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Tutorials : Aug 1, 2011 (Vol. 31, No. 14)

Microplate Reader Absorbance Assays

New Tools Bridge Gap between Single and Multiple Sample Absorbance Instruments
  • E. J. Dell

For generations after Gustav Kirchhoff and Robert Bunsen deduced that almost all molecules in nature absorb and emit light at distinct wavelengths, scientists have used this knowledge of spectroscopy across many scientific disciplines. As a result, spectroscopic instruments that measure light absorption have become a mainstay in biology and chemistry laboratories around the world. One of the primary uses of these instruments is to determine how much of a particular substance (DNA, copper, riboflavin, etc.) is present in a solution.

These spectroscopic instruments use the principals of light transmittance and absorbance. Transmittance is the measurement of light intensity leaving a sample in a solution (I) divided by the incident of light directed at the sample (I0) or T=I/I0. Absorbance (A) is the log of the inverse of transmission or A=log 1/T=log I0/I. Therefore, an instrument that has a defined path length (b), can be used with the Beer-Lambert Law (A=εbc) to determine the concentration (c) of a substance in solution, with a known molar absorptivity (e), or log I0/I=εbc.

Barrier between Single and Multiple Sample Absorbance Instruments

As spectroscopic instruments evolved from single sample test tube instruments to multisample microplate readers, new technological barriers were encountered. One example of such a barrier was in the lamps that provide the incident of light directed at the sample (I0). The first lamps provided less energy over a limited range of wavelengths, thus inhibiting the use of a microplate reader.

Halogen, tungsten, and deuterium lamps had to be used in combination to cover most of the UV to far red spectrum. High energy xenon flash lamps overcame this barrier, providing a single light source with more energy over a broader spectral range. This advancement propelled microplate readers to the next level, allowing for a larger range of assays at lower concentrations.

Another technological barrier in microplate readers is the inability to instantly capture a full absorbance spectrum from the ultraviolet to the near infrared. Recent technology used for wavelength selection, such as filters and monochromators, allows for absorbance measurements of one wavelength or a band of wavelengths in less than one second per well (Figure 1).

But if a broad range of wavelengths or if multiple wavelengths at a high resolution were to be measured, which are both common on a single sample cuvette instrument, the time spent using either technology is greater than two minutes per well. This would equate to more than three hours for a 96-well microplate, thereby making instantaneous, high-resolution full-spectrum measurements impractical on a microplate reader.

New Technology

Using a proprietary spectrometer, BMG LABTECH's SPECTROstar Nano instantaneously captures a full spectrum (220–1,000 nm at resolutions of 1, 2, 5, or 10 nm) in less than one second per well. In addition to being able to measure microplates of up to 1,536-wells, single sample cuvettes and low volume samples for DNA, RNA, or protein can also be measured on the SPECTROstar Nano.

This next-generation absorbance technology breaks down one of the last barriers to adapting single sample measurements to a microplate reader. With the SPECTROstar Nano, data collection does not have to be compromised. Users no longer ask questions such as, “What filter or wavelengths should we use?” or “How many reference points can I capture in one second per well?”

Low Volume Measurements for DNA, RNA, and Protein

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

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.