Microwells

Bioluminescence in vitro chemosensitivity assays are now used to assess the sensitivity of tumor cells (obtained by surgical or needle biopsy) to different dmgs and combinations of dmgs. Cells are grown in microwell plates in the presence of the dmgs at various concentrations. If the tumor cells are sensitive to the dmg then they do not grow, hence total extracted cellular ATP, measured using the bioluminescence firefly luciferase reaction, is low. This method has been used to optimize therapy for different soHd tumors and for leukemias (306). 

J. Strahan, Development and application of an enzyme-linked immunosorbent assay method for the determination of multiple sulfonylurea herbicides on the same microwell plate, in Environmental Immunochemical Methods, ed. J.M. Van Emon, C.L. Gerlach, and J.C. Johnson, American Chemical Society, Washington, DC, pp. 65-73 (1996). 

Chiron provides a microwell plate heater, a luminometer, and data management software. The plate heater is specially designed to provide precise control of the hybridization temperature (0 0.5°C) and to distribute heat evenly throughout the microwell plate. The luminometer maintains a temperature of 37°C and accommodates the 96-well plates. The data management software runs on an IBM PC or compatible computer with a minimum of 80386,16-Mhz microprocessor, 2 Mb of RAM, monitor, mouse, compatible printer, MS DOS (version 5.0 or greater), and Windows (version 3.1 or greater). 

To a set of labeled tubes, add 2 ml of OPA reagent (Thermo Fisher) and 200 pi of the appropriate standard or sample. Mix well. If using a microplate format, scale back these quantities 10-fold to fit in the microwells. 

Brillhart, K.L., and Ngo, T.T. (1991) Use of microwell plates carrying hydrazide groups to enhance antibody immobilization in enzyme immunoassays./. Immunol. Meth. 144, 19-25. 

CombiCHEM System (Fig. 3.9) For small-scale combinatorial chemistry applications, this barrel-type rotor is available. It can hold two 24- to 96-well microtiter plates utilizing glass vials (0.5-4 mL) at up to 4 bar at 150 °C. The plates are made of Weflon (graphite-doped Teflon) to ensure uniform heating and are sealed by an inert membrane sheet. Axial rotation of the rotor tumbles the microwell plates to admix the individual samples. Temperature measurement is achieved by means of a fiber-optic probe immersed in the center of the rotor. 

ELISA (TC Microwell test kit) lOOligkg-1 Muscle tissue [95]... 

An elegant approach is to capture the target DNA or RNA with specific oligonucleotides on to a microwell plate. Synthetic branched DNA bearing multiple alkaline phosphatase-labeled probes hybridizes to the target. A chemiluminescent substrate is added to produce signal. This branched DNA assay has been used in infectious disease detection (W3). 

Karp JM, Yeh J, Eng G, Fukuda J, Blumling J, Suit KY, Cheng J, Mahdavi A, Borenstein J, Langer R, Khademhosseini A (2007) Controlling size, shape and homogeneity of embryoid bodies using poly(ethylene glycol) microwells. Lab Chip 7 786-794. 

Mix and transfer the contents of each microwell to a 1.5-ml microcentrifuge tube. 

Zhang, B., Foret, F, and Karger, B. L. (2001). High-throughput microfabricated CE/ESTMS automated sampling from a microwell plate. Anal. Chem. 73, 2675—2681. 

ELISAs exist in many different formats, such as microwell assays (microtiter plates, strips), dipstick assays, and immunofiltration assays. 

Such thermoplastics have also been used as DNA microarray substrates (Matson et al., 1995 Shchepinov, 1997 Beier and Hoheisel, 1999) and in the construction of protein microarrays in microwells (Matson et al., 2001 Moody, 2001). Pierce (see searchlight perbio.com) introduced the Search-Light series of microarray-based ELISA assays immobilizing capture antibodies in a low density array format into polystyrene microwells. 

Other early work includes that of Moody et al. (2001) who spotted anticytokine monoclonals onto the bottom of polystyrene microtiter plates (Max-isorp, Nalge Nunc) and measured cytokine levels in stimulated peripheral blood mononuclear cells. Finally, although not strictly a microarray, the microwell array system developed by Michael Snyder s group at Yale University to measure kinase activity is a simple and elegant approach (Zhu et al., 2000). The "protein chip" is comprised of microwells fabricated in a flexible elastomer of PDMS [poly(dimethylsiloxane)] substrate by a molding process. 

The PDMS microwell array is mounted onto a glass slide and activated using an epoxysilane to which the protein may be attached. The wells were about 1.4 mm in diameter and 300 pm deep allowing a volume of approximately 300 nL. The microwell protein chip should be widely applicable. 

This author and coworkers at Beckman Coulter first described the use of a low form 96-well plastic microplate for automated micro-ELISA immunoassays (Matson et al., 2001). The polypropylene plate was first modified by a radiofrequency plasma amination process (Matson et al., 1995) followed by conversion to an acyl fluoride surface chemistry for rapid covalent attachment of biomolecules. Proteins (1 to 2 mg/mL) were prepared in 50 mM carbonate buffer, pH 9, containing 4% sodium sulfate (to improve spot uniformity) and printed using a conventional arrayer system. Approximately 200-pL droplets of monoclonal antibodies (anti-cytokine) were deposited into the bottom of the microwells using a Cartesian PS7200 system equipped... 

Figure 6.34 Elastomer-based microwell protein kinase assay array. (From Zhu, H. et al.. Nature Genetics, 26, 283-289, 2000. With permission.)...

The use of a battery of environmental bioassays for the management of hazardous wastes is applied in the Czech Republic [176]. This battery of environmental bioassays has included representatives of producers, consumers, and destructors D. magna (possible substitution by D. pulex), acute, reproduction, chronic test Scenedesmus quadricauda S. capricomutum), as bottle test or in microwell plates Poecillia reticulate (Danio rerio), acute, chronic, embryolarval tests 5. alba (Lactuca sativa), germination test, 72 hours. 

Scenedesmus quadricauda (Selenastrum capricornutum) as bottle test or in microwell plates ... 

Chevrier, D., Rasmussen, S. R., and Gues-Jon, J. L. (1993). PCR product quantification by non radioactive hybridization procedures using an oligonucleotide covalently bount to microwells. Mol. Cell. Probes 7,187-197. 

K6. Kowalski, A., Radu, O., and Gold, B., Colorimetric microwell plate detection of the factor V Leiden mutation, Clinical Chemistry 8, 1195—1198 (2000). 

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