Glycan arrays on ptfe-like aluminum coated glass slides and related methods

Abstract

Aluminum coated glass slides provide a novel glycan array platform. Specifically, aluminum coated glass slides increase sensitivity of fluorescent based assay methods. Additionally, aluminum coated glass slides allows for mass spectroscopic analysis of carbohydrates and provide a platform for examining activity of cellulases. The unique properties of ACG slides include: the metal oxide layer on the surface can be activated for grafting organic compounds such as modified oligosaccharides; the surface remains electrically conductive, and the grafted oligosaccharides can be simultaneously characterized by mass spectrometry and carbohydrate-binding assay; and the slides are more sensitive than transparent glass slides in binding analysis.

What is claimed is: 
     
         1 . A method for characterization of carbohydrates immobilized on a PTFE-like aluminum-coated transparent solid substrate, the method comprising:
 (a) providing an array comprising a plurality of carbohydrates immobilized at discrete locations on a surface of a PTFE-like aluminum-coated transparent solid substrate; and   (b) performing mass spectroscopic analysis to characterize the carbohydrates immobilized at each discrete location.   
     
     
         2 . The method of  claim 1 , wherein the mass spectroscopic characterization of the immobilized carbohydrates comprises a time-of-flight mass spectrometry (MS-TOF). 
     
     
         3 . The method of  claim 1 , further comprising:
 (c) then performing a binding analysis of suspected carbohydrate binding moieties.   
     
     
         4 . The method of  claim 3 , wherein the suspected carbohydrate binding moieties are cellulase proteins. 
     
     
         5 . The method of  claim 3 , further comprising:
 (d) incubating the cellulase proteins with the bound carbohydrates immobilized on the array surface under conditions suitable for the cellulases to hydrolyze the carbohydrates.   
     
     
         6 . The method of  claim 5 , further comprising:
 (e) characterizing the products of the cellulase proteins remaining immobilized on the array surface following hydrolysis by the cellulases.   
     
     
         7 . The method of  claim 5 , wherein the cellulases are selected from the group consisting of 1,4-ß-glucosidases, exoglucanases (1,4-ß-D glucan cellobiohydrolases) and endoglucanases (1,4-ß-D glucan glucanohydrolases). 
     
     
         8 . A method for analysis of binding reactions between the carbohydrates and molecules suspected of specifically binding the carbohydrates, the method comprising:
 (a) providing an array comprising a plurality of carbohydrates immobilized at discrete locations on a surface of an aluminum-coated transparent solid substrate or a PTFE-like aluminum-coated transparent solid substrate;   (b) contacting the array with one or more molecules suspected of binding to one or more of the plurality of carbohydrates immobilized on the array surface; and   (c) identifying the presence or absence of binding reactions at one or more discrete locations on the array surface.   
     
     
         9 . The method of  claim 8 , wherein the molecules suspected of specifically binding the carbohydrates are proteins labeled with a detectable label. 
     
     
         10 . The method of  claim 9 , wherein the protein labels comprise fluorescent dyes. 
     
     
         11 . The method of  claim 10 , wherein the fluorescent dyes comprise amine-reactive cyanine dyes. 
     
     
         12 . The method of  claim 8 , wherein the binding of a molecule to a carbohydrate on the array is representative of a biological process. 
     
     
         13 . The method of  claim 12 , wherein the biological process is selected from the group consisting of protein folding, protein secretion, protein stabilization, viral infection, bacterial infection, cancer metastasis, inflammatory response, innate immunity, adaptive immunity, a receptor-mediated signaling process, and biofuel production. 
     
     
         14 . The method of  claim 8 , wherein the carbohydrates are polysaccharides, or oligosaccharides, or carbohydrate portions of a glycoconjugate, or cellobiose, or cellotriose, or cellotetraose, or GloboH, or Gb5. 
     
     
         15 . The method of  claim 8 , wherein a mass spectroscopic characterization of the carbohydrates immobilized on the array is performed prior to the binding analysis. 
     
     
         16 . The method of  claim 8 , wherein a mass spectroscopic characterization of the carbohydrates immobilized on the array is performed prior to and following the binding analysis,
 wherein the one or more molecules suspected of binding to one or more of the plurality of carbohydrates immobilized on the array surface comprises a cellulose protein enzyme capable of hydrolyzing one or more carbohydrates on the array, and   further wherein the binding reaction is performed under conditions suitable for the cellulose to hydrolyze the carbohydrate.   
     
     
         17 . The method of  claim 16 , wherein the cellulases are selected from the group consisting of 1,4-ß-glucosidases, exoglucanases (1,4-ß-D glucan cellobiohydrolases) and endoglucanases (1,4-ß-D glucan glucanohydrolases). 
     
     
         18 . A method for fabricating an array of carbohydrates immobilized on an aluminum coated transparent solid substrate or a PTFE-like aluminum-coated transparent solid substrate, the method comprising:
 (a) immobilizing a plurality of carbohydrates at discrete locations on a surface of an aluminum coated transparent solid substrate or a PTFE-like aluminum-coated transparent solid substrate, wherein the substrate is conductive or semiconductive of an electrical field,   wherein the array is suitable for performing mass spectroscopic characterization of the immobilized carbohydrates, and   wherein the array is suitable for performing analysis of binding reactions between the carbohydrates and molecules suspected of specifically binding the carbohydrates.   
     
     
         19 . The method of  claim 18 , wherein the carbohydrates are immobilized by a non-covalent bond. 
     
     
         20 . The method of  claim 19 , wherein the carbohydrates are polyfluorinated. 
     
     
         21 . The method of  claim 18 , wherein the carbohydrates are immobilized by a covalent bond. 
     
     
         22 . The method of  claim 21 , wherein the carbohydrates are modified with a phosphonic acid functional group. 
     
     
         23 . The method of  claim 18 , further comprising: (b) performing a characterization of carbohydrates immobilized on the array surface by mass spectrometry, wherein observation of one or more of a high signal/noise (S/N) ratio, low laser fluence rate, or a low fragmentation of signal, in an absence of matrix material is indicative of the array being suitable for performing mass spectroscopic characterization of the immobilized carbohydrates. 
     
     
         24 . The method of  claim 23 , further comprising: (c) performing a carbohydrate binding assay on the array by contacting the array with a carbohydrate-binding protein, wherein detection of specific binding at one or more discrete locations on the array is indicative of the array being suitable for performing analysis of binding reactions between the carbohydrates and molecules suspected of specifically binding the carbohydrates. 
     
     
         25 . The method of  claim 24 , wherein the carbohydrate is selected from a sugar, or a glycoprotein, or a glycolipid, or mannose, each comprising internal or nonreducing terminal alpha-mannosyl groups and the binding molecule is Concanavalin A.