Fluorescence and UV-Vis absorbance are complementary measurement techniques for the quantification of biomolecules. Traditionally seen as separate, and sometimes competing methodologies, this article will discuss the advantages of each as well as new systems that integrate multiple modes into single compact bench-top instruments.
Accurate quantification via absorbance and fluorescence across a broad dynamic range can be achieved by using DeNovix spectrophotometer/fluorometer systems. These systems include a full-spectrum cuvette mode, microvolume mode, and an integrated fluorometer. The microvolume mode incorporates SmartPath® Technology which enables reproducible and precise quantification for 1µL samples. The fluorescence mode includes a proprietary optical core that uses highly sensitive photodiodes and four LED sources (UV, Blue, Green, Red). The integrated fluorometer can detect even trace levels of fluorescence over ranges of four wavelengths.
Basics of Absorbance Measurements
In life science laboratories, measurements of UV-Vis absorbance are traditionally made to quantify purified biomolecules. Using this approach, the concentration of a biomolecule can be quickly determined depending on its absorbance profile at particular wavelengths. Beer’s Law provides a direct method for determining the concentration of a sample if the path length, the extinction coefficient of the biomolecule, and the measured absorbance of the sample are known. Other common absorbance techniques include quantification via cuvette UV-Vis using colorimetric standard curve methods such as the Bradford assay.
Contamination of samples may also be detected through absorbance measurements. When other molecules are present that absorb at or close to the same wavelengths as the target molecule, the structure of the absorbance spectrum will be modified to some extent.
Figure 1. Absorbance and fluorescence quantification. Image credit: DeNovix
Basics of Fluorescence Quantification
Fluorophores are capable of absorbing light at a single wavelength, also known as excitation wavelength, and generate light at another wavelength called emission wavelength. It is possible to leverage the structures of specific fluorophores to fluoresce only when adhered to a particular molecule, such as a double-stranded DNA molecule. This unique binding specificity is used by fluorescence assays to determine a direct relationship between the concentration of the target biomolecule in the solution and the level of fluorescence produced by a sample. Assays are designed so they will not bind with other biomolecules that may be present in a sample such as RNA in a dsDNA sample.
If a known sample concentration is mixed with a fluorophore, and the relative fluorescent units (RFU) is determined, then the correlation between the quantified RFU and the sample concentration can be plotted and the same can then be utilized as a standard curve. Subsequently, the concentration of the sample can be measured by plotting the emission of the same fluorophore against the standard curve. This fluorophore adheres to samples of unknown origin.
Absorbance vs. Fluorescence
Absorbance measurements offer a number of benefits. For instance, there is no need to use reagents and the absorbance, thus quantified, is the direct outcome of the target molecule, which absorbs light at a known wavelength. The amount of the absorbed light directly matches the concentration of the target molecule.
In contrast, fluorescence is an indirect measurement and provides several benefits like specificity and high sensitivity. In terms of specificity, the fluorophore has good binding properties, making this technique quite selective for particular molecules. These assays can be used for samples containing contaminants.
Such impurities can interrupt an absorbance measurement. In terms of sensitivity, fluorescence assays are highly sensitive because the fluorophore is known to have a high extinction coefficient. As a result, molecules can be detected at much reduced concentrations when compared to the conventional absorbance measurements. A typical lower detection limit for microvolume spectrophotometers is 2.0 ng/mL dsDNA. Fluorescence assays such as the DeNovix Ultra High Sensitivity assay can detect dsDNA at concentrations as low as 0.5 pg/mL.
Integrated Instruments
Absorbance quantitation using the cuvette or microvolume-based capabilities as well as measurements via fluorometric methods are available in the DS-11 FX Spectrophotometer / Fluorometer Series of instruments from DeNovix Inc. (Wilmington, DE). These integrated systems allow users to combine all of the modes required for their workflow. Researchers need not choose absorbance or fluorescence or occupy additional bench space. Instead, in a single 20 x 30 cm footprint labs can utilize any method of their choice. The instrument software is pre-configured with dozens of commonly used quantification assays and methods making use extremely quick and simple.
Conclusion
Both fluorescence and absorbance are complementary measurement techniques. Absorbance is often the most rapid manner for quantification. However, quantitation through fluorescence using a secondary reporter fluorophore is often suitable for samples falling below the measurable threshold for UV-Vis absorbance measurements. Additionally, in certain situations, fluorescence measurement techniques can be employed for detecting samples in the presence of buffer elements or contaminants that would otherwise interfere with the UV-Vis measurements.
DeNovix DS-11 FX Spectrophotometer /Fluorometer systems provide UV-Vis as well as fluorescence capability in a compact, benchtop platform. These instruments have the same sample export features and are integrated with EasyApps® software to make simple, fast and intuitive data analysis.
Figure 2. DeNovix DS-11 Series of Spectrophotometers / Fluorometers. Image credit: DeNovix
DS 11 Series | Spectrophotometer | Fluorometer
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