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May 01, 2010 (Vol. 30, No. 9)

Increasing Spectrophotometer Flexibility

Multivolume Accessory Expands Versatility of Analytical System

  • Results and Discussion

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    Figure 1. Microvolume quantification accuracy of dsDNA relative to measurements made at 1 cm pathlength with BioCell accessory

    Figure 1 demonstrates the linearity of a dsDNA calibration curve. Abscissa data is reflective of measurements made with 1 cm pathlength; these measurements are considered actual dsDNA concentrations. Ordinate data reflects microvolume analysis obtained with the Take3 plate accessory using 2 µL sample volumes. Insert is an exploded view of the low dsDNA concentration calibration curve.

    Excellent linearity is available over three orders of concentration magnitude. This dynamic range covers the expected concentrations of isolated dsDNA from genomic DNA isolation kits (20–200 ng/µL) and plasmid DNA isolation kits (500–2,000 ng/µL). The linear regression slope of 1.000 ±0.0038 to a 95% confidence limit can also be used as a measure of microvolume quantification accuracy relative to 1 cm pathlength measurements.

    We have devised a method for the addition of 2 µL protein standards or unknown protein samples, followed by 2 µL BCA working reagent directly to the microspots of the Take3 plate accessory. Sixteen microspots on the plate allow for a six-point calibration curve in duplicate and four microspots for unknown samples.

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    Figure 2. BCA Calibration curve using BSA concentration range from 0.01–1.0 mg/mL

    Figure 2 depicts a BSA standard calibration curve of 0.01–1.0 mg/mL, which was used to quantify two BSA samples of known concentration: 0.037 mg/mL and 0.34 mg/mL. In situ microvolume analysis produced accuracies of 0.99% and 1.5%, attesting to the high accuracy provided by running samples and standards on the same microplate.

    The Epoch Spectrophotometer system can also be used for standard microplate colorimetric assays. An example is provided here for the quantification of thiostrepton’s cytotoxic effects using an MTT assay. Figure 3 demonstrates the viability of mesothelioma cells seeded in a 96-well plate to 20,000 cells/well with increasing thiostrepton dose. As expected, thiostrepton demonstrates cytotoxic effects on the cells with an EC50 of 3.5 µM, consistent with previously published work.

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    Figure 3. Thiostrepton dose-response curve using 20,000 primary mesothelioma cells/well

    The Epoch Spectrophotometer System is flexible for microvolume, cuvette, and microplate-based determinations. Accuracy in direct DNA quantification is comparable to 1 cm pathlength cuvette-based measurements with improved ease of use and sample conservation. The microplate accessory can also be used with reporter molecule-based assays such as BCA in microvolume format. This maintains a simple workflow and conserves sample.

    Six-point calibration curves can be conducted in duplicate with four microspots available for unknown quantification. Measuring both standards and samples on the plate yields accuracies <2% different from 1 cm pathlength measurements. Finally, the spectrophotometer system can be used in myriad microplate-based colorimetric assays, including cytometric experiments using MTT.