阅读说明：本技术 基于激发态分子内质子转移型的化合物及其制备方法和应用 (Compound based on excited state intramolecular proton transfer type and preparation method and application thereof ) 是由 唐本忠 倪侦翔 李美雪 林荣业 于 2018-07-24 设计创作，主要内容包括：本发明涉及一种基于激发态分子内质子转移型化合物及其制备方法和应用。本发明合成的一系列基于激发态分子内质子转移型荧光化合物易于合成,并且显示出良好的聚集诱导发光特性,作为生物荧光探针不仅能对脂滴、内质网、溶酶体细胞器进行特异性标记,在光照条件下还能产生消灭癌细胞的活性氧簇,对于推进生物荧光检测技术和光动力疗法的诊疗一体化,具有相当重要的意义与价值。(The invention relates to an excited state intramolecular proton transfer type compound and a preparation method and application thereof. A series of fluorescent compounds synthesized by the invention based on excited state intramolecular proton transfer are easy to synthesize, show good aggregation-induced emission characteristics, can be used as a biological fluorescent probe for specifically marking lipid drops, endoplasmic reticulum and lysosome organelles, can generate active oxygen clusters for killing cancer cells under the illumination condition, and have important significance and value for promoting the diagnosis and treatment integration of a biological fluorescent detection technology and a photodynamic therapy.)

1. A compound based on an excited intramolecular proton transfer type, wherein the structural formula of the compound is as shown in formula I:

wherein Ar is₁A group selected from any of the following structural formulae:

Ar₂is H or a group selected from any of the following structural formulae:

Ar₃a group selected from any of the following structural formulae:

wherein R is₁And R₂Each independently selected from H, CF₃Cyano, nitro, halogen, hydroxy, amino, optionally substituted alkyl, alkylaminoalkyl, alkoxy, alkylthio, alkenyl, alkynyl, cycloalkyl, cycloalkyloxy, cycloalkylthio, acyl, aryl, heterocyclyl, heteroaryl, heterocycloalkyl, monoalkylamino, or dialkylamino; the anion X is selected from halide ion, hydroxyl ion, trifluoromethanesulfonate ion, nitrate ion and hexafluorophosphate ion.

2. The compound of claim 1, wherein Ar is Ar₁、Ar₂And Ar₃Each independently selected from the group of any of the following structural formulae:

3. the compound of claim 2, wherein Ar is Ar₁、Ar₂And Ar₃Each independently selected from:

4. the compound of claim 1, wherein the compound is selected from one of the structural formulas of formulae I 'to III':

5. the compound of claim 4, wherein the compound is selected from one of the following structural formulas:

6. a process for the preparation of a compound according to any one of claims 1 to 5, comprising: placing a ketone or aldehyde structural formula compound shown in a formula II and a m-hydroxy aldehyde hydrazone aromatic compound shown in a formula III in an organic solvent, and stirring at room temperature to obtain a reaction product shown in a structural formula I; wherein formula II and formula III are as follows:

7. the preparation method of claim 6, wherein the obtained reaction product of the structural formula shown in formula I is subjected to ice bath, filtered to obtain a solid crude product, washed by using a cold organic solvent, and filtered to obtain a solid finished product.

8. Use of a compound according to any one of claims 1 to 5 for the preparation of a bioluminescent probe for the specific labelling of an organelle; the organelles include lipid droplets, lysosomes, and the endoplasmic reticulum.

9. Use of a compound according to any one of claims 1 to 5 for the preparation of a bio-fluorescent probe for labeling organelles of tumor cells, normal cells, freshwater algae and saltwater-like seaweeds, a fluorescent probe for recognizing bacteria, a light emitting diode, a photoelectric amplifier, an optical information storage, a liquid crystal display, an optical waveguide material, a biosensor or logic gate, a bio-probe for nondestructive readout.

10. Use of a compound according to any one of claims 1 to 5 for the preparation of an anticancer preparation for photodynamic therapy or an integrated diagnostic preparation for bioluminescence detection techniques and photodynamic therapy.

Technical Field

The invention relates to the technical field of detection and analysis, in particular to a compound based on excited intramolecular proton transfer and a preparation method and application thereof.

Background

Excited-state intramolecular proton transfer (ESIPT) refers to a proton transfer process that occurs between a proton donor and a proton acceptor in proximity inside an Excited-state molecule after the probe molecule is Excited by light. At this time, the enol structure of the excited state is rapidly converted to the keto isomer of the excited state by the ESIPT process, resulting in an emission of one large stokes shift. After decay to the ground state, the keto-isomer reverts to the previous enol structure by a reverse proton transfer process. The mechanism by which the emission of ESIPT-type dyes in the short wavelength region is fluorescence generated by a normal excited state and the emission in the long wavelength region is fluorescence of tautomers generated by the ESIPT process is well suited for designing an Aggregation-induced emission (AIE) -type fluorescent material. The main theoretical basis of the design strategy of the structure of the AIE luminescent material (AIE molecules, AIE fluorophores and AIEgen) which hardly emits light in solution and enhances the light in a solid state or an aggregation state is an intramolecular motion limited (RIM) mechanism model, namely, the intramolecular motion causes the energy of excited-state molecules to be attenuated in a non-radiative mode, and weak fluorescence emission is generated. When these molecules aggregate, the pinning action of each other restricts the movement inside the molecule, reduces the dissipation of energy, increases the energy ratio of light output, and thus exhibits a phenomenon of fluorescence enhancement. Through the development of the last two decades, the material with AIE characteristic is almost applied in the field of many luminescent materials, including intelligent materials for stimulus response and reversible sensing, liquid crystal or polarized light materials with tunable refractive index, high-efficiency organic light emitting diode devices and lighting materials, optical waveguide materials, biological sensing probes, and fluorescent probes for imaging organelles, viruses, bacteria or blood vessels in biological systems.

The organelles constitute the basic structure of the cell, which enables the cell to work and operate normally, and mainly include cell nucleus, mitochondria, endoplasmic reticulum, lipid drop, Golgi apparatus, ribosome and the like. For example, Lipid drop (Lipid drop) is an organelle for dynamically storing fat, the main function is to dynamically regulate the energy balance of cells, and the Lipid drop is closely related to organelles such as endoplasmic reticulum for synthesizing Lipid molecules, mitochondria between energy-powered vehicles and the like, provides energy for cells and biological organisms, and is an important energy reservoir in the cells. Various metabolic diseases, such as obesity, fatty liver, cardiovascular diseases and diabetes, neutral lipid storage diseases and type C niemann's disease, are often accompanied by abnormal lipid storage. Lysosomes (Lysosomes) are produced by Golgi body breakage, mainly digestion, and contain various hydrolases inside to decompose aged or damaged organelles, phagocytize and kill invading viruses or bacteria. The digestion organs within the cell, cellular autolysis, defense, and utilization of certain substances are all associated with the digestion of lysosomes. In recent years, the relationship between lysosomes and tumors has attracted increasing attention, including (1) the functions of carcinogens to regulate cell division and chromosomal aberrations; (2) some substances (such as croton oil) affecting the permeability of lysosome membranes have initiating factors promoting carcinogenesis and abnormal cell division; (3) certain products of lysosomal metabolic processes are the material basis for tumor cell proliferation; (4) before the carcinogenic substance enters cells and is integrated with chromosomes, the carcinogenic substance is always stored in lysosomes, and the like, and further research is needed. (III) the Endoplasmic reticulum (Endoplasmic reticulum) is a communicated lamellar gap-shaped or tubular system formed by biological membranes, mainly used as a pathway for intracellular substance transportation and provides a reaction area required by enzyme reaction in cells. The primary function of the rough endoplasmic reticulum is to synthesize secreted proteins; the synovial endoplasmic reticulum is mainly involved in the synthesis and transport of steroids and lipids, carbohydrate metabolism, and inactivation of hormones. In recent years, biological studies on important organelles have been highlighted, and the main focus of the research is to find and determine specific markers for various organelles (e.g., lipid droplets, lysosomes, endoplasmic reticulum), or for specific species, organs and tissues. Therefore, the method has many problems of high preparation cost, low stability, low sensitivity and the like in the prior art for the bioluminescent labeled probe, such as a commercialized dipyrrole probe. The invention provides a high-performance biological fluorescent probe which is simple, high in yield and capable of being produced in large scale, and the AIE characteristic shown by the ESIPT effect can be effectively applied to the diagnosis and treatment integrated research of a biological fluorescent detection technology and photodynamic therapy and the future market.

Disclosure of Invention

The invention aims to provide an excited-state intramolecular proton transfer type compound and a preparation method and application thereof, and solves the problems of high preparation cost, high self-absorption, low stability, low sensitivity and the like of a commercial labeled probe in the prior art.

The technical scheme adopted by the invention for solving the technical problem is as follows: a compound based on an excited intramolecular proton transfer type, the structural formula of the compound is shown as formula I:

wherein Ar is₁A group selected from any of the following structural formulae:

Ar₂is H or a group selected from any of the following structural formulae:

Ar₃a group selected from any of the following structural formulae:

wherein R is₁And R₂Each independently selected from H, CF₃Cyano, nitro, halogen, hydroxy, amino, optionally substituted alkyl, alkylaminoalkyl, alkoxy, alkylthio, alkenyl, alkynyl, cycloalkyl, cycloalkyloxy, cycloalkylthio, acyl, aryl, heterocyclyl, heteroaryl, heterocycloalkyl, monoalkylamino, or dialkylamino; the anion X is selected from halide ion, hydroxyl ion, trifluoromethanesulfonate ion, nitrate ion and hexafluorophosphate ion.

In the compound of the present invention, Ar is₁、Ar₂And Ar₃Each independently selected from the group of any of the following structural formulae:

in the compound of the present invention, Ar is₁、Ar₂And Ar₃Each independently selected from:

in the compounds of the present invention, the compound is selected from one of the structural formulae shown in formulae I 'to III':

in the compounds of the present invention, the compound is selected from one of the following structural formulae:

the invention also provides a preparation method of the compound, which comprises the following steps: placing a ketone or aldehyde structural formula compound shown in a formula II and a m-hydroxy aldehyde hydrazone aromatic compound shown in a formula III in an organic solvent (called mother solution), and stirring at room temperature to obtain a reaction product shown in a structural formula I; wherein formula II and formula III are as follows:

the specific process comprises the following steps:

further, in the preparation method of the invention, the obtained mother liquor of the reaction product with the structural formula shown in formula I is subjected to ice bath, and then filtered to obtain a solid crude product and a filtrate A, and then slightly washed by a cold organic solvent, and then filtered to obtain a solid finished product and a filtrate B. Among them, the organic solvent may be ethanol, methanol, acetone, etc., preferably ethanol.

Further, the crude product obtained by concentrating the filtrate A and the filtrate B under reduced pressure is added with cold organic solvent for washing, and then another batch of pure ESIPT type solid compound is obtained by filtering, thereby improving the yield of the product. Among them, the organic solvent may be ethanol, methanol, acetone, etc., preferably ethanol.

The invention also provides application of the compound in preparing a bioluminescent probe for specifically marking an organelle.

In this application, the organelles include lipid droplets, lysosomes, and the endoplasmic reticulum.

The invention also provides application of the compound in preparing a biological fluorescent probe for marking organelles of tumor cells, normal cells, freshwater algae and salt water algae, a fluorescent probe for identifying bacteria, a light-emitting diode, a photoelectric amplifier, an optical information memory, a liquid crystal display, an optical waveguide material, a biosensor or a logic gate and a biological probe for nondestructive reading.

The invention also provides the application of the compound in preparing an anticancer product for photodynamic therapy or a diagnosis and treatment integrated product for bioluminescence detection technology and photodynamic therapy, wherein the product can be a medicament, a pharmaceutical composition, a kit and the like.

The invention also provides an in vitro detection method for the cell specific markers, such as lipid droplets, lysosomes and endoplasmic reticulum of tumor cells such as HeLa cells and the like, which comprises the following steps:

A. treating cells with the above compounds;

B. cell imaging was detected by fluorescence microscopy or confocal laser scanning microscopy.

Terms and definitions

The term "halogen" represents fluorine, chlorine, bromine and iodine, in particular fluorine or chlorine, preferably fluorine.

The term "alkyl" represents a class of straight or branched alkyl groups containing only two atoms of carbon and hydrogen, for example C1-10 alkyl refers to straight or branched alkyl groups having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms, for example methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, hexyl, heptyl, octyl, nonyl, decyl, 2-ethylbutyl, 2-ethylhexyl, 2-butyloctyl. Preferably, the alkyl group contains 1, 2, 3 or 4 carbon atoms (C1-4 alkyl), such as methyl, ethyl, n-propyl or n-butyl.

The term "alkenyl" generally means straight or branched chain, containing from 2 to 20 carbon atoms and containing 1 or more double bonds, such as vinyl, propenyl, (E) -2-methylvinyl or (Z) -2-methylvinyl.

The term "alkynyl" generally means straight or branched chain, containing 2 to 12 carbon atoms and containing 2 or more double bonds, such as ethynyl, propynyl, 2-butynyl or 2-pentynyl.

The term "alkoxy" generally means a straight-chain or branched alkyl group bonded through an oxygen atom, wherein the term "alkyl" has the above definition, e.g., methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, isobutoxy, pentoxy or isomers thereof. Preferably, the alkoxy group contains 1 or 2 carbon atoms (C1-2 alkoxy), such as methoxy or ethoxy.

The term "alkylthio" generally means a straight or branched chain alkyl group bonded through a sulfur atom, wherein the term "alkyl" has the meaning as set forth above.

The term "cycloalkyl" generally means a straight or branched chain, saturated monocyclic hydrocarbon ring containing 3 to 8 carbon atoms, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl. Preferred cycloalkyl groups contain 5, 6 or 7 carbon atoms (C5-7 cycloalkyl), for example cyclopentyl, cyclohexyl or cycloheptyl.

The term "cycloalkyloxy" generally means a straight or branched chain cycloalkyl group bonded through an oxygen atom, where the term "cycloalkyl" has the definition as set forth above.

The term "cycloalkylthio" generally means a straight or branched chain cycloalkyl group bonded through a sulfur atom, where the term "cycloalkyl" has the definition as set forth above.

The term "aryl" generally means an aromatic or partially aromatic monocyclic, bicyclic or tricyclic hydrocarbon ring containing 6 to 14 carbon atoms, in particular a ring having 6 carbon atoms (e.g. phenylcyclyl or biphenylcyclyl), a ring having 9 carbon atoms (e.g. indenyl), a ring having 10 carbon atoms (e.g. dinaphthyl or naphthyl), a ring having 13 carbon atoms (e.g. fluorenyl) or a ring having 14 carbon atoms (e.g. onilyl). Preferably, aryl is a phenyl ring substituted with an "alkoxy" group containing 1 to 4 carbon atoms.

The term "heterocyclyl" generally means a saturated or partially saturated monocyclic or bicyclic hydrocarbon ring containing 5 to 8 carbon atoms and containing 1 to 3 heteroatom-containing groups selected from oxygen, sulfur or nitrogen. Such as furyl, thienyl, pyrrolyl, thiazolyl, thiadiazolyl, oxazolyl, imidazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyridazinyl or pyranyl. Preferably, the heterocyclyl is thienyl, pyridyl or a heterocyclyl substituted therewith having 1 to 4 carbon atoms (C1-4 alkyl).

The term "heteroaryl" generally means a group of compounds in which a benzene ring is fused with a heterocyclic ring, such as quinolinyl, benzothiazolyl, benzothiadiazolyl, benzoxazolyl, indolyl or purinyl.

The term "heterocycloalkyl" generally means a heterocyclic group containing from 3 to 7 carbon atoms and from 1 to 3 oxygen, sulfur or nitrogen heteroatoms, such as piperidinyl, hydroxypiperidinyl, phenanthrolinyl, piperazinyl, N-methylpiperazinyl, tetrahydropyrrolyl, tetrahydrofuranyl, tetrahydrothienyl, morpholino or thiazinyl. Preferred heterocycloalkyl groups are piperidinyl, N-methylpiperazinyl or morpholino.

The term "monoalkylamino" generally means an amino group (NH)₂) Wherein the term "alkyl" has the meaning as described above, is substituted for 1 hydrogen of (a). Such as methylamino, ethylamino, propylamino, butylamino or isomers thereof.

The term "dialkylamino" generally means an amine group (NH)₂) Wherein the term "alkyl" has the meaning as described above. Such as dimethylamino, diethylamino, dipropylamino, dibutylamino or isomers thereof. Preferably, the dialkylamino group is a dimethylamino group or a diethylamino group.

The term "heteroatom" is generally meant to contain 1 or more oxygen, sulfur or nitrogen atoms.

The term "optionally substituted" generally means that a hydrogen in the structure is replaced by the substituent. Unless otherwise specified, an optionally substituted group may have a substituent at each substitutable position of the group, or more than one position in the structure may be substituted.

As used herein, the numerical range "C1-10" and its included sub-ranges generally means having a defined number of 1-10 atoms, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 atoms; "C1-10" and the sub-ranges contained therein, inclusive, of the number of carbon atoms, generally means a group having a defined number of 1-10 carbon atoms, i.e., a group containing 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms.

Suitable substituents are typically selected from halogen, C1-6 alkyl, C1-6 alkoxy, C1-6 alkylthio, nitro, cyano, hydroxy, C1-6 dialkylamino, N-methylpiperazinyl or morpholino.

The Excited State Intramolecular Proton Transfer (ESIPT) based compound, the preparation method and the application thereof have the following beneficial effects: the simple high-yield mass-production serial characteristic-based fluorescent compounds provided by the invention are easy to synthesize, show good AIE characteristics, have specific markers with high overlapping efficiency on organelle lipid droplets, lysosomes and endoplasmic reticulum compared with commercialized marker probes as a biological fluorescent probe, can generate active oxygen clusters for killing cancer cells in an illumination environment, have potential value as a photodynamic therapy, and have important significance and value for the biological fluorescent detection technology in the field of diagnosis and treatment integration.

Drawings

FIG. 1 is a schematic illustration of a functional AIE targeting probe that is easy and mass to manufacture;

FIG. 2 is a normalized ultraviolet-visible light absorption spectrum and Photoluminescence (PL) spectrum of AP-DEA in a THF solution (concentration: 10. mu.M, excitation wavelength: 390 nm);

FIG. 3 is a normalized ultraviolet-visible absorption spectrum and Photoluminescence (PL) spectrum of AP-ML in THF solution (concentration: 10. mu.M, excitation wavelength: 360 nm);

FIG. 4 is a normalized ultraviolet-visible absorption spectrum and Photoluminescence (PL) spectrum of AP-PZ in THF solution (concentration: 10. mu.M, excitation wavelength: 360 nm);

FIG. 5 is a schematic view showing intramolecular proton transfer of AP-DEA in an excited state;

FIG. 6 is a graph of relative energy levels and molecular orbitals of AP-DEA in absorption, enol-emission and keto-emission;

FIG. 7 shows the ethanol content (f) of AP-DEA at different ratios_EVol%, vol%) in THF/ethanol mixture (concentration: 10 μ M, excitation wavelength: 380 nm);

FIG. 8 is the ketone emission vs. AP-DEAPL Peak intensity in enol-type emission (I/I)₀) Ethanol content (f) with THF/ethanol mixture_EVolume%) (concentration: 10 μ M, excitation wavelength: 380 nm);

FIG. 9 shows the ratio of ethanol (f) in AP-ML_EVol%, vol%) in THF/ethanol mixture (concentration: 10 μ M, excitation wavelength: 380 nm);

FIG. 10 is PL peak intensity (I/I) of keto versus enol emission of AP-ML₀) Ethanol content (f) with THF/ethanol mixture_EVolume%) (concentration: 10 μ M, excitation wavelength: 380 nm);

FIG. 11 shows the AP-PZ ratios (f) at different ethanol contents_EVol%, vol%) in THF/ethanol mixture (concentration: 10 μ M, excitation wavelength: 380 nm);

FIG. 12 is the PL peak intensity (I/I) of keto versus enol emission for AP-PZ₀) Ethanol content (f) with THF/ethanol mixture_EVolume%) (concentration: 10 μ M, excitation wavelength: 380 nm);

FIG. 13 is a graph of fluorescence decay curves for AP-DEA, AP-ML and AP-PZ in THF (IRF is the instrument response function);

FIG. 14 is a graph showing fluorescence decay curves of AP-DEA, AP-ML and AP-PZ in a solid powder state (IRF is an instrument response function);

FIG. 15 is a crystal structure of a single molecule in AP-DEA crystal analysis;

FIG. 16 is a diagram showing a structure of a package in the analysis of AP-DEA crystals;

FIG. 17 is a diagram showing arrangement between molecules in an AP-DEA crystal analysis;

fig. 18 is an intermolecular force diagram in AP-DEA crystal analysis (bond length unit:)；

FIG. 19 is the crystal structure of a single molecule in AP-ML crystal analysis;

FIG. 20 is a diagram of a crystal pack structure in AP-ML crystal analysis;

FIG. 21 is a diagram of intermolecular alignment in AP-ML crystal analysis;

fig. 22 is an intermolecular force diagram in AP-ML crystal analysis (bond length unit:)；

FIG. 23 is the crystal structure of a single molecule in the AP-PZ crystal analysis;

FIG. 24 is a diagram of a crystal pack structure in AP-PZ crystal analysis;

FIG. 25 is a view showing arrangement between molecules in AP-PZ crystal analysis;

fig. 26 is an intermolecular force diagram in AP-PZ crystal analysis (bond length unit:

)；

FIG. 27 is a data analysis diagram of photodynamic therapy with AP-DEA, AP-ML and AP-PZ; DCFH is an indicator of reactive oxygen species; excitation wavelength: 480 nm; emission wavelength: 525 nm;

FIG. 28 is a graph of confocal laser scanning microscopy of HeLa cells in bright and dark fields co-stained with AP-DEA and a commercial dipyrrole probe; excitation wavelength: 405nm (AP-DEA) and 488nm (dipyrrole); an emission filter: 490-600nm (AP-DEA) and 570-700nm (dipyrrole); the scale used for the images was 20 μm;

FIG. 29 is a graph of confocal laser scanning microscopy of HeLa cells in the bright and dark fields of AP-ML co-staining with a commercial ER-Tracker Red probe; excitation wavelength: 405nm (AP-ML) and 560nm (ER-Tracker Red); an emission filter: 490-600nm (AP-ML) and 570-700nm (ER-Tracker Red); the scale used for the images was 20 μm;

FIG. 30 is a graph of confocal laser scanning microscopy of HeLa cells in bright and dark fields co-stained with AP-PZ and a commercial LysoTracerDeep Red probe; excitation wavelength: 405nm (AP-PZ) and 560nm (LysoTracerDeep Red); an emission filter: 490-600nm (AP-PZ) and 570-700nm (LysoTracker Deep Red); the scale used for the images was 20 μm;

FIG. 31 is a block diagram of cell viability of HeLa cells incubated under light and dark using different concentrations (0-10 μ M) of AP-DEA probe;

FIG. 32 is a block diagram of cell viability of HeLa cells incubated under light and dark conditions using different concentrations (0-10 μ M) of AP-ML probe;

FIG. 33 is a block diagram of cell viability of HeLa cells incubated under light and dark conditions using different concentrations (0-10 μ M) of AP-PZ probe;

FIG. 34 is a graph showing the change in PL of AP-DEA and commercial dipyrrole probe-labeled HeLa cells with increasing irradiation time; excitation wavelength: 405nm (AP-DEA) and 488nm (dipyrrole); an emission filter: 490-600nm (AP-DEA) and 570-700nm (dipyrrole); irradiation time: 22.4 seconds/scan; laser power: 0.3. mu.W.

Detailed Description

The excited state based intramolecular proton transfer type compound of the present invention, its preparation method and application are further described below with reference to the accompanying drawings and examples:

the invention adopts an m-hydroxy aldehyde hydrazone aromatic structure as a substrate of an Excited State Intramolecular Proton Transfer (ESIPT) type compound, as shown in figure 1, develops an ESIPT type compound system which is easy to synthesize and has aggregation-induced emission characteristics, successfully performs a specific labeling technology on organelle lipid drops, lysosomes and endoplasmic reticulum as a bioluminescent probe, provides a fluorescent probe with good light stability, good specific labeling and good biocompatibility in the technical field of bioluminescence detection, and has important significance and value in the fields of bioluminescence detection technology and the like.

The following is a detailed description of specific examples.