Apparatus for assay, synthesis and storage, and methods of manufacture, use, and manipulation thereof
The invention features methods of making devices, or “platens”, having a high-density array of through-holes, as well as methods of cleaning and refurbishing the surfaces of the platens. The invention further features methods of making high-density arrays of chemical, biochemical, and biological compounds, having many advantages over conventional, lower-density arrays. The invention includes methods by which many physical, chemical or biological transformations can be implemented in serial or in parallel within each addressable through-hole of the devices. Additionally, the invention includes methods of analyzing the contents of the array, including assaying of physical properties of the samples.
1 - 72 . (canceled)
73 . A cell chip comprising first and second platens, each having a plurality of through-holes, and a porous membrane, wherein the platens are aligned such that the through-holes of the first platen are substantially aligned with the through-holes of the second platen and the membrane is sandwiched in between the two platens.
74 . The cell chip of claim 73 , wherein the membrane comprises pores that are no more than half the through-hole diameter.
75 . The cell chip of claim 73 , wherein the membrane comprises pores of between about 0.2-250 μm.
76 - 77 . (canceled)
78 . The cell chip of claim 77 , wherein the membrane comprises aluminum oxide or polycarbonate.
79 - 80 . (canceled)
81 . The cell chip of claim 79 , wherein the polycarbonate is coated with fibronectin, laminin, collagen, or another substrate that supports cell adhesion.
82 . The cell chip of claim 73 , wherein the platens comprise polystyrene or a metal selected from the group consisting of gold, Tungsten or stainless steel.
83 - 84 . (canceled)
85 . The cell chip of claim 73 , wherein the chip further comprises a gasket that seals off individual wells.
86 . The cell chip of claim 85 , wherein the gasket is removable.
87 . The cell chip of claim 73 , wherein the chip further comprise a hydrophobic compound that prevents lateral diffusion.
88 . (canceled)
89 . The cell chip of claim 73 , wherein one platen comprises a flexible biocompatible material selected from the group consisting of silicone, polypropylene, or rubber and the other platen is a rigid platen that supports the flexible platen.
90 . (canceled)
91 . The cell chip of claim 73 , wherein the two platens are attached by raised surfaces on one platen that fit into a recessed surface on the other platen.
92 - 94 . (canceled)
95 . The cell chip of claim 73 , further comprising a solid support in contact with the first platen.
96 . The cell chip of claim 95 , wherein the solid support is a microscope slide.
97 . The cell chip of claim 96 , further comprising a coverslip in contact with the second platen.
98 . The cell chip of claim 97 , wherein the coverslip, microscope slide, and cell chip are secured together.
99 . A cell chip comprising in order from top to bottom:
(a) a coverslip in contact with a spacer;
(b) a spacer that separates the covership from a first platen;
(c) a first platen having a plurality of through-holes;
(d) a gasket comprising a plurality of through-holes that provides a seal between the first platent and the membrane;
(e) a porous membrane comprising aluminum oxide and having pores between 0.1 and 1 μm sandwiched between the gasket and the second platen;
(f) a second platent having a plurality of through holes;
(g) a solid support in contact with the second platen.
100 . The cell chip of claim 99 , wherein the chip further comprises a fastener that holds the various components together.
101 . The cell chip of claim 99 , wherein the membrane comprises a uniform structure of pores that are 0.2 μm in diameter.
102 . The cell chip of claim 99 , wherein the platens comprise tungsten, gold, or stainless steel.
103 . A method of culturing a cell on a cell chip, the method comprising:
(a) providing a cell chip of claim 73 or 99 comprising cell culture medium;
(b) contacting the porous membrane with a cell; and
(c) incubating the cell under conditions suitable for cell survival.
104 . The method of claim 103 , wherein the conditions comprise contacting the cell chip a gas permeable liquid.
105 . The method of claim 104 , wherein the gas permeable liquid is perfluorodecalin.
106 . The method of claim 104 , wherein the cell chip further comprises a hydrophobic fluid in contact with the cell culture medium, wherein the hydrophobic liquid is selected from the group consisting of perfluorodecalin, silicone oil or and mineral oil
107 . (canceled)
108 . A method of constructing a cell chip of claim 73 or 99 the method comprising:
(a) filling a first platen having a plurality of through-holes with cell culture medium;
(b) contacting the first platen with a porous membrane;
(c) contacting the membrane with a second platen having a plurality of through-holes, such that the through-holes are substantially aligned, thereby constructing a cell chip.
109 . The method of claim 108 , the cell chip further comprising a solid support in contact with the first platen.
110 . The method of claim 109 , wherein the cell chip further comprises a spacer in contact with the second platen, wherein the spacer is in contact with a cover slip.
111 . The method of claim 108 , wherein the cell chip further comprises a gasket sandwiched between the first platen.
112 . The method of claim 108 , wherein the gasket comprises a flexible material or a biocompatible elastomer selected from the group consisting of teflon, silicone, or rubber.
113 - 114 . (canceled)
115 . The method of claim 108 , wherein the filling is accomplished by placing the first platen on a solid support and centrifuging the platen and solid support.
116 . The method of claim 108 , wherein the first platen is contacted with a gasket and cell medium is then overlayed on the platen.
117 . A method for identifying an agent having a desired biological activity, the method comprising:
(a) contacting a cell chip of claim 73 or 99 comprising a cell with a platen comprising an agent;
(b) contacting the cell with the agent; and
(c) detecting an alteration in the cell, thereby identifying an agent having a desired biological activity.
118 . The method of claim 117 , wherein the agent is present in cell growth medium, or is contacted with the cell using a slotted pin or syringe, or by adding the cell to a well comprising the agent.
119 . The method of claim 117 , wherein the agent is a polypeptide, nucleic acid molecule, or small compound.
120 . The method of claim 117 , wherein the nucleic acid molecule is an siRNA, microRNA, or an aptamer.
121 . The method of claim 117 , wherein the alteration is an alteration in gene expression, polypeptide expression, cell growth, proliferation or survival, in the intracellular localization of a cellular component, morphological change, or change in motility.
122 . The method of claim 117 , wherein the alteration is detected in an immunoassay, an enzymatic assay, highthroughput gene expression profiling, reverse transcriptase polymerase chain reaction (RT-PCR), quantitative PCR, real time PCR, methylation, or high content screening (HCS) using quantitative fluorescence microscopy and automated image acquisition.
123 . The method of claim 117 , wherein the high content screening detects alterations in protein translocation.
124 . The method of claim 117 , wherein the cell is lysed and the proteins or nucleic acid molecules are bound on a binding surface.
125 . The method of claim 117 , wherein the binding surface is a weak cationic exchange medium.
126 . The method of claim 122 , wherein the bound proteins or nucleic acid molecules are analyzed for a characteristic selected from the group consisting of sequence, molecular weight, binding characteristic, and expression level.
127 . The method of claim 122 , wherein the binding characteristic is detected in an immunoassay or by polypeptide binding.