Enabling Technology for High-Throughput Assays
Flexibility (available detection modes), performance (sensitivity, throughput), and cost are prime considerations in microplate reader selection, although the order of preference may differ for each market. Detection modes define the instrument&rsquo
Flexibility (available detection modes), performance (sensitivity, throughput), and cost are prime considerations in microplate reader selection, although the order of preference may differ for each market. Detection modes define the instrument’s 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 (TRFRET), AlphaScreen, absorbance, and luminescence. Absorbance and fluorescence intensity are the most widely used detection techniques, constituting more than half of all applications.
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.
According to Xavier Amouretti, product manager at BioTek (Winooski, VT), users 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), chargecoupled 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. 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.
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 singlepoint and kinetic assays, as the “workhorse” detection mode for Microplate Readers.
“Absorbance readers are popular due to their ease of use and intuitiveness, and are by far the most economical way to perform ELISA assays,” says Pekelis. “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, however, they can detect only one wavelength per well.
To provide multi-parameter 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, and 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.
• Easily upgrade instrument functionality in < 2 minutes
• Excellent sensitivity and performance
• Up to 1,536- and 3,456-well plate format
• High-speed detection
• Includes four filters that cover wavelengths for most common absorbance assays
•A choice of software packages enables the user to connect to a PC and run both endpoint and kinetic assays with ease
•Single or dual wavelength measurements plus kinetic measurements maximize analysis options
Synergy™ H1 Hybrid
• Monochromator-based reader for UV-Vis absorbance, top/ bottom fluorescence and luminescence assays can be upgraded to a “hybrid” reader with the addition of a highperformance filter module
• Independent filter module with dichroic mirrors supports FP, TRF, TR-FRET and BRET assays
• Both radiometric and non-radiometric assays can be carried out with ease
• Seven measurement technologies are available, such as fluorescence intensity
• Capable of reading Microtiter plates from 6 to 384 wells
• Includes MikroWin Lite 2000 instrument control software