Fluorescent Dye Probes for Biomarkers

Fluorescent dye probes or fluorophores are often described as fluorescent chemical sensors because they can absorb light at a given wavelength and emit light at a higher wavelength to produce fluorescence in various colors. Generally, they could be divided into organic dyes (e.g., fluorescein, rhodamine, AMCA), biological fluorophores (e.g., green fluorescent protein, phycoerythrin, allophycocyanin), and quantum dots. Due to the high sensitivity, technical flexibility, and quick response time of fluorescent dyes, they have become widely-used tools for quantitative detection and visual imaging.

Compositions of Fluorescence Probes

Biomolecules require to be marked by fluorescent labels before they can be observed through fluorescence microscopy. That's to say, researchers need to attach fluorescent dyes to organic compounds inside the molecules since fluorescent labels are sensitive even at low concentrations, durable for extended durations of time, and therefore do not affect the functionality of the protein targets.

A fluorescence probe consists of three necessary components, including a signaling unit (chromophore), a separator (chemical bridging), and an attaching unit (receptor), the alteration of which enables researchers to design and tailor probes for particular targets. Chromophores usually are organic pigments that can only capture visible light. Diverse auxochromes and functional groups that can change the ability of chromophores to collect light are often added to these organic pigments. Auxochromes can be added to alter the spectrum position and brightness of the generated color.

Specific sensors with red or green fluorescence, which are two of the most sought hues in the range of the electromagnetic spectrum, are widely used in biochemistry, immunofluorescence, immunostaining, and a series of processes. A symbolic application of fluorescent sensors is in the field of imaging living tissue, which can reduce cellular fluorescence intensity and specific binding in multicolored confocal microscopy.

Fluorescence Imaging Techniques to Visualize Target Protein

There are two techniques that can be used to visualize target proteins by taking advantage of fluorescence imaging. One is the aforementioned method that biologically attaches a fluorescent dye to a target molecule to form an intrinsic chromophore, and the other is using fluorescently tagged antibodies to selectively bind to a targeted protein. But a final test is usually required in practice, especially in cases where fluorescent proteins are not applicable, for example, in histology samples.

Advantages & Disadvantages of Fluorescent Proteins

The fact that fluorescent proteins are proteins themselves could be a further drawback given certain proteinous properties inside a cell. Specifically, it may lead to malfunction or incorrect interpretations when researchers investigate associated or particular proteins of interest. Moreover, if target antibodies are available, immunofluorescence would be more time-effective than luminescent proteins that require desired gene replication or DNA insertion into the target cell.

But it's undeniable that fluorescent proteins are useful gene function sensors in various applications as well as promising indicators to monitor specific cellular components, identify proteins in human tissues, and track enzyme dispersion and intracellular pathways. In addition, fluorescent proteins complexed with antibodies or other biochemicals are also important in immunocytochemistry, electron microscopy, Western blot analysis, immunohistochemistry, and fluorescence-based immunoassay assay (FLISA).

Development of New Fluorescence Probes

Novel fluorophores have enhanced the potential for the specific detection of enzymatic activities and their interactions. And analyses of cellular procedures and systems, which were previously unachievable, are now accessible due to improved fluorescent dyes. What's more, fluorescent dyes have various benefits including high specificity, high sensitivity, as well as the capacity to fine-tune optical characteristics, such as lifetime, emitted and activation spectra, brightness, and anisotropy. And they may be used in model organism research, including such tissues and cells.

Therefore, it's necessary to design new fluorescence probes with improved characteristics, functions, and structures.