What are Fluorescent Dyes?
Fluorescent dyes are tools used throughout biotechnology and medicine that offer a unique method of detecting and quantifying the presence of a target molecule, cell, or tissue within an even more complex biological sample. They are used within many realms of research and development, and are often conjugated to other molecules as micro- or nanocarriers for bioassays. In a clinical setting, they can be used to monitor drug delivery to target tissues or for precise imaging and diagnostics applications.
By nature, each fluorescent dye exhibits a unique absorbance and emission spectra, resulting from the change in energy states of electrons when the fluorophore is excited. These absorbance and emission spectra can not only exist within the visible spectrum, but also in ultraviolet (UV) and near infrared (NIR). Additionally, fluorescent dyes are highly sensitive, selective, and have extremely low toxicity which makes them useful for in vivo, in vitro, and in situ applications.
Fluorescent Detection Compared to Other Methods
Outside of fluorescence, colorimetry is another method of detecting target compounds or cells within a sample. In colorimetric assays, the target substrate or activity often produces a colored byproduct, which is quantifiable by absorbance spectroscopy and which is directly related to the presence and/or concentration of the target within the sample.
One application of colorimetric assays is to assess the total cell viability within a sample. For example, in the trypan blue exclusion assay, cells that have lost membrane integrity will appear blue through the binding of trypan blue to macromolecules and intracellular proteins, highlighting non-viable cells. The CCK-8 (cell counting kit-8) is another cell viability assay; it relies on the reduction of a tetrazolium salt, WST-8, by dehydrogenase to produce a yellow-colored formazan dye. Since active dehydrogenases are involved, it correlates with viable cells. In a similar vein, the MTT assay can also be used to indirectly determine cell viability by assessing cell metabolism. This assay relies on the oxidoreductase of NADH in cells to quantitate total metabolic activity within a cell population.
Another common use of colorimetric assays includes total protein quantification. For example, the BCA assay is one that combines the reduction of Cu2+ into Cu1+ by proteins in an alkaline medium, with the detection of the cuprous cation by bicinchoninic acid (BCA). A second, related assay is the Bradford assay, which relies on the reaction between acidified Coomassie blue to proteins to undergo a quantifiable color change.
