Enabling Technology for High-throughput Assays
Microplate readers are used extensively in the pharmaceutical industry for high-throughput drug screening and in biology labs for enzyme assays, immunoassays, binding assays, protein and cell assays, ELISAs, and biomolecule concentration measurements.
Flexibility (available detection modes), performance (sensitivity, throughput), and cost are prime considerations in Microplate readers selection, although the order of preference may differ for each market. Pharmaceutical screeners typically value throughput as their top criterion, says Xavier Amouretti, product manager at BioTek (Winooski, VT), while academic researchers may be more satisfied with a lower throughput but high flexibility, particularly if it means a lower overall cost.
Microplate Reader detection modes define the instruments experimental capabilities, while the optics determine spectral selectivity. Detection modes include top- and bottom-read fluorescence, fluorescence polarization, time-resolved fluorescence (TRF), time-resolved fluorescence energy transfer (TR-FRET), AlphaScreen, absorbance, and luminescence. Absorbance and fluorescence intensity are the most widely used detection techniques, constituting more than half of all applications.
According to a 2009 market study by HTStec (Cambridge, UK), about 60 percent of microplate readers employ optical filters only. Filters are selective for excitation and emission light, so two are required. Thirty percent of readers use monochromators, which tune in excitation and emission wavelengths through diffraction gratings controlled by the instrument software. Approximately 10 percent of readers are hybrid systems incorporating both monochromators and filters. Filters provide the highest sensitivity and read speed, while monochromators afford flexibility, wavelength scanning, and lower operating costs; hybrid systems constitute the best of both worlds.
In 2007 HTStec estimated market growth of Microplate Readers at about 6 percent per year, with an average highthroughput system costing between $65,000 and $110,000. (Note: simple absorbance readers for individual plates cost significantly less). Large pharmaceutical and biotech companies purchased 23 percent of systems, academic labs 25 percent, and small pharma/biotech 52 percent.
The more recent HTStec survey listed on-site demonstration as the single most important factor in Microplate reader purchase decisions. According to Amouretti, users also value flexibility, add-on functionality, and upgrade capability. Other desirable features include a full complement of detection modes, sample throughput, advanced optics, additional photomultiplier tubes for reading two wavelengths simultaneously (useful in FRET, TR-FRET, and fluorescence polarization), charge-coupled device cameras for imaging portions of plates or whole plates, and application-specific light sources such as pulsed lasers.
Many researchers are investigating multiplexed assays, where more than one assay is performed in the same well, Amouretti notes. For example, we have monitored the small molecule induction of cytochrome P450 isozymes in hepatocytes using luminescence readout while simultaneously monitoring cell viability by fluorescence. Multiplexing saves time, provides better data quality, and ensures that all assays are conducted under identical conditions. But this requires the reader to have good performance across a number of detection modes.
Absorbance: the sweetspot
Bio-Rad Laboratories (Hercules, CA), offers only absorbance-based readers to serve the large and growing market for ELISA assays. Marina Pekelis, senior product manager, describes absorbance, which is suitable for both single-point and kinetic assays, as the “workhorse” detection mode for Microplate Readers.
“Absorbance readers are popular due to their ease of use and intuitiveness,” Pekelis tells Lab Manager Magazine, “and are by far the most economical way to perform ELISA assays. Users don’t want to spend a lot of time figuring out an instrument when all they want to do is protein detection.” Price is another positive: simple desktop absorbance MPRs cost less than $10,000.
Absorbance techniques have the inherent limitation of detecting only one wavelength per well. To provide multiparameter assays, Bio-Rad licensed the Luminex xMAP bead-based assay technology and turned it into a proprietary product, Bio-Plex. Experiments are run in 96-well plates, but instead of being a result of optical reading, detection occurs through flow cytometry.
Like MPRs with multiple detection modes, absorbance readers can analyze one well at a time, which is slow but more reliable, or use a detector array that reads an entire row on the plate at once. In this mode reader, system software will automatically calculate an average well reading and generate statistics.
In addition to using system software that has limited functionality, users typically control Microplate readers and setup methods through dedicated personal computer applications. Add-on software provides advanced statistical calculations, concentrations, ratios, threshold or qualitative readings, report generation, method development and storage, export to popular spreadsheet programs, and other capabilities through a familiar Windows interface. BioRad’s Microplate Manager PC software, like packages sold by other vendors, is proprietary, but Ms. Pekelis says that workarounds exist that permit software and systems from different vendors to coexist.