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Substrates, devices, and methods for cellular assaysRelated Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test StripSubstrates, devices, and methods for cellular assays description/claimsThe Patent Description & Claims data below is from USPTO Patent Application 20060141446, Substrates, devices, and methods for cellular assays. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] This application claims the benefit of provisional application 60/382,446, filed May 22, 2002. FIELD OF THE INVENTION [0002] The present invention relates to the fields of molecular biology, cellular biology, developmental biology, stem cell differentiation, immunology, oncology, general laboratory sciences and microbiology, and in particular to methods and compositions based on liquid crystal assays and other biophotonically based assays for detecting and quantifying the number of cells present on a test surface or within a test substrate and the proliferation, death or movement of cells under control conditions and in response to chemotactic and other cytoactive (including compounds that are chemokinetic but not chemotactic and agents that inhibit cell migration) agents. Additionally, the present invention describes a novel biophotonic approach for the detection and quantification of enzymatic activity. BACKGROUND OF THE INVENTION [0003] Every year cancer claims the lives of hundreds of thousands of people worldwide. The populations of many of the heavily industrialized countries are particularly susceptible to cancer induced morbidity and mortality. In fact, cancer is the second leading cause of death in industrialized nations. For example, prostate cancer is the second most common malignancy in men. It is estimated that in 2002 in the United States nearly 180,000 men will be diagnosed with prostate cancer. Breast cancer is the most common female malignancy in most industrialized countries, and in the United States it is estimated that breast cancer will affect about 10% of women during their lives. Approximately 30 to 40% of women with operable breast cancer eventually develop metastases distant from the primary tumor. [0004] Metastasis, the formation of secondary tumors in organs and tissues remote from the site of the primary tumor, is the main cause of treatment failure and death for cancer patients. Indeed, the distinguishing feature of malignant cells is their capacity to invade surrounding normal tissues and metastasize through the blood and lymphatic systems to distant organs. Cancer metastasis is a complex process by which certain cancer cells acquire substantial genetic mutations and perturbed signal cascades that allow them to leave the primary tumor mass and establish secondary tumors at distant sites. Metastatic cancer cells break adhesions with neighboring cells, dissolve the extracellular matrix, migrate and invade surrounding tissue, travel via the circulatory system, invade, survive and proliferate in new sites. Unfortunately, the molecular mechanisms that promote and restrain the metastatic spread of cancer cells have yet to be clearly identified. [0005] Medical researchers have made considerable efforts to understand whether chemotactic agents are involved in metastasis and why particular cancers preferentially metastasize to certain sites. Breast cancer, for example, favors metastasizing to regional lymph nodes, bone marrow, and lung and liver tissues. Prostate cancer favors metastasizing to bone marrow. Several theories have been advanced to explain the preferential metastasis of certain cancers. [0006] It has recently been shown that one important property of highly metastatic cells is their ability to respond to chemotactic agents such as paracrine and autocrine motility factors. For example, recent work done by Muller et al. provides evidence for chemotactic homing of breast cancer to metastatic sites. (Muller et al. "Involvement of chemokine receptors in breast cancer metastasis," Nature, 410:50-56 [2001]); See also, M. More, "The role of chemoattraction in cancer metastases," Bioessays, 23:674-676 [2001]). Muller et al. findings indicate that CXCR4 and CCR7 chemokine receptors are found on breast cancer cells and that ligands for these receptors are highly expressed at sites associated with preferential breast cancer metastases. [0007] Many conventional assay methods have been adapted for studying the effects of chemotactic agents on cancer and other cells of interest (e.g., densitometric, analyses of membrane filters, visible spectrum or spectrophotometric ELISA microplate readers, fluorescence microplate readers, scintillation counters, and photoluminescence readers). Each of these methods has particular advantages and disadvantages. One disadvantage found in each of these methods is the requirement that the cells of interest be "tagged" with dyes, fluorescing agents, or radioisotopes, in order to observe the cellular responses to chemical agents. Extrinsic cell labeling techniques add to the expense and complexity of the existing assay methods and often requires the expertise of highly skilled technicians. [0008] An important property of metastatic cells is their ability to produce proteases, such as Matrix Metalloproteinases (MMPs) that are capable of digesting constituents of the extracellular matrix. The elaboration of these proteases facilitates their invasion of tissues. The role of proteases in the metastatic process using in vitro and in vivo systems as well as their quantification for use as a prognostic indicator for metastatic potential has been widely reported. The amount of a given protease present can be measured using ELISA but this requires a specific antibody capable of reacting with the protease from a given species. Another drawback of ELISA is that it measures the total amount of a given protease and does not discriminate between proenzyme, activated enzyme or inhibitor complexed enzyme. For example, the activation state of MMP's in the cellular environment is tightly regulated by Tissue Inhibitors of Metalloproteinases (TIMPs). Zymography (to measure proteases) and reverse zymography (to measure TIMPs), are widely used methods that involve gel electrophoresis combined with enzymatic digestion of an appropriate substrate. Both the proenzyme and active forms of proteases can be distinguished on the basis of molecular weight. Unfortunately, standard zymographic methods are laborious requiring many preparative steps (Hawkes S P, Li H, Taniguchi T. Zymography and reverse zymography for detecting MMPs and TIMPs. In Matrix Metalloproteinase Protocols. Volume 151 of Methods in Molecular Biology. Ian Clark ed. Humana Press. Totowa N.J. 2001. pp 399-410). [0009] Other assays used include a variety of protease assays including quantifying radiolabelled collagen fragments released by enzymatic cleavage of a radiolabelled substrate, and the measurement of fluorescence produced when an fluorescently autoquenched fluorescent substrate undergoes digestion and creating an increase in quantifiable fluorescvent signal. These methods do not allow discrimination between proteases however (Cawston T E, Koshy P, Rowan A D. Assay of matrix metalloproteinases against matrix substrates. In Matrix Metalloproteinase Protocols. Volume 151 of Methods in Molecular Biology. Ian Clark ed. Humana Press. Totowa N.J. 2001. pp 389-397). [0010] What are needed are assay devices and systems for detecting quantifying cell number and identifying their spatial location as well as identifying and quantifying proteases and protease inhibitors that do not require extrinsic cell labeling techniques that are robust and easier to use which allows for enhanced evaluation of samples. SUMMARY OF THE INVENTION [0011] The present invention relates to the fields of molecular biology, cellular biology, immunology, oncology, developmental biology, stem cell differentiation, general laboratory sciences and microbiology, and in particular to methods and compositions based on liquid crystal assays and other biophotonically based assays for detecting and quantifying the number of cells present on a substrate (allows for the quantitation of cell adhesion and cell proliferation) as well as direct quantification of proliferation, cell death, differentiation, or cell migration on a surface or through an extracellular matrix (cell invasion) under control conditions and in response to the presence of chemotactic, growth, differentiation enhancing and other cytoactive (accounts for chemokinetic agents and agents that inhibit cell migration) agents. [0012] In some embodiments, the present invention provides an analyte assay apparatus comprising a substrate having a surface, the surface having thereon at least one non-specific binding assay region configured to orient mesogen. In some embodiments, the mesogens are oriented by anisotropic features on the surface. In other embodiments, the assay region promotes homeotropic orientation that can be disrupted by presence of an analyte. In some preferred embodiments, the non-specific binding assay region is substantially free of recognition moieties specific for the analyte. The assays are useful for detecting a variety of analytes, including, but not limited to, cells, particulate matter and microorganisms. The present invention is not limited to the detection of any particular microorganism. Indeed, the detection of a variety of microorganisms is contemplated, including, but not limited to, bacteria, viruses, and fungi. The present invention is not limited to any particular non-specific assay region configured to orient mesogens. Indeed, a variety of such assay regions are contemplated, including but not limited to regions comprising rubbed proteins, rubbed polymeric surfaces, and ordered polymeric surfaces. The present invention is not limited to any particular rubbed polymeric surface. Indeed, a variety of rubbed polymeric surfaces are contemplated, including, but not limited to, polyurethane, polyimide, tissue culture polystyrene and polystyrene. The present invention is not limited to the use of any particular ordered surface. Indeed, a variety of ordered surfaces are contemplated, including, but not limited to, micromolded polymeric surfaces, micro/nanoabraded polymeric surfaces, photolithographically created polymeric surfaces, and obliquely deposited metallic films. In still other embodiments, the at least one non-specific assay region configured to orient mesogens comprises particles arranged on the surface. The present invention is not limited to any particular method of orienting the particles. Indeed, a variety of orienting methods are contemplated, including, but not limited to, electric fields, a magnetic fields, shear fields, fluid flow, and mechanical transfer. The present invention is not limited to any particular number of assay regions. Indeed, a variety of assay region configurations are contemplated, including assays comprising 6, 12, 24, 36, 96, 384, or 1536 assay regions, wherein the assay regions are readable by a plate reader. In some particularly preferred embodiments, the 6, 12, 24, 36, 96, 384, or 1536 assay regions are arranged in an array. [0013] In other embodiments, the present invention provides an assay apparatus comprising a surface, the surface having thereon particles, wherein the particles are ordered to orient mesogens. The present invention is not limited to any particular method of orienting the particles. Indeed, a variety of orienting methods are contemplated, including, but not limited to, electric fields, a magnetic fields, a shear fields, fluid flow, and mechanical transfer. The present invention is not limited to the use of any particular particles. Indeed, the use of a variety of particles is contemplated, including, but not limited to metallic, charcoal, chalk, soapstone, graphite, and pumice particles. [0014] In still further embodiments, the present invention provides an assay apparatus comprising an assay region, the assay region comprising an extracellular matrix configured to orient mesogens. In some preferred embodiments, the assay apparatus further comprises mesogens. The present invention is not limited to the use of any particular mesogens. Indeed, the use of a variety of mesogens is contemplated, including, but not limited to thermotropic and lyotropic liquid crystals. [0015] In other embodiments, the present invention provides an assay apparatus comprising an assay region, the assay region comprising a matrix having mesogens embedded therein. In some preferred embodiments, the assay apparatus further comprises mesogens. The present invention is not limited to the use of any particular mesogens. Indeed, the use of a variety of mesogens is contemplated, including, but not limited to thermotropic and lyotropic liquid crystals. [0016] In some embodiments, the present invention provides an assay apparatus comprising a substrate having a surface, the surface having thereon at least one assay region that is contacted by mesogens, the surface configured to orient the mesogens, and at least one cell seeding region associated with the at least one assay region. In some preferred embodiments, the substrate has therein at least one reservoir for containing a test compound and the at least one cell seeding region comprises a well. In some other preferred embodiments, one or more of the at least one cell seeding regions contains at least one cell. In further embodiments, the reservoir contains a test compound. The present invention is not limited to any particular type of cell. Indeed, the use of a variety of cells is contemplated, including, but not limited to, prokaryotic cells and eukaryotic cells. The present invention is not limited to any particular non-assay region configured to orient mesogens. Indeed, a variety of such assay regions are contemplated, including but not limited to regions comprising rubbed proteins, rubbed polymeric surfaces, and ordered polymeric surfaces. The present invention is not limited to any particular rubbed polymeric surface. Indeed, a variety of rubbed polymeric surfaces are contemplated, including, but not limited to, polyimide, polyurethane, tissue culture polystyrene and polystyrene. The present invention is not limited to the use of any particular ordered surface. Indeed, a variety of ordered surfaces are contemplated, including, but not limited to, micromolded polymeric surfaces, micro/nanoabraded polymeric surfaces, photolithographically created polymeric surfaces, and obliquely deposited metallic films. In still other embodiments, the at least one assay region configured to orient mesogens comprises a surface having particles thereon, the particles configured to orient liquid crystals. In other preferred embodiments, the surface is decorated with at least one molecular recognition moiety. In some preferred embodiments, the recognition moieties are attached to the surface by covalent bonds. The present invention is not limited to the use of any particular recognition moiety. Indeed, the use of a variety of recognition moieties is contemplated, including, but not limited to, metals, amino acids, peptides, polypeptides, antibodies, nucleic acids, lipids, phospholipids, fatty acid derivatives, steroids, transcription factors, saccharides, cellular receptors, and receptor recognition sequences. The present invention is not limited to the use of any particular polypeptides. Indeed, the use of a variety of polypeptides is contemplated, including, but not limited to collagens, fibronectin, laminins, osteopontin, thrombospondin, vascular cell adhesion molecule-1, intracellular adhesion molecule-1, intracellular adhesion molecule-2, chondroitin sulfate, von Willebrand factor, entactin, fibrinogen, tenascin, mucosal addressin cell adhesion molecule, C3b, MDC proteins and vitronectin. The present invention is not limited to the use of any particular receptor recognition sequences. Indeed, the use of a variety of receptor recognition sequences is contemplated, including, but not limited to, cadherin, the immunoglobulin superfamily, selectins, mucins and integrin binding sequences. The present invention is not limited to the use of any particular integrin binding sequences. Indeed, the use of a variety of integrin binding sequences is contemplated, including, but not limited to, RGD, EILDV, LDV, LDVP, IDAP, PHSRN, SLDVP, GRGDAC and IDSP. The present invention is not limited to any particular configuration of assay regions. In some embodiments, an assay apparatus of the present invention comprises 6, 12, 24, 36, 96, 384, or 1536 assay regions. In some preferred embodiments, the plurality of assay regions are arranged in an array. In other embodiments, the array of assay regions is configured to correspond to the reading positions of commercial plate reading devices. In some embodiments, an assay apparatus of the present invention comprises at least one microfluidic channel. In other embodiments, the at least one microfluidic channel is configured to be fluidically connected to the reservoirs. [0017] In some embodiments of the present invention, methods are provided for analyzing an analyte comprising: a substrate having a surface, an analyte, and mesogens, wherein the analyte is not specifically bound to the surface; and applying the mesogens to the surface on the substrate so that the analyte is detected. In some embodiments, the mesogens are applied under conditions such that regions of order and disorder on the surface are identified. In some embodiments, the surface is configured to orient the mesogens. In other embodiments, the surface does not orient the mesogens. In some embodiments, the presence of the analyte disrupts ordering of the mesogens. The methods of the present invention are not limited to the analysis of any particular analyte. Indeed, the analysis of a variety of analytes is contemplated, including, but not limited to, prokaryotic cells, eukaryotic cells, viruses, and particulate matter. In some embodiments, migration of the cells across the surface creates regions of disorder. In other embodiments, migration of the cells across the surface creates regions of order. In still further embodiments, the presence of cells on the surface causes ordering or disordering of mesogens, thereby allowing detection of the presence of the cells. In further embodiments, the methods of the present invention comprise the step of quantifying the number of the cells on the surface by comparing areas of disorder to areas of order. [0018] In still other embodiments of the present invention, methods are provided for analyzing cells comprising: providing mesogens and apparatus comprising a substrate having a surface, the surface having thereon at least one assay region configured to orient mesogens and at least one cell seeding region associated with the at least one assay region; applying cells to the at least one cell seeding region; and assaying the movement of the cells into the at least one assay region by applying the mesogens to the surface. In some embodiments, the apparatus further comprises a reservoir for containing a test compound. In further embodiments, the effect of the test compound on the cells is analyzed by movement of the cells into the at least one assay region. In some embodiments, the test compound is suspected of affecting cell motility. In other embodiments, the test compound is suspected of effecting cell proliferation or cell adhesion. [0019] The present invention also provides methods for detecting the effects of test compounds on cell motility, comprising, providing: an assay apparatus comprising a substrate having a surface, the surface having thereon at least one assay region configured to orient mesogens and at least one cell seeding region associated with the at least one assay region, wherein the at least one cell seeding region contains at least one cell; a test compound suspected of inhibiting or promoting cell motility; and contacting the at least one cell, with the test compound; and analyzing the effect of the test compound on the at least one cell. The methods of the present invention are not limited to the analysis of any particular cell. Indeed, a variety of cells can be analyzed, including eukaryotic (e.g., mammalian) and prokaryotic cells. In some embodiments, the mammalian cell is suspected of being cancerous. In other embodiments, the mammalian cell is a human cell. In some embodiments, the analysis is performed with a liquid crystal. In still other embodiments, the orientation of the mesogens within a liquid crystal film is analyzed on a plate reader. [0020] The present invention further provides methods for detecting the effects of test compounds on the production of cellular secretory products, comprising: providing mesogens and an assay apparatus comprising a substrate having a surface, the surface having thereon at least one assay region comprising recognitions moieties that recognize the cellular secretory product and at least one cell; contacting the at least one cell with the at least a first test compound; and applying the mesogens to the surface to analyze the production of the at least one cellular secretory product. The present invention is not limited to the analysis of any particular cell secretory product. Indeed, the analysis of a variety of cell secretory products is contemplated, including, but not limited to, trophic factors, cytokines, chemokines, and electrolytes. The present invention is not limited to the analysis of any particular trophic factor. Indeed, the analysis of a variety of trophic factors is contemplated, including, but not limited to, epidermal growth factors, fibroblast growth factors, neurotrophins, platelet derived growth factors, hepatocyte growth factors, growth hormone, prolactin, angiotensin, kinins, neurodifferentiation factor, insulin, insulin like growth factors (IGF) I and II, keratinocyte growth factor, amphiregulin, heregulin, TGF-alpha, TGF-beta, neuropeptides, and neurotransmitters. The present invention is not limited to the analysis of any particular cytokine or chemokine. Indeed, the analysis of a variety of cytokines and chemokines is contemplated, including, but not limited to interleukins, erythropoietin, tumor necrosis factor, colony stimulating factors, interferons and cell migration factors. The present invention is not limited to the analysis of any particular electrolytes. Indeed, the analysis of a variety of electrolytes is contemplated, including, but not limited to, calcium, magnesium, sodium and potassium. The present invention is not limited to the analysis of any particular type of cell. Indeed, the methods of the present invention are useful for analysis of a variety of cell types, including, but not limited to, eukaryotic cells (e.g., mammalian cells including human cells, and cancerous cells as well as cells derived from other vertebrate and invertebrate species). In some embodiments, the analyzing step is performed with mesogens. In further embodiments, the mesogens are analyzed on a plate reader. [0021] The present invention also provides methods for detecting production of cellular secretory products, comprising: providing mesogens, at least one cell, and an assay apparatus comprising a substrate having a surface, the surface having thereon at least one assay region comprising recognitions moieties that recognize the cellular secretory product; and applying the mesogens to the surface to analyze the production of the at least one cellular secretory product. In some embodiments, the cellular secretory product is a natural secretory product, while in other embodiments, the cellular secretory product is a recombinant secretory product. Continue reading about Substrates, devices, and methods for cellular assays... 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