Sample: In general, the number of samples and the sample volume determine the number of wells and the volume required. In addition, certain bottom types (U-bottom, V-bottom) are advantageous if small volumes are present. Depending on the type of sample and whether it will be subjected to downstream processing following the initial analysis, the plate may need to meet specific requirements with respect to purity grade. For example, RNA analyses involve safeguarding an RNase-free environment, whereas work with cells requires sterile plates. There are certain circumstances where work with cells also needs to be free of endotoxins.
Process: Before or after the analysis, which also places specific demands on the material, additional processing steps, such as centrifugation or incubation at different temperatures, may be carried out in the plate, or specific chemicals may be added to the plate. These steps may place additional demands on the robustness of the plate. In case higher chemical resistance or centrifugation stability is required, a plate made of polypropylene (PP) will be better suited than a plate made from polystyrene (PS). The bottom shape may also have an influence on the application: A round bottom (U-bottom) is the preferred choice when mixing or washing steps are also carried out inside the plate. In contrast, the conical V-shape is beneficial if substances are precipitated or if recovery needs to be maximized when the sample is removed from the plate.
Analysis: The analysis of many assays is based on the measurement of light inside a plate reader using three basic techniques: UV/Vis spectrophotometry measures absorbance via the weakening of the intensity of the light as it passes through the sample. The fluorescent molecules employed in fluorescence assays are excited by light with a specific wavelength. The molecules then emit light, and the resulting signal is measured through a detector. Detection of luminescence is comparable. However, light is generated via a chemical or biochemical reaction.
While the microplate used must be compatible with the instrument, its optical properties must also fit the application. In the case of absorbance measurements, light traverses the sample. This demands that the bottom of the plate be transparent and flat. Plates made of polystyrene comply with these requirements. However, the material transparency is limited to wavelengths above 340 nm. Since these plates are thus mainly suitable for assays that are carried out with light in the visible range, they are also known as “Vis-plates.” For absorbance measurements in the UV range (for example, for the quantification of nucleic acids), plates need to provide a UV-transparent bottom (“UV-Vis plates”) (figure 3).