Arrays and methods for guided cell patterning

Abstract

Guided cell patterning arrays for single cell patterning, methods for making the arrays, and methods for using the arrays.

1 . An array for guided cell patterning, comprising a plurality of cell adhesion sites, each site being individually isolated on an inert surface,
 wherein each cell adhesion site comprises one or more ligands having an affinity to a cell surface receptor; and   wherein the inert surface is resistant to cell adhesion.   
     
     
         2 . The array of  claim 1 , wherein each cell adhesion site further comprises a single cell immobilized thereto by the interaction of the one or more ligands and one or more cell surface receptors of the immobilized cell. 
     
     
         3 . The array of  claim 1 , wherein each cell adhesion site further comprises two or more cells immobilized thereto by the interaction of the one or more ligands and one or more cell surface receptors of the immobilized cells. 
     
     
         4 . The array of  claim 1 , wherein the one or more ligands are covalently coupled to a self-assembly monolayer on a metal surface on the inert surface. 
     
     
         5 . The array of  claim 1 , wherein the one or more cell ligands are adsorbed to a self-assembly monolayer on a metal surface on the inert surface. 
     
     
         6 . The array of  claim 1 , wherein the one or more ligands are cell adhesion peptides. 
     
     
         7 . The array of  claim 1 , wherein the inert surface comprises a silicon surface having polyalkylene oxide moieties covalently attached thereto. 
     
     
         8 . The array of  claim 1 , wherein the inert surface comprises an oxidized silicon surface having polyalkylene oxide moieties covalently attached thereto. 
     
     
         9 . The array of  claim 1 , wherein the inert surface comprises a silicon surface with from about 40% to 65% by weight Si, from about 5% to about 20% by weight SiO x<2 , and from about 20% to about 40% by weight SiO 2 . 
     
     
         10 . A method for making an array of cell adhesion sites, comprising:
 (a) providing a metal-patterned silicon substrate having an array of metal surfaces disposed on a silicon surface;   (b) forming a self-assembly monolayer on each metal surface to provide an array of monolayers disposed on the silicon surface;   (c) passivating the silicon surface by covalently coupling polyalkylene oxide moieties to the silicon surface to provide a surface resistant to cell adhesion isolating each self-assembly monolayer of the monolayer array; and   (d) attaching a plurality of ligands to each self-assembly monolayer to provide an array of cell adhesion sites.   
     
     
         11 . The method of  claim 10  further comprising immobilizing a single cell at each cell adhesion site through the interaction of the ligands and one or more cell surface receptors of the cell. 
     
     
         12 . The method of  claim 10  further comprising immobilizing two or more cells at each cell adhesion site through the interaction of the ligands and one or more cell surface receptors of the cells. 
     
     
         13 . The method of  claim 10 , wherein the metal-patterned silicon substrate comprises a p-type silicon substrate with (100) orientation having an array of metal squares patterned thereon. 
     
     
         14 . The method of  claim 10 , wherein forming a self-assembly monolayer on each metal surface comprises reacting the metal surfaces with a thiol-terminated alkanoic acid to provide a carboxylic acid-terminated monolayer. 
     
     
         15 . The method of  claim 10 , wherein passivating the silicon surface by covalently coupling polyalkylene oxide moieties to the silicon surface comprises exposing the silicon surface to a reactive silane terminated-polyalkylene oxide. 
     
     
         16 . The method of  claim 10 , wherein attaching a plurality of ligands to each self-assembly monolayer comprises covalently coupling the ligands to each self-assembly monolayer. 
     
     
         17 . The method of  claim 10 , wherein attaching a plurality of ligands to each self-assembly monolayer comprises adsorbing the ligands to each self-assembly monolayer. 
     
     
         18 . The method of  claim 10 , wherein metal surfaces are selected from the group consisting of gold, platinum, and silver surfaces. 
     
     
         19 . The method of  claim 10 , wherein the ligands are cell adhesion peptides. 
     
     
         20 . An array of cell adhesion sites obtainable by the methods of  claim 10 . 
     
     
         21 . A method for analyzing a plurality of single cells immobilized in an array, comprising:
 (a) subjecting one or more cells individually immobilized in an array to a stimulus to provide an array comprising individually treated cells, the array comprising a plurality of cell adhesion sites, each site isolated on an inert surface, wherein each cell adhesion site comprises a single cell immobilized thereto by the interaction of one or more ligands attached to the site and one or more cell surface receptors of the immobilized cell, and wherein the inert surface is resistant to cell adhesion; and   (b) individually addressing one or more of the treated cells to measure the effect of the stimulus on the treated cells.   
     
     
         22 . The method of  claim 21 , wherein the stimulus is a therapeutic drug. 
     
     
         23 . The method of  claim 21 , wherein the stimulus is a toxin. 
     
     
         24 . The method of  claim 21 , wherein individually addressing one or more of the treated cells comprises individually addressing the treated cells optically. 
     
     
         25 . The method of  claim 21 , wherein individually addressing one or more of the treated cells comprises individually addressing the treated cells electrically. 
     
     
         26 . An array for guided cell patterning, comprising a plurality of individually immobilized cells isolated on an inert surface resistant to cell adhesion, wherein the inert surface comprises a silicon oxide surface having polyalkylene oxide moieties covalently coupled thereto, and wherein the silicon oxide surface comprises from about 40% to 65% by weight Si, from about 5% to about 20% by weight SiO x<2 , and from about 20% to about 40% by weight SiO 2 . 
     
     
         27 . The array of  claim 26 , wherein each cell is immobilized through the interaction of one or more ligands and one or more cell surface receptors of the immobilized cell. 
     
     
         28 . The array of  claim 26 , wherein the silicon oxide surface comprises from about 50% to 60% by weight Si, from about 10% to about 15% by weight SiO x<2 , and from about 25% to about 35% by weight SiO 2 . 
     
     
         29 . The array of  claim 26 , wherein the silicon oxide surface comprises about 58% by weight Si, about 12% by weight SiO x<2 , and about 30% by weight SiO 2 . 
     
     
         30 . The array of  claim 26 , wherein the ligands are cell adhesion peptides. 
     
     
         31 . A method for making an array for guided cell patterning, comprising:
 (a) providing a metal-patterned silicon substrate having an array of metal surfaces disposed on a silicon surface;   (b) exposing the substrate to an oxide etch to remove native oxide from the silicon oxide surface to provide a native oxide depleted silicon surface;   (c) oxidizing the native oxide depleted silicon surface with an oxidizing agent to provide a silicon oxide surface; and   (d) passivating the silicon oxide surface by covalently coupling polyalkylene oxide moieties to the silicon oxide surface to provide a surface resistant to cell adhesion isolating each metal surface of the metal surface array.   
     
     
         32 . The method of  claim 31  further comprising forming a self-assembly monolayer on each metal surface to provide an array of monolayers isolated on the silicon oxide surface. 
     
     
         33 . The method of  claim 32  further comprising attaching a plurality of ligands to each self-assembly monolayer to provide an array of cell adhesion sites. 
     
     
         34 . The method of  claim 33  further comprising immobilizing a single cell at each cell adhesion site through the interaction of the ligands and one or more cell surface receptors of the cell. 
     
     
         35 . The method of  claim 33  further comprising immobilizing two or more cells at each cell adhesion site through the interaction of the ligands and one or more cell surface receptors of the cells. 
     
     
         36 . The method of  claim 31 , wherein the oxide etch comprises H 2 SO 5  or a mixture of hydrogen fluoride and ammonium fluoride. 
     
     
         37 . The method of  claim 31 , wherein the oxidizing agent is selected from a group consisting of dry oxygen, ozone, hydrogen peroxide, and chromic acid. 
     
     
         38 . The method of  claim 31 , wherein oxidizing the native oxide depleted surface comprises oxidizing the native oxide depleted surface with dry oxygen at about 300° C. to about 500° C. for about 5 to about 24 hours. 
     
     
         39 . The method of  claim 31 , wherein the silicon oxide surface comprises from about 40% to 65% by weight Si, from about 5% to about 20% by weight SiO x<2 , and from about 20% to about 40% by weight SiO 2 . 
     
     
         40 . The method of  claim 31 , wherein the silicon oxide surface comprises from about 50% to 60% by weight Si, from about 10% to about 15% by weight SiO x<2 , and from about 25% to about 35% by weight SiO 2 . 
     
     
         41 . The method of  claim 31 , wherein the ligands are cell adhesion peptides. 
     
     
         42 . An array of cell adhesion sites obtainable by the methods of  claim 31 .