Fluorescent Dyes for Protein Labeling

Fluorescent dyes are a class of chemicals that can emit fluorescence when excited by light of a specific wavelength. They have high sensitivity, high selectivity, and good photostability, and are widely used in various analytical techniques in biomedicine and biological research. There are many types of fluorescent dyes, including organic small molecule dyes, fluorescent proteins, and quantum dots, each with different spectral characteristics and applicable ranges. In terms of protein labeling, fluorescent dyes bind to target proteins through covalent or non-covalent bonds, and the localization and dynamic changes of proteins can be visually observed under a microscope. This technology is widely used in cell biology, molecular biology, and drug development.

Fluorescent Dye

Fluorescent dyes are a class of organic compounds with luminescent properties. Their molecular structure and conjugated system determine their specific luminescent color and optical properties. Specifically, fluorescent dyes are chemical substances that can absorb photons of a specific wavelength and re-emit photons with a longer wavelength and lower energy. In biomedicine, organic fluorescent dyes are widely used in fields such as cell imaging, molecular probes, and drug delivery systems. Currently, the more common organic luminescent molecules include coumarins, fluoresceins, rhodamines, diimide derivatives, fluoroborane derivatives, cyanines, and porphyrin derivatives.

• Coumarin

Coumarin has a benzopyrone structure and is the parent nucleus of a large class of coumarin compounds found in the plant kingdom. Coumarin fluorescent dyes have a very small molecular weight and can be excited by ultraviolet light. They generally emit blue fluorescence (emission range ~410 to 470 nm) and are often used as blue light dyes in multicolor fluorescence detection experiments. Due to their low price and small molecular weight, coumarin dyes are often used to label enzyme substrates, thereby detecting enzyme activity in cells and solutions. Currently reported coumarin derivatives have a wide range of applications in laser dyes, organic light-emitting diodes, photodynamic therapy, bioimaging, and fluorescent probes.

• Fluorescein

Fluorescein, also known as fluorescent yellow, is the first batch of synthetic fluorescent dyes. Fluorescein is a fluorescent dye with tautomerism in molecular structure, namely, an open-ring quinone structure (I) and a closed-ring lactone structure (II). Quinone-structured fluorescein has strong absorption and fluorescence in the visible light region, while lactone-structured fluorescein has poor molecular conjugation, so it only has certain absorption and fluorescence in the ultraviolet region, which is usually difficult to observe. In the field of analysis, fluorescein is an important fluorescent reagent. This is because although fluorescein is easily soluble in water, it also has a good affinity with cell membrane lipids, and is easy to penetrate into cells for biological imaging. At the same time, through molecular design, a receptor that has a specific response to a certain detection object can be connected to fluorescein. By utilizing the different luminescence properties of fluorescein in different forms, the purpose of effectively identifying the detection object can be achieved. Therefore, fluorescein has a wide range of applications in the field of fluorescent probes. When fluorescein is attached to a protein as a fluorescent probe, changes in the protein's conformation will cause changes in the microenvironment in which the fluorescein is located, thereby causing changes in its spectral properties. Therefore, changes in the spectral position, intensity, and fluorescence lifetime of fluorescein can be used to detect changes in the protein's conformation.

• Rhodamine

Rhodamine dyes have the same fluorescent core as fluorescein. Rhodamine is a 3',6'-diaminated xanthene fluorescent dye. Compared with fluorescein dyes (hydroxylated xanthene), the fluorescence of rhodamine (aminated xanthene) has better pH stability. In addition, aminated dyes are more likely to have diversified structures, thus obtaining more fluorescent dyes with different excitation/emission wavelengths. Therefore, rhodamine dyes have more choices. Many dyes have longer fluorescence wavelengths than fluorescein, which is a powerful supplement to fluorescein dyes. Similar to fluorescein, rhodamine has a high fluorescence quantum yield in aqueous solution, and its structure is easy to modify. It can prepare and synthesize various types of fluorescent probes for detecting ions, small biological molecules and enzymes. In addition, the absorption and emission wavelengths of rhodamine are both above 530 nm. Through molecular modification, the absorption and emission spectra can be further red-shifted to the near-infrared region. It has good water solubility and biocompatibility and is widely used in biological imaging.

• Cyanine

Cyanine dyes (Cy) are a common type of fluorescent dyes. The core structure of cyanine is formed by two nitrogen-containing heterocyclic rings connected by an odd number of methine groups. They are usually prepared by reacting aromatic quaternary ammonium salts with corresponding condensation reagents. Cyanine dyes have a strong resonance structure, and the charge is located at the tail end of the chromophore. Changing the length of the conjugated chain between the two nitrogen atoms can significantly change the absorption and emission spectra of cyanine. Studies have shown that each additional vinyl group will cause the spectrum to red-shift by about 100 nm. For example, Cy3 exhibits yellow-green fluorescence with a maximum emission wavelength of 570 nm, while the maximum emission wavelength of Cy5 is red-shifted to 670 nm, reaching the near-infrared region. The most commonly used cyanine dye is indocyanine green (ICG), which has near-infrared absorption and emission properties. Although this type of dye has poor photostability and is greatly affected by the acidity and alkalinity of the solvent, it is still widely used in fields such as fluorescent probes and biological imaging.

Protein Definition

Proteins are biological macromolecules formed by amino acids linked by peptide bonds. They are important components of cells and key molecules that perform multiple functions in living organisms. Their basic structural units are 20 different amino acids, each of which consists of an amino group, a carboxyl group and a specific side chain. Proteins are divided into primary structure, secondary structure, tertiary structure and quaternary structure, which are determined by the sequence of amino acids and their spatial conformation. The primary structure is the linear arrangement of amino acids; the secondary structure includes α helix and β fold; the tertiary structure is the three-dimensional conformation of the entire peptide chain; and the quaternary structure refers to the polymer of multiple peptide chains.

Protein Function

The functions of proteins are diverse, covering catalysis, structural support, signal transduction, immune defense and other aspects. For example, enzymes are proteins with catalytic functions that can speed up the speed of biochemical reactions; actin and myosin in muscles provide structural and dynamic support; antibodies, as part of the immune system, help resist foreign pathogens. Due to their diversity and complexity, protein research not only plays an important role in basic biology, but also plays a key role in applied fields such as medicine, agriculture and bioengineering. Among them, protein labeling is a technology widely used in biology, chemistry and medicine to track and study the behavior, location and interaction of proteins in cells. This technology visualizes and detects target proteins under various experimental conditions by attaching specific chemical tags or fluorescent markers to them. By labeling specific proteins, their localization and dynamic changes in cells can be observed to understand protein functions and cellular processes. For example, fluorescently labeled proteins are often used to study the cytoskeleton, endomembrane system and signal transduction pathways. In the field of biochemistry, protein labeling is used to identify protein-protein, protein-nucleic acid or protein-small molecule interactions. By labeling specific proteins, co-precipitation, mass spectrometry or fluorescence resonance energy transfer (FRET) and other techniques can be used to detect protein-protein interactions and reveal their mechanisms of action in biological processes.

Protein Labeling

Protein labeling is a widely used technique in molecular biology and biochemistry, which is used to introduce one or more detectable markers at specific sites of protein molecules. These markers can be fluorescent molecules, radioisotopes, metal ions, biotin, enzymes or other reporter molecules, and are intended to study the structure, function, interaction, localization and dynamic changes of proteins in cells. At present, the methods of labeling proteins can be mainly divided into two categories: chemical labeling and biological labeling.

Fig. 1. Chromophores in fluorescent proteins (Biochem Biophys Res Commun. 2022, 633: 29-32).

• Chemical Labeling

Chemical labeling methods use chemical reactions to directly connect markers to proteins. Commonly used chemical labeling reagents include N-hydroxysuccinimide esters (NHS esters), maleimides, azides and alkynes. These reagents are usually based on specific chemical groups, such as amino groups, sulfhydryl groups or carbon-hydrogen bonds, and covalently react with corresponding groups in proteins for labeling. The advantage of chemical labeling is its flexibility, the ability to introduce various types of markers into proteins, and the fast labeling speed. However, it sometimes affects the structure and function of proteins, thereby interfering with subsequent experiments.

• Biolabeling

Biolabeling methods use biotechnology to label proteins to avoid functional effects on proteins. Common biomarker methods include fusion expression of green fluorescent protein (GFP) and its derivatives, enzyme-catalyzed labeling (such as HIS tag, FLAG tag) and gene editing as labeling (such as CRISPR-Cas9 system). GFP labeling is one of the most well-known biomarker methods. By fusing the GFP gene with the coding gene of the target protein, the expressed fusion protein is fluorescently labeled, and the dynamic changes of the protein can be observed in real time in living cells. The advantage of the biomarker method is that it can be studied in real time in living cells or organisms, and has little effect on the natural structure and function of the protein.

Fluorescent Dye Labeling to Protein

Fluorescent dye-labeled protein technology is an important tool widely used in biomedical science and biotechnology research. It uses the binding of fluorescent dyes to specific sites or functional groups of proteins, allowing researchers to visualize and track the distribution, dynamic changes and interactions of proteins in complex biological systems. Labeled fluorescent proteins can be used in a variety of experiments. Such as fluorescence microscopy observation, flow cytometric analysis, protein interaction research, etc. In the experiment, fluorescent dye labeling of proteins generally goes through the following basic steps:

• Selecting fluorescent dyes: It is crucial to choose the right fluorescent dye. Common fluorescent dyes include FITC (fluorescein isothiocyanate), TRITC (tetramethylrhodamine isothiocyanate), and various quantum dots and new dyes. Each dye has different spectral characteristics and chemical properties, and researchers need to choose the right fluorescent dye according to experimental needs.
• Protein labeling: The binding of fluorescent dyes to proteins is usually completed through covalent bonds. Taking FITC as an example, it can react with amine groups in proteins to form stable covalent bonds. This process needs to be carried out under suitable pH and temperature conditions to ensure that the binding efficiency of the dye to the protein is maximized.
• Purify the marker: After the fluorescent dye is bound to the protein, a typical experimental step may include purification and detection of the labeling efficiency. The purification step usually uses methods such as gel filtration or affinity chromatography to remove unbound dye. Detection of labeling efficiency can be done by colorimetry or fluorescence spectroscopy.
• Validation and characterization: Characterize and validate the labeled protein through techniques such as UV-Vis spectrophotometry, fluorescence spectrophotometry, or chromatography to ensure that the fluorescent dye is successfully labeled on the target protein.

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Reference:

1. Lukyanov, K.A. Fluorescent proteins for a brighter science. Biochem Biophys Res Commun. 2022, 633: 29-32.

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