Choosing the correct assay plate
Choosing the correct microplate for your application can mean the difference between indifferent and great results. There are three basic methods of obtaining useful optical data from microplate-based samples. The simplest method is absorbance measurement. Where greater sensitivity is required, fluorescence measurements are preferred. Sensitivity can be ten times greater than simple absorbance measurements. The third method involves luminescence, a naturally occurring phenomenon exhibited by certain animal and plant species which can emit light. This is a biological adaptation of a process which can also be seen as purely chemically-driven reactions, in which case it is referred to as
chemiluminescence. Microplate readers are designed to read from either the top or the bottom of a microplate. Top reading instruments rely on measuring reflected light above the wells.
A good solid bright white plate is best for these absorbance measurements and a solid black plate for fluorescence readings. Bottom reading units illuminate the sample from above and then use detectors placed below the plate to measure the absorption or fluorescence/luminescence emission. This necessitates the use of clear-bottomed plates. The requirement is to transmit the light wavelengths of interest. Visible wavelength range (900-350nm) measurements require only clear plastic bases, whilst readings between 220nm and 350nm will require a UV-transparent material. This can be Quartz sheet or a modern polymer such as Cyclo-Olefin Co-Polymer (COP/COC). Optical glass sheet is used where visible range detection is combined with confocal optics or whole plate imaging which requires a very clear uniformly-flat base.
Simple 96-well microplates for ELISA type assays are made from solid clear polystyrene with no additives. These are adequate for clinical and diagnostic tests, ELISA assays and any colour endpoint determination with relatively high absorbance.Typically they are available with flat well bottoms, giving high surface area, round well bottoms for good mixing or V-wells for high liquid recovery. Crosstalk can also be an issue in bottom-reading absorbance and
fluorescence measurements. To address this applications challenge, Porvair Sciences has developed the Krystal 2000 zero-crosstalk plates in which individual clear wells are moulded into either a white or black matrix. The black or white base material also projects down below the clear well bottom to further reduce the possibility of crosstalk. By carefully selecting the correct plate type for the assay, it is possible to significantly improve results. By following the simple guidelines set out here, those tasked with assay development can ensure that their final assay has the best possible chance of success. The table summarizes the choices available for Assay Plate selection and indicates the plate type most likely to give the best results.
Porvair Sciences will be happy to supply samples of their plates for evaluation free of charge, as different detection systems and differing assay development may lead to variances in results between ostensibly similar plate types.