Fluorescent Probes: Definition, Structure, Types and Application

The organism contains a multitude of chemical substances, including enzymes, proteins, and various cations and anions, which are of significant importance to human life activities and health. Additionally, there are numerous substances in nature that await testing, including antibiotics, disease markers, and pollutants, which merit further exploration by researchers. Conducting tests on these substances has positive implications, and various methods have been developed to detect specific analytes, such as chromatography, mass spectrometry, and biological methods. Each of these methods has its characteristics but tends to be time-consuming and more costly, and they cannot provide real-time information feedback. Therefore, finding alternative and convenient detection methods is crucial. Fortunately, fluorescence detection has gained widespread recognition among researchers for detecting specific analytes due to its sensitivity, convenience, cost-effectiveness, and visualization features. Fluorescence detection relies on the construction of fluorescent sensors (also known as fluorescent probes). The preparation of high-quality fluorescent probes and the exploration of suitable detection mechanisms can ensure superior quality in fluorescence detection.

What are Fluorescent Probes?

Fluorescent probes are molecular measurement devices based on spectroscopic chemistry and optical waveguide and measurement technology. They selectively convert the chemical information of the analyte into a fluorescence signal that can be easily measured by analytical instruments. Fluorescent probes are influenced by their surrounding environment. Within a certain system, after undergoing chemical or physical interactions with a particular substance, the fluorescence signal of the molecule can change accordingly, allowing for the detection of specific information present in the surrounding environment. By contrasting the signal changes of specific molecular probes with the intrinsic properties of the tissues in vivo, imaging effects can be obtained, and fluorescence imaging can achieve high spatial resolution within the body. Fluorescent probes have a wide range of applications in practical life. They can detect cations and anions present in real samples, measure the content of enzymes or small molecules in living organisms, and are also used in medical imaging to distinguish whether tumor cells have undergone pathological changes. Thanks to these advantages of fluorescent probes, the design of molecules with good selectivity and high sensitivity has garnered widespread attention from many researchers today.

Fluorescent Probe Structure

Generally, a designed fluorescent chemical sensor can be roughly composed of three parts (Fig. 1), including ion recognition unit (Receptor), signal unit (Fluorophore), and linking group.

Fig. 1. Structure of fluorescent probe.

• Receptor: The recognition part primarily ensures the specificity and selectivity of the probe, making it the most critical component in disease-targeted design. The higher the interference resistance of the recognition part, the more accurate the signal will be.
• Fluorophore: The fluorophore serves as the signal generation component, mainly composed of inorganic fluorescent materials and organic small molecule fluorescent dyes. Inorganic fluorescent materials primarily include rare earth fluorescent materials, such as the representative lanthanide luminescent elements-Europium (Eu), Samarium (Sm), Erbium (Er), Neodymium (Nd), and luminescent quantum dots like CdSe and CdTe. There is a wide variety of organic molecular fluorescent materials, which often have easily adjustable structures and a broad wavelength range. Common fluorescent moieties used in fluorescent probes include fluorescein dyes, rhodamine series, coumarin and its derivatives, naphthalimide, BODIPY, and aggregation-induced emission (AIE) fluorescent molecules like tetraphenylethylene. The excitation and emission wavelengths of the fluorophore are key considerations in probe design. The near-infrared (NIR) wavelength range (700 nm-1700 nm) lies outside the tissue's intrinsic fluorescence range, with tissue penetration depths of 5-10 mm. Therefore, almost all optical imaging agents used clinically are NIR materials. Many commercially available dyes cover the NIR wavelength range, and research has shown that longer wavelengths are more favorable for improving the signal-to-noise ratio.
• Linker: The linking part serves as a bridge connecting the recognition portion to the fluorophore, typically consisting of a carbon chain or chemical bond. The linker can control the overall size of the probe, thus affecting its overall performance, such as in vivo diffusion rate, cellular permeability, and plasma circulation time.

Types of Fluorescent Probes

Fluorescent probes have attracted the attention of researchers due to their effective applications in biological, chemical, medical, and environmental processes. The response mechanism of fluorescent probes to analytes involves changes in fluorescence signals through the interaction of probe molecules with specific analytes, leading to the design of various types of fluorescent probes. When the free probe binds with the analyte, photophysical processes occur, resulting in changes in fluorescence phenomena (such as excitation/emission wavelengths, quantum yield, fluorescence intensity, fluorescence lifetime, etc.). Common recognition mechanisms include: (1) Photoinduced Electron Transfer (PET); (2) Intramolecular Charge Transfer (ICT); (3) Fluorescence Resonance Energy Transfer (FRET); (4) Excited State Intramolecular Proton Transfer (ESIPT); (5) Monomer/Excimer Association; (6) Aggregation-Induced Emission (AIE), among others. Commonly used fluorescent probes are of the following types:

• Fluorescein Probes

Fluorescein probes are composed of fluorescein or its derivatives, which can bind to specific biomolecules or ions, thereby altering their fluorescence intensity or wavelength. For example, fluorescein isothiocyanate (FITC) is a commonly used fluorescein probe that can covalently bind to biomolecules such as proteins or antibodies, making it useful for labeling and detecting biomolecules.

• Ion Fluorescent Probes

These probes are made up of inorganic ions or their complexes, which can react with specific metal ions or other inorganic ions, resulting in changes to their fluorescence intensity or wavelength. For instance, rhodamine B is a commonly used inorganic ion fluorescent probe that can form a complex with copper ions, leading to enhanced fluorescence.

• Fluorescent Quantum Dots

These probes are composed of semiconductor nanoparticles that exhibit high fluorescence quantum yield and stability, along with narrow emission peaks and broad excitation spectra. Fluorescent quantum dots can be surface-modified to bind with different biomolecules or ions, enabling their use in multicolor fluorescent labeling and detection. For example, cadmium sulfide (CdS) is a commonly used fluorescent quantum dot material that can be modified to bind with biomolecules such as DNA or antibodies, facilitating the labeling and detection of genes or cells.

• Molecular Beacons

These probes consist of semiconductor nanoparticles and possess high sequence and structural specificity, allowing them to hybridize with target DNA or RNA, which alters their spatial conformation and fluorescence properties. Molecular beacons typically contain a fluorescent group and a quencher group. When in a single-stranded state, the two groups are in close proximity, resulting in fluorescence quenching; upon hybridization with target DNA or RNA, the two groups move apart, leading to fluorescence recovery. Thus, molecular beacons can be used for the specific detection of the presence and expression of DNA or RNA.

What are Fluorescent Probes Used For?

Fluorescent probes are widely used in both daily life and scientific research. In addition to their application in environmental ion detection, they are also extensively utilized in fields such as optoelectronic devices, biological imaging, biomedicine, dyes, fluorescent whitening agents, fluorescent coatings, and organic electroluminescent devices.

• Biological Imaging

One of the primary applications of fluorescent probes is in biological imaging. They enable researchers to visualize cellular processes in real time, providing insights into dynamic biological systems. For instance, fluorescent dyes are commonly used in fluorescence microscopy to stain specific cellular components, such as membranes, organelles, and proteins. Techniques such as live-cell imaging allow scientists to track cellular activities and interactions in living organisms, leading to breakthroughs in understanding cellular dynamics, disease mechanisms, and drug effects.

• Molecular Diagnostics

Fluorescent probes play a crucial role in molecular diagnostics, particularly in detecting specific nucleic acids and proteins associated with diseases. Fluorescently labeled probes can hybridize to target DNA or RNA sequences, allowing for the identification of genetic mutations, viral infections, or cancer biomarkers. Techniques like quantitative PCR (qPCR) and fluorescence in situ hybridization (FISH) utilize fluorescent probes for sensitive and specific detection, contributing to early diagnosis and personalized medicine.

• Environmental Monitoring

In environmental science, fluorescent probes are used to detect and quantify pollutants, such as heavy metals and organic compounds, in water and soil samples. Fluorescent sensors can provide real-time monitoring of environmental changes, helping to assess the impact of contaminants on ecosystems. These probes can also be engineered to respond to specific environmental conditions, such as pH changes or the presence of certain ions, enhancing their applicability in environmental monitoring.

• Drug Discovery and Development

Fluorescent probes are valuable in drug discovery, particularly in high-throughput screening assays. They enable the monitoring of drug interactions with target proteins, cellular pathways, or biological systems. For example, fluorescent probes can be used to study receptor-ligand interactions, enzyme activity, or the effects of drug candidates on cell viability and proliferation. Their ability to provide quantitative data makes them essential for identifying potential drug candidates and optimizing their pharmacological properties.

• Imaging of Disease Processes

Fluorescent probes are instrumental in studying various disease processes, including cancer, neurodegenerative disorders, and infectious diseases. In cancer research, fluorescent probes can be used to visualize tumor microenvironments, monitor cancer cell proliferation, and assess the efficacy of therapeutic interventions. In neurobiology, they enable the study of neuronal activity, synaptic interactions, and the effects of neurotoxic compounds, contributing to the understanding of neurodegenerative diseases. Additionally, fluorescent probes can be designed to target specific pathogens, aiding in the visualization and characterization of infectious agents.

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