix located between two electrodes.

1 - 50 . (canceled)  
     
     
         51 . A method for separating molecules having different charges utilizing a system for separating molecules having different charges and capturing a molecule of interest for detection, comprising a microstructure plate comprising at least one microstructure, each microstructure comprising a series of microstructure sections and channels, wherein each microstructure section is directly interconnected to at least one other microstructure section by at least one channel, the series comprising: at least one sample accepting microstructure section, wherein the sample accepting section is fluidly connected to the exterior of the microstructure plate; at least one first electrode microstructure section; at least one second electrode microstructure section; at least one capture microstructure section containing a capture matrix, wherein the capture microstructure section is between the first and second electrode microstructure sections in the series; wherein the microstructures in the microstructure plate are formed by at least two layers of material, wherein at least one layer is a sealing plate layer which seals at least one channel or microstructure section in the assembled microstructure plate; and an electrode assembly, the electrode assembly having at least one first and at least one second electrode, wherein each first electrode microstructure section is in electrical contact with at least one first electrode, and wherein each second electrode microstructure section is in electrical contact with at least one second electrode 
 wherein the method comprises the steps of    (a) filling the microstructure with a liquid,    (b) introducing a sample into a sample-accepting microstructure section of the apparatus,    (c) energizing the electrode assembly for a sufficient period of time to allow a charged molecule of interest in the sample to migrate towards an electrode of the electrode assembly and to be caught in the capture matrix, and    (d) detecting the charged molecule of interest caught in the capture matrix.    
     
     
         52 . The method of  claim 51 , further comprising the preparatory step of placing at least one capture matrix in at least one capture microstructure section of a microstructure of the apparatus.  
     
     
         53 . The method of  claim 52 , wherein the capture matrix is a hydrogel matrix.  
     
     
         54 . The method of  claim 53 , wherein the hydrogel is polymerized after being placed in the capture microstructure section, the method further comprising the step of subjecting a hydrogel precursor to UV irradiation after being placed in the capture microstructure section.  
     
     
         55 . The method of  claim 51 , wherein the capture matrix is a membrane.  
     
     
         56 . The method of  claim 51 , wherein the liquid is an aqueous buffer.  
     
     
         57 . The method of  claim 56 , wherein the aqueous buffer is selected from the group consisting of: tris hydrochloride buffers, tris borate buffers, histidine buffer, alanine buffers, adipic dihydrazide buffers, and HEPES buffers.  
     
     
         58 . The method of  claim 56 , wherein the microstructure is filled with an aqueous buffer by introducing the buffer into the sample microstructure section under pressure.  
     
     
         59 . The method of  claim 51 , wherein the microstructure is filled with liquid by an automated pipettor.  
     
     
         60 . The method of  claim 51 , wherein the sample is introduced into the sample-accepting microstructure section by an automated sample transfer device, and wherein the sample is transferred from the well of a microtiter plate.  
     
     
         61 . The method of  claim 51 , wherein the electrode assembly is separate from the microstructure plate in step (c), further comprising the step of lowering the electrode assembly into electrical contact with the first and second microstructure sections before energizing the electrode assembly in step (d).  
     
     
         62 . The method of  claim 51 , wherein the electrode assembly is in place when the sample is loaded into the sample-accepting microstructure section in step (b).  
     
     
         63 . The method of  claim 51 , wherein the electrodes are energized to apply 1 μAmp to 10 mAmp of current through the microstructure.  
     
     
         64 . The method of  claim 51 , wherein the electrodes are energized to apply 1 μAmp to 5 mAmp of current through the microstructure.  
     
     
         65 . The method of claim  5   1 , wherein the electrodes are energized to apply 5 μAmp to 1 mAmp of current through the microstructure.  
     
     
         66 . The method of claim  5   1 , wherein the electrodes are energized to apply a potential of 0.1 V to 500 V across the microstructure.  
     
     
         67 . The method of  claim 51  wherein the electrodes are energized to apply a potential of 0.5 V to 100 V across the microstructure.  
     
     
         68 . The method of  claim 51  wherein the electrodes are energized to apply a potential of 1.0 V to 40 V across the microstructure.  
     
     
         69 . The method of  claim 51 , wherein the detection in step (e) is by a method selected from the group consisting of fluorometry, colorimetry, luminometry, mass spectrometry, electrochemical detection, and radioactivity detection.  
     
     
         70 . The method of  claim 69 , wherein the detection in step (e) is by fluorometry.  
     
     
         71 . The method of  claim 69 , wherein the charged molecule of interest is detected by placing at least a portion of the apparatus containing the microstructure plate into a microtiter plate reader.  
     
     
         72 . The method of  claim 51 , wherein the charged molecule of interest is the product of a substrate reaction wherein the net charge of a substrate is changed in the enzymatic reaction.  
     
     
         73 . The method of  claim 72 , wherein the charged molecule of interest and the substrate both comprise a detectable labeling moiety.  
     
     
         74 . The method of  claim 73 , wherein the labeling moiety is a fluorescent moiety.  
     
     
         75 . The method of  claim 72 , wherein the method is capable of detecting the enzymatic conversion of at least 10% of the substrate.  
     
     
         76 . The method of  claim 72 , wherein the method is capable of detecting the enzymatic conversion of at least 1.0% of the substrate.  
     
     
         77 . The method of  claim 72  wherein the method is capable of detecting the enzymatic conversion of at least 0.1% of the substrate.