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

Microplate Reader Absorbance Assays

New Tools Bridge Gap between Single and Multiple Sample Absorbance Instruments

  • 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

    Click Image To Enlarge +
    Figure 1. In the same amount of time a traditional absorbance microplate reader captures one wavelength or wavelength band (left), the new ultra-fast spectrometer captures full UV-Vis spectra at resolutions of 1 nm (right).

    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?”



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