Fluorescence is a luminescence phenomenon of photoluminescence. When certain substances are excited by light, electricity, magnetism, chemical energy, the electrons absorb energy and transition from the ground state to the excited state, and the electrons in the excited state are unstable. It will return to the ground state through radiation transition and non-radiation transition. The decay process of radiation transition is accompanied by the emission of photons, fluorescence, and phosphorescence are produced. Non-radiation transition includes vibrational relaxation, internal conversion, and intersystem crossing. Non-radiative transitions will cause energy loss, the energy of the emitted photon is generally less than the absorbed photon’s energy. Accordingly, the emission spectrum wavelength of fluorescent substances is usually greater than the absorption spectrum wavelength.
一 Conditions that fluorescent probes need to mee
1.Easy to synthesize and purify, high yield, safe and non-toxic.
2.Good stability and solubility, especially fat solubility through the membrane.
3.It can specifically bind to the labeled substance through the physical-chemical effect, the labeling conditions are mild. The residue and by-products are easy to remove.
4.High fluorescence quantum yield, large molar extinction coefficient, strong anti-bleaching ability. Fluorescence has a clear contrast with the background. In addition. The excitation and emission wavelength can effectively avoid the background interference of cell autofluorescence.
二 Advantages of Near Infrared (NIR) Fluorescent Probes
1.Near-infrared light detection sample has strong penetrability, high imaging resolution, high detection sensitivity, and high signal-to-noise ratio.
2.In the visible light region, certain components of biological tissues will self-excited to produce autofluorescence. And the scattered light intensity of the sample is relatively high, which seriously interferes with fluorescence detection and imaging tracing. Near-infrared fluorescence autofluorescence background is low.
三 NIR method for detecting intracellular active small molecule (RSM)
RSMs in cells tend to have a short lifespan, high reactivity, very low concentration, sensitivity to the environment, and non-fluorescence. Therefore, it is must be aided by the specific capture label of the fluorescent probe to have near-infrared fluorescence properties.
四 The reason fluorescent probes widely used for biomacromolecule study
Molecular fluorescent probes are widely used in the study of surface-induced conformational changes on the contact surface of proteins and other biological macromolecules. Among the direct analysis methods, the fluorescence analysis method is widely used and superior to the indirect analysis method because it does not destroy the balance of binding and closest to the true existence of the reaction.
五 Fluorescent probes as energy donors for photosensitizers drugs
The fluorescent nanoprobe can not only use as a drug carrier to combine with the photosensitizer drug molecule to form a multifunctional nanomaterial, but also can be used as an energy donor of the photosensitizer drug molecule to improve its singlet oxygen generation efficiency.
Absorption, fluorescence emission spectra of the photosensitizer, and the fluorescent dye are less overlapped. It effectively avoids fluorescence quenching caused by energy transfer between molecules, reduced singlet oxygen generation efficiency, fluorescence imaging, etc. At the same time as fluorescence imaging, while the singlet oxygen generated by the forced excitation of the photosensitizer may also photochemically react with the excited fluorescent probe and cause fluorescence quenching. Or it may expose the potentially toxic side effects of quantum dots during imaging. Therefore, designing an appropriate nanocarrier configuration to physically isolate the photosensitizer drug molecule from the fluorescent probe is an effective way to avoid such photochemical reactions.
六 Composition and function of fluorescent molecular probe
1.Receptor
It selectively binds with the object (Analyte) and causes changes in the chemical or biological microenvironment where the probe is.
2.Fluorophore
The chemical or biological microenvironment change caused by the combination of the recognition group and the analyte is converted into a signal that is easy to perceive(color changes)or detectable by the instrument.
Small-molecule fluorescent probes generally use organic small molecule fluorophores, which include Anthracene, Coumarin, Fluorescein, BODIPY, Naphthalimide, Rhodamine, Cyanine, and more. The emission wavelength range of their derivatives covers almost all visible light regions(400-800 nm). And the coverage of blue and green light to red light and near-infrared light (650-900 nm)can be achieved by appropriately modifying these fluorophores. In addition, light-emitting quantum dots, up-conversion nanomaterials, macromolecule polymer fluorescent materials, and fluorescent proteins can also be used as signal groups in fluorescent probes.
3.Spacer
Connect the fluorophore and the recognition group to effectively convert the identification information into a fluorescent signal such as the change of fluorescence intensity, the shift of fluorescence spectrum, the change of fluorescence lifetime. so as to realize the treatment Effective detection of test objects. Not all probes have linking groups.
七 Detectable of biosome substance with the fluorescent probe
1.Proton(H+).
2.Free radicals and other reactive nitrogen and oxygen species (ROS, RNS).
3.Gas signal molecules(NO, CO, H2S,…)
4.Heavy metal pollution (Cd2+. Hg2+.Pb2+…)
5.Anions(Cl-, HCO3-, H2PO4-, HPO42-…)
6.Transition metal ions(Fe2+/Fe3+, Zn2+, Cu+/Cu2+…)
7.Alkali metal and alkaline earth metal ions( K+, Na+, Ca2+, Mg2+…)
8.Biological macromolecules such as DNA, RNA, and protein.
9.Small organic molecules such as peptides, glucose, maltose, etc.
八 Types of fluorescent probes
According to the change of the fluorescent signal after interacting with the guest, it can be divided into intensity change type fluorescent probe and proportional meter type fluorescent probe. The intensity change type probe is divided into quenching type (ON-OFF) and enhanced type (OFF-ON) Fluorescent probe.
The intensity change type fluorescent probe realizes the detection of the guest species according to the change of fluorescence intensity. Since the fluorescence intensity is also affected such as the concentration of the probe, the efficiency of the excitation light source, and the environment where the probe is, etc this type of probe also has obvious limitations in the quantitative detection of guest species. At present, most fluorescent probes are enhanced fluorescent probes.
The ratiometric fluorescent probe itself also emits a certain wavelength of fluorescence. Its obvious advantage is that it can eliminate the interference of most environmental factors by the ratio of the fluorescence intensity at the two wavelengths, so as to realize the quantitative detection of the tested species when the probe concentration is unknown.
九 Design mechanism of fluorescent probe
Traditional molecular probe design principles include Photoinduced Electron Transfer (PET), Intramolecular Charge Transfer (ICT), Twisted Intramolecular Charge Transfer (TICT), Metal to Ligand Charge Transfer(MLCT), Electron Energy Transfer (EET), Fluorescence Resonance Energy Transfer (FRET), Excited-State Intramolecular Proton Transfer (ESIPT), Excited-state Excimer/Exciplex Formation, etc. Emerging mechanisms include Aggregation induced emission (AIE), Upconversion Luminescence (UCL), etc.
十 Photoelectron transfer (PET)
Generally speaking, the photo-induced electron transfer (PET) process is divided into two types. One is the transfer of electrons from the electron donor to the excited state fluorophore (electron acceptor), and the excited state fluorophore is reduced to cause fluorescence quenching. The other is the transfer of electrons from the excited state fluorophore (electron donor) to the excited state fluorophore (electron acceptor). The electron acceptor, the excited state fluorophore is oxidized to cause fluorescence quenching. When the object is not bound, the PET between the fluorophore and the acceptor will quench the fluorescence. After the object is bound, the PET process is inhibited, and the fluorophore emits fluorescence.
十一 Intramolecular Charge Transfer (ICT)
Intramolecular charge transfer is also called Photo-induced Charge Transfer (PCT), and it is also an important method for designing a ratiometric fluorescent probe. The recognition group of this type of fluorescent probe is directly connected to the fluorophore, and it can also be understood that certain atoms or groups that make up the fluorophore directly participate in the recognition of the guest.
十二 Fluorescence resonance energy transfer (FRET)
Fluorescence resonance energy transfer (FRET) is a type of energy transfer (ET). Energy transfer refers to the process of energy transfer from the donor chromophore to the acceptor chromophore in the molecule. FRET efficiency is often changed by adjusting the degree of spectral overlap and the donor-acceptor distance.
十三 Excited Intramolecular Proton Transfer (ESIPT)
ESIPT phenomenon is means a compound molecule from ground-state transition to the excited state, and then proton transferred to adjacent N, S, O heteroatom in the molecule by an intramolecular hydrogen bond to form a process of the corresponding tautomer.
十四 Excimer/Exciplex Formation
Excimer(Er)can be defined as an association formed by the interaction of an excited fluorophore and a ground state fluorophore of the same structure. Similarly, if a fluorophore in an excited state and a fluorophore with a different structure in the ground state form a complex, it is called an excited state complex.