Chemistry: molecular biology and microbiology – Apparatus – Including condition or time responsive control means
Reexamination Certificate
2001-05-15
2004-11-16
Redding, David A. (Department: 1744)
Chemistry: molecular biology and microbiology
Apparatus
Including condition or time responsive control means
C435S286700, C435S287200, C435S287300, C435S288700
Reexamination Certificate
active
06818435
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
This invention relates to methods and apparatus for performing microanalytic analyses and procedures. In particular, the present invention provides devices and methods for the performance of miniaturized cell based assays. These assays may be performed for a variety of purposes, including but not limited to screening of drug candidate compounds, life sciences research, and clinical and molecular diagnostics.
2. Background of the Related Art
Recent developments in a variety of investigational and research fields have created a need for improved methods and apparatus for performing analytical, particularly bioanalytical assays at microscale (i.e., in volumes of less than 100 &mgr;L). In the field of pharmaceuticals, an increasing number of potential drug candidates require assessment of their biological function. As an example, the field of combinatorial chemistry combines various structural sub-units with differing chemical affinities or configurations into molecules; in theory, a new molecule having potentially unique biochemical properties can be created for each permutation of the sub-units. In this way, large libraries of compounds may be synthesized from relatively small numbers of constituents, each such compound being a potential drug lead compound of usually unknown biological activity and potency. Similarly, increasingly large numbers of targets for these putative therapeutic compounds are being discovered, many as a result of the growing information derived from such large-scale biological research as the sequencing of the human genome.
As the first phase of drug discovery, compounds that represent potential drugs are screened against targets in a process known as High Throughput Screening (HTS) or ultra-High Throughput Screening (uHTS). An advantage of these screening methods is that they usually consist of simple solution phase biochemical assays that can be performed quickly and with small amounts of expensive compounds and reagents. However, a significant drawback to HTS is that the targets do not provide a functional assessment of compounds' effects on the complex biochemical pathways inherent in the normal and abnormal (mutant or disease-state) functioning of cells, tissues, organs, and organisms. As a result, compounds that have shown biochemical activity of interest in initial screens are usually put through cell-based screens, in which the affect of the compounds on cellular function is independently assayed.
There are a wide range of assays that may be performed using living cells. Assays that involve the use of living cells include gene expression, in which levels of transcription in response to a drug candidate are monitored; cell permeability assays, in which the ability of drugs to traverse membranes of cells is monitored; and functional assays designed to investigate both macroscopic effects, such as cell viability, as well as biochemical effects and products produced in and by the cells as a result of treatment with the drug lead compound.
These assays include cytotoxicity and cell proliferation to measure the viability of a population of cells, often in the presence of a putative therapeutic compound (drug candidate). A variety of methods have been developed for this purpose. These include the use of tetrazolium salts, in which mitochondria in living cells use dehydrogenases to reduce tetrazolium salts to colored formazan salts. Soluble or insoluble precipitates may be formed, depending on the nature of the tetrazolium salt used. A typical assay procedure is to culture the cells, add a solution of tetrazolium salt, phenazine methosulfate and DPBS, incubate, and determine absorbance at 490 nm. The absorbance measured is larger for viable cell populations that have metabolized the salt. Another such assay uses alamarBlue, which uses a fluorometric/colorimetric growth indicator that is reduced to a membrane-soluble, red, fluorescent form by the products of metabolic activity. A variety of other indicators are either taken up by living cells, dead cells, or both; for example, neutral red is taken up only by live cells, while trypan blue is excluded by live cells. Dyes that bind to or intercalate with DNA can be used to visualize or quantitate the number of live or dead cells, since DNA synthesis only occurs in living cells.
Another important class of cell based assays in reporter gene assays. These assays are used to study the control of gene transcription. They can also be used as a secondary detection method for a number of other molecules present in or acting on a cell. Pharmaceutical companies and others involved in drug development commonly utilize reporter gene assays to determine the effects of their compounds on transcription of specific genes whose promoter sequences are known. For example, the production of proteins associated with a condition of interest can be quantified by using a reporter gene operatively linked to the promoter of the gene encoding the protein. The method employed in reporter gene assays varies with the type of reporter gene used and the application. Initially, the promoter from the gene of interest in operable combination with the reporter gene is inserted into a commercially available plasmid comprising an antibiotic resistance gene, which is then transfected into the cells. Cells that have been successfully transfected can be selected by addition of the antibiotic, thereby eliminating the cells that have not been successfully transduced with the plasmid. When studying gene transcription, the cells are subsequently plated, compound(s) to be tested are introduced, and the assay for the reporter protein is conducted. These assays range from extremely simple to complex, with reporter proteins ranging from enzymes to hormones and photoproteins. Typically, enzymes are assayed using rate assays, hormones are detected using immunoassays, and photoproteins (e.g., green fluorescent protein, aequorin) are imaged optically.
Cell permeability assays measure the transport of compounds across cells. The commonly-used example is the CaCo-2 cell line derived from human intestinal endothelial cells. When grown to confluency over a porous membrane, these cells form a “biologically active” filter: Transport of compound through the cell layer is accepted in the art to be correlated with absorbsion by the digestive system.
The compounds available for such cell-based testing have increased dramatically in recent years. In the decade from 1985 to 1995, drug library development through methods such as combinatorial chemistry and the discovery of new targets have created an explosive growth in both the number of compounds with promising biochemical properties. In order to effectively assay these “hits” using cell-based assays, an equivalent system of high throughput screening for such cell-based assays is needed.
To achieve the primary need of high throughput for cell based assays, a number of secondary features are desirable. First, it is advantageous to have a high degree of process automation, such as fluid transfer, cell plating and washing, and detection. It is also advantageous for the processes to be integrated so as to require a minimum of human intervention. Compound consumption (non-specific adsorption onto the materials comprising the assay apparatus) must be minimized, in order to prevent depletion of rare and/or expensive components of the compound libraries. This is most readily addressed through miniaturization of assays from their current scale of hundreds of microliters to ten microliters or less. A goal in the art is to provide automated, integrated and miniaturized apparatus for performing assays that are reliable and produce results consistent with the results produced by current, more laborious, expensive and time-consuming assays.
In addition to these advantages, miniaturization itself can confer performance advantages. At short length scales, diffusionally-limited mixing is rapid and can be exploited to create sensitive assays (Brody et al., 1996
, Biophysical J
. 7
Carvalho Bruce L.
Feakes Christina
Kellogg Gregory J.
Sheppard, Jr. Norman F.
McDonnell & Boehnen Hulbert & Berghoff
Redding David A.
Tecan Trading AG
LandOfFree
Microfluidics devices and methods for performing cell based... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Microfluidics devices and methods for performing cell based..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Microfluidics devices and methods for performing cell based... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-3300929