May 1, 2008 (Vol. 28, No. 9)

The Addition of Absorbance-Measurement Capabilities Enhances Filter-Based Systems

An ever-increasing pool of potential therapeutic targets is now available, largely as a result of the successful deciphering of the human genome five years ago. Hence, researchers in life science and pharmaceutical companies have set out to better understand the basic mechanisms underlying acute and chronic diseases such as diabetes, Alzheimer’s, and chronic pain.

To broaden their knowledge base, scientist are scrutinizing reporter genes, protein-protein binding, and DNA content and incorporating high-throughput strategies in the primary stages of target validation.

Microplate readers are central to this work. Their utilization in basic research and drug development has benefitted tremendously as a result of automation and miniaturization of microplates. The implementation of 384- and 1,536-well plates along with new technologies have definitely streamlined the use of assays for particular targets. As a result, kinase profiling, protein-protein binding, the study of GPCRs, and cell-based assays are increasingly subject to refinement and thus becoming more and more sophisticated.

As microplate-reading technology has advanced, so has the user’s preference for multidetection readers. Recent reports suggest that absorbance measurements and TR-FRET-based assays are, and will continue to be, the most widely used applications. The challenge then is to provide multidetection readers that handle all modes of measurement for fluorescence intensity, fluorescence polarization, time-resolved fluorescence, FRET/TR-FRET, luminescence, and BRET as well as UV/Vis full-spectrum absorbance.

Microplate readers can typically be broken down into two classes: filter based and monochromator based. Optical filters provide the best sensitivity when used in microplate readers. Advanced optics, photomultiplier tubes, light guides, and their sophisticated combination provide an excellent platform for fluorescence, luminescence, and time-resolved fluorescence assays.

Figures 1, 2, 3, 4, and 5
Filters offer many advantages over monochromators including more light transmission and excellent blocking of undesired wavelengths, higher sensitivity, precise control over transmitted peak shape, fast switching to other wavelengths when more than one filter pair is employed, and lower cost.

BMG Labtech’s FLUOstar Optima and POLARstar Optima readers are capable of measuring a range of assays and offering simultaneous dual emission for FRET, BRET, and fluorescence polarization measurements. Full-spectrum absorbance measurements, however, are not possible.

To address that shortcoming, BMG has incorporated Tandem Technology into its Omega microplate-reader series (Figure 1). Tandem Technology combines an ultrafast UV/Vis full-spectrum absorbance spectrometer built into a microplate reader and high-performance optical filters.

The ultrafast UV/Vis absorbance spectrometer in the Omega series features efficient optical grating and a solid-state array detector that allows the measurement of light intensity throughout the UV and visible parts of the spectrum.

With this instrument, full-spectral absorbance can be captured at high speeds from 220–850 nm at a resolution of 1 nm. Unlike a monochromator, the spectrometer allows users to capture the entire UV/Vis spectrum of a sample in approximately one second per well—no scanning needed. If users choose to reduce data-set size, up to eight discrete wavelengths can be selected and data will be collected simultaneously.

Further, microplate data analysis and reduction software (MARS) automatically creates a standard curve and calculates the concentration for unknown samples. If the option “path length correction” is used, the measured data is normalized to a path length of 1 cm, thereby allowing a comparison to be made between absolute data obtained from a microplate reader and data obtained from a cuvette-based spectrometer.

Validation of Technology

The validation of Tandem Technology was performed using the FLUOstar/ POLARstar Omega reader. The Bradford Assay (protein binding) and the determination of DNA content (A260/A280 ratio) were used to validate the absorbance capacity of the UV/Vis spectrometer.

The Bradford Assay is based on the binding of protein to a dye, leading to a shift in the absorbance maximum of the dye. The Bradford reagent was purchased ready to use, and a stock solution of bovine serum albumin was diluted to 1 mg/mL and served as the protein standard.

Bradford reagent, 290 µL, was pipetted into a transparent 96-well microplate. The addition of 10 µL of the protein dilution was followed by mixing in the wells. The Bradford assay was performed according to the manufacturer’s protocol (Figure 2).

The protein assay is linear in the range of 0.1–1.4 mg/mL. Because of its homogeneous and fast nature, the Bradford Assay is a preferred method to determine the protein concentration of samples (Figure 3). Data analysis and calculation of the standard curve was performed with the MARS software (Figure 4).

One of the most common methods for nucleic acid detection is the measurement of solution absorbance at 260 nm (A260), since nucleic acids have an absorption maximum at this UV wavelength. DNA purity can be calculated by performing ratio absorbance measurements at A260/A280.

DNA stock solution from calf thymus was dissolved to yield concentrations from 0.1 to 100 µg/mL. Four replicates of 350 µL aliquots of each standard were pipetted into a 96-well UV plate. Replicates of 350 µL aliquots of distilled water served as a blank.

Double-stranded and single-stranded DNA or RNA possess different extinction coefficients. The MARS software offers ready-to-use templates, so data analysis for different nucleic acids was simply done by executing the appropriate template (Figure 5).

In conclusion, the built-in UV/Vis absorbance spectrometer in the Omega microplate series from BMG Labtech was successfully validated by performing two different assays. Tandem Technology ensures that high-quality data can be obtained with a single, multidetection microplate reader and thus offers the user maximum flexibility at minimal cost.

Franka Ganske, Ph.D., is application specialist, Michael Fejtl is international sales and marketing specialist, and Marjan Orban, Ph.D., is international sales and marketing manager at BMG Labtech. Web: Phone: +49-781-969680. E-mail: [email protected].

Previous articleInvestigators Generate Sequence-Based Map of Structural Variations
Next articleTorreyPines Licenses Glutamate-Receptor IP from Johns Hopkins