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August 01, 2011 (Vol. 31, No. 14)

Microplate Reader Absorbance Assays

New Tools Bridge Gap between Single and Multiple Sample Absorbance Instruments

  • Low Volume Measurements for DNA, RNA, and Protein

    Click Image To Enlarge +
    Figure 2. Spectra (220–400 nm) for low volume dsDNA samples (2 µL) were measured using BMG LABTECH’s LVis Plate (reference peaks 600 and 970 nm not shown). Inset shows 10 blank measurements. Corrected values (260–340 nm) have lower standard deviations than raw data. Since the LOD = 3*SD[Blank], this simple correction allows for greater sensitivity in DNA measurements.

    When measuring low volume samples of two microliters or less, variables such as path length (b), concentration (c), and purity become factors that can be easily accounted for using fast, full-spectrum analysis.

    As Figure 2 shows, measurements at 230 nm (phenol), 260 nm (DNA/RNA), 280 nm (protein), 340 nm (background), 600 nm (bacteria), and 970 nm (water) can all be taken with one reading. As the inset illustrates, blank correcting for the buffer at 340 nm decreases the standard deviation. This is one example of how this technology allows for lower limits of detection, greater sensitivity, and more reliability in each DNA measurement.

  • HTS Compound Library Management

    To avoid contamination when using an HTS compound library, a working library is often created. With this new microplate reader, the purity and concentration of the compounds in the working library are quickly and easily confirmed.

    For instance, if a researcher has a library in a 384-well microplate and wants to verify each well's concentration and purity by capturing a full absorbance spectrum (220–1,000 nm), it can be accomplished in 6 minutes at a 1 nm resolution on the SPECTROstar Nano.

    Traditionally, using a monochromator with 10 nm increments, this would take more than six hours per 384-well plate. In addition to being more than 60 times faster the resolution is 10 times better, allowing for unprecedented high-resolution, full-spectrum scans of each well.

  • Fast, Full-Spectrum ELISAs Allow for Better Data Collection

    Click Image To Enlarge +
    Figure 3. If the peak wavelength is chosen (430 vs. 450 nm), the standard curve has almost a 20% larger dynamic range. If a nonsaturated wavelength is chosen (495 vs. 430 nm), then all of the standards are used, thereby increasing the dynamic range of the assay.

    Typically for ELISAs, one or two wavelengths per well are measured, which takes about three minutes per 96-well plate. With the SPECTROstar Nano, the whole spectrum can be taken in half of the time, allowing for optimal wavelength selection in each assay.

    For instance, standards not measured at the optimal wavelength can decrease the range of the standard curve (Figure 3, Example 1). Conversely, saturated standards that would be discarded at 450 nm can now be used at 495 nm, thus extending the range of the standard curve (Figure 3, Example 2). Choosing the correct wavelength creates standard curves with larger dynamic ranges, helping to save time, money, and data.

  • Conclusion

    Ultrafast full-spectrum analysis is now possible on a multisample absorbance instrument. As Kirchhoff and Bunsen redefined the way we use light more than a century ago, this new technology will help to redefine microplate reader absorbance assays as we know them today.

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