Quantitative analysis of carbohydrate-protein interactions using glycan microarrays: determination of surface and solution dissociation constants

Abstract

A method, system and device to identify, study and/or mimic carbohydrate-protein interactions on cell surfaces and in solution measured by a glycan microarray. In some instances the method, system and device uses very small quantities of carbohydrate as low as attomol. In some instances the system, method and device is high-throughput. The small quantity senstivity may allow for close placement of carbohydrate array members wherein due to close proximity multivalent interactions with proteins may be identified.

What is claimed is: 
     
         1 . A carbohydrate microarray for protein measurements, comprising:
 a solid substrate configured to provide a supporting substance; and   a reactive layer fabricated on the solid substrate, the reactive layer comprising at least one group of small molecules wherein the small molecules comprise at least one of carbohydrates and glycolipids wherein the small molecules are disposed relative to each other to allow the at least one protein to multivalently bond with the small molecules;   wherein the at least one group of small molecules is configured to bind with at least one protein wherein the at least one protein is immobilized on the solid substrate via the reactive layer;   wherein the carbohydrate microarray is configured to perform quantitative measurement of binding with the at least one protein; and   wherein at least one protein of a given concentration is applied whereby the binding affinity between the small molecules and the at least one protein can be measured.   
     
     
         2 . The device of  claim 1 , wherein an average spatial separation between the small molecules ranges from about 65 Angstrom to about 100 Angstrom. 
     
     
         3 . The device of  claim 1 , wherein the proteins comprise at least one of carbohydrate binding proteins and lipid binding proteins. 
     
     
         4 . The device of  claim 1 , wherein the proteins are fluorescence labeled or can be detected by a secondary labeled protein. 
     
     
         5 . The device of  claim 1 , wherein each of the at least one reactive group has at least about 10 −18  mole of carbohydrate. 
     
     
         6 . The device of  claim 1 , wherein carbohydrates with reactive groups are immobilized on the surface of the device wherein the arrays comprise about 2 to 50 subarrays wherein each subarray contains one or more carbohydrates. 
     
     
         7 . A kit comprising:
 a carbohydrate microarray comprising carbohydrate deposited on a solid substrate, each carbohydrate comprising a reactive group of at least one of carbohydrate and glycolipid, wherein the carbohydrates are configured to react with and bind to at least one protein; wherein the reactive groups are disposed relative to each other to allow the at least one protein to multivalently bond with the reactive groups;   a microarray reader; and   instructions for use.   
     
     
         8 . The kit of  claim 7 , wherein the carbohydrates of the microarray are deposited at least about 10 −18  mole of carbohydrate molecule per spot. 
     
     
         9 . The kit of  claim 7 , wherein the carbohydrates include at least one glycan. 
     
     
         10 . The kit of  claim 7 , wherein the carbohydrates include at least one glycolipid.