Use of a certified test plate can help to increase efficiency when calibrating microplate readers.
A microplate reader’s moving parts are subject to wear. In addition to these parts, optical components may be exposed to a variety of chemical spills and vapors. Assay controls and software correction factors normalize and compensate for day-to-day and assay-to-assay variability from environmental conditions, operator technique, and reagent variations. Equally important, instrument testing serves as a quality control check to ensure that the instrument hardware and applicable software are working properly. Indeed, GLP and other controlled research and clinical laboratories require routine, documented testing confirming the calibration of the microplate reader.
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Figure 1a. (top) Data generated using a Universal Test Plate on the Synergy H4 Hybrid Microplate Reader (BioTek Instruments) correlates with expected values of the NIST traceable neutral density filters with an R2 of 1.0.
Figure 1b. (middle) Fluorescence Test Plate data generated on the Synergy H4 correlates with expected values of the solid-state fluorescence wells with an R2 of 0.999.
Figure 1c. (bottom) Luminescence Test Plate data generated on the Synergy H4 illustrates the dynamic range of the reader and its linearity with an R2 of 0.998.
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Instrument testing can be carried out with manual tests or by using a certified test plate. Manual methods involve pipetting prepared solutions into microplate wells, reading, and comparing the results to the manufacturer’s specified test result criteria. Although the per-test cost may be inexpensive, it takes time to prepare fresh solutions, pipette, and interpret the results. Fluctuations due to pipetting technique, pipette calibration, and reagent integrity may significantly influence results.
A more efficient and cost effective alternative over the microplate reader’s lifetime is use of a certified test plate. These plates are filled with glass, crystals, solid-state materials, gels, or sealed liquids with fixed optical properties, and are manufactured and certified to NIST (National Institute of Standards and Technology) standards. The plate is read and results compared to values within the manufacturer’s specified test result criteria (Figures 1a-c). Since operator technique, pipette calibration, and reagent variability are removed from the process, results are more representative of the reader’s performance. Test plates can also detect any mechanical variations in the reader.
It should be noted that in a microplate reader’s vertical optics path configuration, the light beam interacts differently on a flat, solid surface (i.e. test plate) compared to a curved liquid surface (i.e. manual test). This difference is negligible for most applications, but may become more pronounced at extreme ends of the absorbance spectrum. Each microplate reader has slightly different optics, so manufacturer specifications should not be broadly applied to multiple readers.
Test Plate Types
Test plates are available for absorbance, luminescence, and fluorescence detection modes. Test results are compared to the manufacturer’s performance control limits, and each test plate should be recertified to NIST or other strict standards every year.
Absorbance test plates often use Neutral Density (ND) filters to confirm performance accuracy over a range of wavelengths as well as a holmium or didymium glass filter to test wavelength accuracy for the microplate spectrophotometer’s monochromators. These filters are set into a machined aluminum block for stability. Precision-machined holes in the corners of the aluminum block test the reader’s physical alignment. Absorbance test plates may also be used to measure any drift over time.
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Example of an absorbance test plate. |
Luminescence test plates use stable luminescent light sources instead of glass filters to test the sensitivity of the reader’s luminescence optics. When the luminescence plate is powered on, a range of luminescent light sources glow with fixed and known intensity. The amount of light emitted by these light sources has been correlated with the light emitted by ATP in a luminescence assay. Detection of these light sources compared with a background reading can provide a limit of detection in terms of the number of attomoles of ATP that could be detected in a luminescence assay. A secondary light source on the plate verifies the integrity of the internal chargeable battery.
Finally, fluorescence test plates contain up to three inert and stable fluorescent compounds (green, red, blue) embedded within a polymerized matrix or gel, or added to a solid quartz or glass surface. Fluorescent standards may also be available in a sealed liquid format, although if any evaporation occurs over time, readings may be significantly skewed; therefore, application of liquid standards is limited. As with any fluorescent application, photobleaching is a concern, so it is critical that the test plate be recertified every year. The fluorescence test plate can measure crosstalk, physical alignment, signal to noise, repeatability and linearity.
Summary
Regular microplate reader testing is critical to verify proper performance and functionality over the instrument’s life. Using a test plate is a fast and cost-effective method to measure stability and additional test parameters compared to liquid test methods.