Fluorophores are molecules that can absorb light of a specific wavelength and emit fluorescence after excitation. These molecules usually contain conjugated double bonds or aromatic ring structures, which enable them to effectively absorb light energy and release energy through radiative transitions, thereby emitting visible light. The luminescent properties of fluorophores make them widely used in biomarkers, medical diagnostics, and materials science.
Types of Fluorophores
Common fluorophores include fluorescein, rhodamine, and Cy5. These fluorophores exhibit different spectral properties; for example, Cy5 is a long-wavelength dye known for its significant advantage of low autofluorescence in biological samples within this spectral region. Additionally, there are gene-encoded fluorescent proteins such as green fluorescent protein (GFP), blue-shifted green fluorescent protein, and cyan-shifted green fluorescent protein, which possess high stability and brightness.
Fluorescein
Fluorescein is a widely used organic fluorescent dye known for its bright fluorescence and good water solubility. It plays an important role in fields such as biomedicine, chemical labeling, and environmental monitoring.
Rhodamine
Rhodamine is a resonance dye with narrow absorption and emission bands, primarily used for labeling biological molecules like proteins, DNA, and RNA. It is extensively applied in biological research methods such as flow cytometry and immunofluorescence microscopy. Moreover, rhodamine dyes exhibit good pH stability and structural diversity, making them excel in fluorescence labeling and detection.
BODIPY
BODIPY (boron-dipyrromethene) is a fluorescent dye with high fluorescence quantum yield, good stability, and ease of modification. It is widely used in tumor cell detection, biological labeling, metal ion detection, and photodynamic therapy. Furthermore, BODIPY dyes have extensive applications in biomedicine, materials science, and optoelectronics.
Biological Fluorophores
Biological fluorophores can be classified into endogenous and exogenous categories. Endogenous fluorophores are naturally present in biological structures, such as reduced nicotinamide adenine dinucleotide phosphate (NADPH) and flavin adenine dinucleotide (FAD), commonly referred to as autofluorescence. Exogenous fluorophores are externally added to biological systems, including small molecules, nanomaterials, and fluorescent proteins. These fluorophores offer higher signal-to-noise ratios and can specifically label samples.
ICG Dyes
Indocyanine green (ICG) is a near-infrared fluorescent dye with an absorption peak at 800 nm and an emission peak at 810 nm. It is characterized by high absorption, low autofluorescence, and low toxicity. ICG is FDA-approved for various clinical applications, including tumor detection, lymph node localization, retinal and choroidal vascular imaging, and angiography in reconstructive surgeries. Additionally, ICG is used in robotic-assisted urologic surgeries to accurately reflect critical information such as excision margins and blood supply, effectively reducing intraoperative risks and postoperative complications.
Fluorescence Labeling Methods
Fluorescence labeling methods involve the use of fluorescent dyes or proteins to bind with target molecules, widely applied in biomedical research. During the fluorescence labeling process, fluorescent substances are typically covalently bonded or physically adsorbed to the molecules being studied, such as proteins and nucleic acids.
Covalent Labeling
Covalent labeling involves chemically bonding the fluorophore to the target molecule in a stable manner. This method ensures long-term association of the fluorophore with the target molecule, making it suitable for long-term observation and dynamic studies. Common covalent labeling reactions include:
Reactive group reactions: For example, reacting amino-fluorescent dyes with carboxyl-containing target proteins to form amide bonds. This method is applicable to various biomolecules, such as proteins and nucleic acids.
Click chemistry: Utilizing "click reactions," such as the reaction between azides and alkynes, to efficiently label biological molecules with fluorophores. This method is highly selective and efficient, making it suitable for labeling complex samples.
Non-Covalent Adsorption
Non-covalent adsorption methods utilize electrostatic forces, hydrogen bonding, or van der Waals forces to attach fluorophores to target molecules. This method is simple to operate and maintains mild reaction conditions, making it suitable for labeling without damaging the target molecule's structure. Common non-covalent adsorption methods include:
Electrostatic adsorption: Electrostatic interactions occur between oppositely charged target molecules and fluorophores. Studies show that this method is suitable for charged proteins and polymers.
Hydrophobic interactions: Interacting hydrophobic fluorophores with hydrophobic target molecules to form stable complexes. This method is suitable for labeling membrane proteins.
Biotin-Avidin System
The biotin-avidin system is a highly specific and sensitive labeling method widely used for the detection of proteins and nucleic acids. The basic process of this system includes:
Biotin labeling: First, biotin labels the target molecule. Biotin is a small molecule that can chemically bind to functional groups such as amino or carboxyl groups on the target molecule.
Avidin binding: Then, avidin (or streptavidin) is combined with fluorescent dyes. Avidin has high specificity and can form stable complexes with biotin, thereby enhancing the strength of the fluorescence signal.
Genetic Engineering
Genetic engineering methods introduce fluorescent protein genes into cells through recombinant DNA technology, allowing cells to express fluorescent proteins. This method enables cell imaging and dynamic observation, with the specific process as follows:
Transfection: Using transfection reagents to introduce plasmids carrying fluorescent protein genes into target cells. After transfection, cells synthesize fluorescent proteins autonomously.
Observation and analysis: Using fluorescence microscopy to observe cells expressing fluorescent proteins, studying intracellular processes, protein interactions, and molecular localization.
Photosensitive Labeling
Photosensitive labeling is a method that utilizes photosensitizers for labeling. Under light of specific wavelengths, photosensitizers can generate fluorophores upon excitation. The process of this method includes:
Selecting photosensitizers: Choosing appropriate photosensitizers for the target molecules and mixing them with the target molecules.
Light excitation: Using lasers or other light sources to excite the photosensitizers, generating fluorophores and labeling them to the target molecules. This method shows good prospects for application in live-cell imaging.
Applications of Fluorophores
Biomedical Imaging: Fluorophores play a crucial role in biomedical imaging, enabling high-contrast sample imaging and fluorescence labeling. For instance, fluorophores are used in DNA sequencing, cell imaging, super-resolution microscopy, and longitudinal studies of diseases. Moreover, fluorophores are also employed in flow cytometry as detection reagents coupled with antibodies for cell detection and analysis.
Environmental Monitoring: Fluorophores can be used to create fluorescent sensors for monitoring environmental factors such as air and water quality.
Organic Light-Emitting Diodes (OLEDs): The application of fluorophores in organic light-emitting diodes has garnered attention, particularly for high-definition displays, with thermally activated delayed fluorescence (TADF) materials receiving notable interest for their promising applications.
Fluorescent RNA Imaging: Fluorescent RNA technology utilizes RNA aptamers to recognize and bind fluorophore molecules, allowing for the visualization of RNA. This technique permits imaging of mRNA stress granules and small non-coding RNAs in mammalian cells.
Chemical Sensors: The application of fluorophores in chemical sensors includes the detection of metal ions, small molecules, nucleic acids, and proteins.
Smart Phase Change Materials: Advances have also been made in the application of fluorophores in smart phase change materials, such as Rb2MnBr4(H2O)2 crystals that exhibit high-temperature thermochromic fluorescence and optical reversibility.
Near-Infrared Fluorescent Probes: New design concepts for near-infrared (NIR-II) fluorescent materials and their applications in biomedicine, such as the use of the organic small molecule fluorophore FM1210 for deep tissue imaging.
