Microfluidic device with multiple cognitive agents immobilized thereon and methods for its fabrication and method of use

Abstract

A microfluidic device allowing for multiple discrete reactions sites and allowing for sequential reactions and sample analysis along with methods for device fabrication and use is provided. The microfluidic device provides a micro-total analysis system on a single substrate and has multiple reaction sites allowing for cascade reactions and analysis on a single chip using micro-quantities of sample and reagents. The microfluidic device provides discrete sites for immobilization of cognitive agents including enzymes, binding proteins, nucleic acids and the like as well as methods for quantitative analysis. The invention also provides methods for the fabrication of the device.

1 . A microfluidic device comprising a reaction channel fabricated from a reaction layer on a substrate, including at least two reaction channel networks, wherein the at least two reaction channel networks are immobilized with at least one cognitive agent allowing for a sequential reaction along the reaction channel. 
     
     
         2 . The microfluidic device of  claim 1 , wherein said cognitive agent is selected from a group of enzymes, antigens, antibodies, proteins, receptors, nucleic acids or combinations thereof. 
     
     
         3 . The microfluidic device of  claim 1 , wherein the at least two reaction channel networks have different cognitive agents immobilized therein. 
     
     
         4 . The microfluidic device of  claim 3 , wherein the enzymes immobilized in the reaction channel networks are invertase, mutarotase, and glucose oxidase. 
     
     
         5 . The microfluidic device of  claim 1 , wherein the reaction layer is made from a polymer 
     
     
         6 . The microfluidic device of  claim 5 , wherein the reaction layer is made from plastic, polydimethyl siloxane, polycarbonate, acrylic resin, polyvinyl chloride, polyacrylic amide or polyacrylonitrile. 
     
     
         7 . The microfluidic device of  claim 1 , wherein the substrate is made from glass, silicon, polyvinyl chloride, polyester, polyimide, acrylic resin, polycarbonate, cellulose acetate, polyacrylic amide, polyacrylonitrile, polydimethyl siloxane or plastic. 
     
     
         8 . The microfluidic device of  claim 1 , wherein the substrate has a sensor for measurement. 
     
     
         9 . The microfluidic device of  claim 8 , wherein said sensor is a thermal sensor, electromagnetic sensor, mechanical sensor, chemical sensor, optical sensor, ionizing radiation sensor or acoustic sensor. 
     
     
         10 . A method of fabrication of a microfluidic device comprising the steps of:
 providing a substrate;   attaching an immobilization layer having one or more channel networks formed therein to the substrate;   immobilizing one or more cognitive agents onto the substrate surface at the one or more channel networks;   flowing one or more solution of cognitive agents into the channel networks;   detaching the immobilization layer from the substrate; and   positioning a reaction layer including a single reaction channel to the substrate;   wherein the single reaction channel encompasses the one or more channel networks.   
     
     
         11 . The method of fabricating the microfluidic device of  claims 10 , wherein the solution of cognitive agents is an enzyme solution, an antigen solution, an antibody solution, a protein solution, a receptor solution, a nucleic acid solution or combinations thereof. 
     
     
         12 . The method of fabricating the microfluidic device of  claim 10 , further comprising a step of substrate surface pre-treatment, prior to the step of attaching the immobilization layer. 
     
     
         13 . The method of forming the microfluidic device of  claim 10 , wherein the pre-treatment step is preformed prior to flowing the cognitive through the channel network. 
     
     
         14 . The method of  claim 11  wherein the pre-treatment step comprises deposition of a gold layer. 
     
     
         15 . A method of component analysis comprising a step of introducing a solution into the reaction channel of the device of  claim 1  and allowing a component in the solution to flow through the at least two reaction channel networks with the one or more cognitive agents in sequence, and measuring an amount of a reaction product.