Mass Spectrometry & Spectroscopy
UV-Vis and fl uorescence spectroscopy illuminate biological applications Dr Ursula Tems, Pharma/BioPharma Marketing Director, Molecular Spectroscopy, Agilent Technologies
In the intricate landscape of biological research, understanding the behaviour and properties of biomolecules - those tiny building blocks of life - is paramount. UV-Vis (Ultraviolet-Visible) spectroscopy and fl uorescence spectroscopy are two powerful techniques that shed light on the otherwise invisible interactions within cells, tissues, and organisms. In this article, we delve into their applications, principles, and the advantages they offer over other methods.
About the technologies
UV-Vis spectroscopy involves shining light in the ultraviolet (UV) and visible (Vis) wavelength regions onto a sample and measuring how much light it absorbs, to provide information about the electronic transitions of molecules. As well as being non-destructive, UV-Vis is a well-established, easy, and fast technique that requires minimal consumables and so is also cost effi cient. UV-Vis is used in many biological applications for providing the concentration of a sample, or for characterisation of a sample. For example, protein concentration can be determined by observing the maximum absorbance at a specifi c wavelength, and the quality of the sample determined by observing the spectra to ensure that there are no contaminants.
The other key use is to measure changes that occur over time, or over different temperatures, for example, monitoring an enzyme assay or a thermal denaturation experiment. Recent advances in UV-Vis technology have enhanced its usability and accuracy, for example the Cary 3500 UV-Vis spectrophotometer can measure eight samples simultaneously, and at four different temperatures simultaneously, ideal for monitoring biological enzymes. In addition to this, some accessories exist that allow users to directly quantify very low volume samples of purifi ed nucleic acid or protein samples without the need for sample dilution.
Fluorescence spectroscopy takes advantage of the intrinsic fl uorescence of many biological molecules, such as proteins and nucleic acids. These molecules absorb light (excitation) and emit light (emission) at specifi c wavelengths. The strengths of fl uorescence spectroscopy are specifi city, sensitivity, and the ability to analyse molecular interactions, but fl uorescence spectroscopy has many additional biological applications and is a widely used workhorse in life science laboratories.
A recent and unique example of how fl uorescence spectroscopy can be used in biological laboratories is for screening monoclonal antibodies that are prone to aggregation.
Eight samples at four temperatures simultaneously with the Cary 3500 UV-Vis Multizone Peltier Spectrophotometer.
A fl uorescence spectrophotometer is fl exible, able to be combined with many types of sample presentation accessories, and in this case a plate reader is used for high throughput. The properties of a fl uorescent dye are used to indicate which monoclonal antibody samples aggregate and are therefore not useful for further analysis [1].
The wavelengths at which many biomolecules fl uoresce is well characterised, and this can be used to measure the concentration of one type of molecule very specifi cally in a mixture.
Flexible fl uorescence spectroscopy with plate reader.
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