Long afterglow luminescent organic microspheres, preparation method and application thereof

Abstract

The present invention relates to a long afterglow luminescent organic microsphere, which comprises A) at least one light absorber and B) at least one luminescent agent, wherein the luminescent agent is a monomeric non-polymeric compound and has a molecular weight of less than 10000 g mol ‑1 , C) at least one photochemical buffer of formula (I), and #imgabs0#D) a carrier medium for absorbing components A) to C); wherein the light absorber and the luminescent agent are compounds with different structures, and the content of the carrier medium is 30% to 99%, more preferably 35% to 95% and most preferably 40% to 90% based on the total mass of the four components A) to D). The organic microspheres according to the present invention are particularly suitable for immunochromatographic detection technology. In addition, the present invention also relates to test paper, probe and detection method for immunochromatographic detection.

Description

Technical Field

[0001] The invention relates to a long afterglow luminescent organic microsphere, a preparation method and application thereof, and in particular to application of the long afterglow luminescent organic microsphere in immunochromatographic detection technology. Background Art

[0002] Immunochromatography assay (ICA) or lateral flow assay (LFA) was first used to detect human chorionic gonadotropin. With the development of labeling technology, it is also widely used in medical testing, environmental monitoring and food safety. Immunochromatography effectively combines chromatography technology with antigen-antibody immune reaction technology. In immunochromatography, an immunochromatography test strip is often used. Its main structure is a polyvinyl chloride base plate as a support, on which a sample pad (such as glass cellulose membrane), a binding pad (such as glass cellulose membrane), a nitrocellulose membrane (NC membrane) and a water-absorbing pad are arranged. When the sample flows under capillary action, the antigen first binds to the probe on the binding pad to form an immune complex. As the liquid continues to flow, the immune complex will be enriched and retained on the detection line (T line) of the NC membrane. The probe that has not formed an immune complex will be retained by the quality control line (C line), and finally read by the naked eye or instrument.

[0003] The widely used immunochromatographic test strips mainly include colloidal gold immunochromatographic test strips and fluorescent immunochromatographic test strips. Traditional immunochromatographic detection technology mainly uses colloidal gold as the output signal. Due to the insufficient optical density of the colloidal gold probe, the detection sensitivity is low, and it is difficult to quantify, which cannot meet the requirements of clinical diagnosis. Subsequently, fluorescent probes have been developed in immunochromatographic detection, among which luminescent probes based on fluorescent dyes are the most widely used. Fluorescent dyes are usually directly labeled on antibodies or microspheres coated with antibodies, or embedded in microspheres and then modified with antibodies, and light sources such as ultraviolet light are used to excite the dye to emit light. However, in the detection process of luminescent signals, samples such as blood have spontaneous fluorescent signal interference, and the irradiation of the excitation light source will also produce interference with the light signal. These unfavorable factors will seriously affect the accuracy and stability of the detection signal.

[0004] Long afterglow luminescent materials are a special type of luminescent materials that can continue to emit light for a long time after the excitation light source is removed. In the prior art, the luminescence lifetime of long afterglow luminescent materials is usually greater than one hundred milliseconds, and they have important application value in the fields of biomedicine and life sciences. At present, commercialized long afterglow luminescent materials are generally inorganic long afterglow materials such as aluminates, silicates, titanates or sulfides doped with rare earths or transition metals. Using long afterglow luminescent materials as signal indicator probes in immunochromatographic detection can avoid interference from excitation light and background fluorescence. For example, CN105929155A discloses an immunochromatographic test paper based on long afterglow and a detection method thereof, which uses inorganic long afterglow luminescent materials to effectively eliminate interference signals and improve the detection sensitivity and quantitative accuracy of the object to be detected.

[0005] These inorganic long afterglow luminescent materials based on rare earth or transition metal doping are usually prepared by high-temperature solid-phase calcination. High-temperature solid-phase synthesis is the most common and effective production method for this type of material, mainly because high temperature is conducive to obtaining better long afterglow luminescence properties. The luminescence performance of inorganic long afterglow materials synthesized by other non-high-temperature methods is significantly reduced and difficult to obtain widespread application. However, the high-temperature solid-phase reaction conditions are harsh and energy-intensive, it is difficult to control the uniform morphology of the material, and the particle size of the material is generally large. Although the materials synthesized by the high-temperature solid-phase method can be further reduced by grinding and screening, the luminescence brightness drops sharply after the particle size is reduced to the nanometer level (for example, when the particle diameter is less than 1000nm). For example, if the commercial micron-sized inorganic long afterglow powder is ground to the 100nm level, the luminescence brightness of the microspheres may be reduced by two orders of magnitude.

[0006] Therefore, it is difficult to prepare inorganic long afterglow luminescent microspheres, and the luminescence of inorganic long afterglow luminescent microspheres is weak, making it difficult to obtain long afterglow luminescent signals that are clearly visible to the naked eye. The detection of weak signals requires the use of complex professional equipment, and the effect is also limited when applied to immunochromatographic detection. Summary of the invention

[0007] In view of the above-mentioned defects of the prior art, the present invention provides a long afterglow luminescent organic microsphere with higher brightness and unaffected afterglow time or even longer afterglow time.

[0008] Different from the inorganic long afterglow luminescent materials based on photophysical processes in the prior art, the long afterglow luminescent materials of the present invention are based on an organic system, which utilizes the characteristics of photochemical reactions to introduce photochemical reactions between light energy input and light energy output, and organically integrates photophysics and chemistry. In the long afterglow luminescent materials based on the organic system, the luminescence process may involve photochemical interactions between a variety of chemical substances, wherein after a series of photochemical energy conversion and metabolic processes, the input excitation light energy is finally released in the form of luminescence, thereby achieving long afterglow luminescence. The energy conversion and metabolic processes include energy input, energy caching, energy extraction, energy transfer and energy release. Through photochemical reactions, the originally very rapid photon radiation transition process (nanoseconds to microseconds) is changed, and the energy is slowly released and finally emitted in the form of light energy, thereby obtaining an ultra-long luminescence time (milliseconds to hours), which greatly improves the limitation of the short luminescence life of organic molecules.

[0009] Furthermore, the inventors found that long afterglow materials based on organic systems can not only obtain afterglow with longer duration and higher brightness, but also some long afterglow luminescent organic materials are particularly suitable for making long afterglow luminescent microspheres without losing afterglow brightness and duration, and are therefore particularly suitable for immunochromatographic detection technology. In many cases, the afterglow brightness can be increased to a level visible to the naked eye or above, and the long afterglow luminescent signal can be collected and analyzed by common electronic devices such as mobile phones, which greatly increases the practicality of the material.

[0010] In this application, the term photochemical reaction refers to a series of chain reactions, including photochemical addition, photooxidation, photochemical dissociation and bond-breaking and recombination processes.

[0011] In this application, unless otherwise specified, the terms "long afterglow luminescent organic microspheres", "long afterglow materials" and "long afterglow microspheres" have the same meaning and can be used interchangeably.

[0012] Therefore, in the first aspect, the present invention provides a long afterglow luminescent organic microsphere comprising

[0013] A) at least one light absorber,

[0014] B) at least one luminescent agent, said luminescent agent being a monomeric, non-polymeric compound and having a molecular weight of less than 10,000 gmol -1 ,

[0015] C) at least one photochemical buffering agent of formula (I),

[0016]

[0017] wherein formula (I) is described in detail below, and

[0018] D) a carrier medium for adsorbing components A) to C);

[0019] The light absorber and the luminescent agent are compounds with different structures, and the content of the carrier medium is 30% to 99%, more preferably 35% to 95% and most preferably 40% to 90% based on the total mass of the four components A) to D).

[0020] Preferably, the long afterglow luminescent organic nanoparticles consist of components A) to D).

[0021] In a second aspect, the present invention provides a probe comprising the above-mentioned long afterglow luminescent organic microspheres.

[0022] In a third aspect, the present invention provides a method for preparing the above-mentioned long afterglow luminescent organic microspheres.

[0023] In a fourth aspect, the present invention provides a method for preparing a probe comprising the long afterglow luminescent organic microspheres.

[0024] In a fifth aspect, the present invention provides a test strip for immunochromatographic detection.

[0025] In a sixth aspect, the present invention provides a method for immunochromatographic detection using the long afterglow luminescent organic microspheres as described above.

[0026] Further aspects of the invention are presented in the further independent and dependent claims.

[0027] In addition to the above advantages, the composition of the long afterglow luminescent microspheres according to the present invention is flexible, and the composition and properties of the materials can be designed according to actual needs, and flexible and diverse nanostructures can be obtained, and the luminescent properties can be tailored. The wavelength of the charged excitation light and the wavelength of the long afterglow luminescence can be adjusted separately, and the combination of the light absorber and the luminescent agent can be adjusted and replaced very conveniently, so as to efficiently achieve colorful long afterglow luminescence.

[0028] Preferably, the long afterglow luminescent nanomaterial according to the present invention does not contain or contains very little inorganic long afterglow components such as SrAl2O4:Eu 2+ ,Dy 3+ , for example not more than 0.1 wt. %, based on the material mixture.

[0029] The particle size of the long afterglow luminescent microspheres of the present invention can reach 5nm-1000nm, more preferably 50nm-800nm, and most preferably the nanoparticle size is 100nm-500nm. In the context of the present invention, the morphology and particle size of all particles of the microspheres can be characterized by electron microscope images, and the average diameter of the microspheres obtained by multiple measurements is recorded as the particle size. Such characterization methods of microspheres are known to technicians and can be measured, for example, using scanning electron microscopes (SEM) and transmission electron microscopes (TEM) instruments.

[0030] Under the same test conditions, the luminescence intensity of the long afterglow luminescent organic microspheres according to the present invention can far exceed that of the nanoscale commercial inorganic long afterglow material SrAl2O4:Eu 2+ ,Dy 3+ In particular, the long afterglow luminescent organic microspheres according to the present invention can continue to emit light after the excitation light is turned off, and the long afterglow luminescence time can reach 100ms-3600s, preferably 500ms-1200s, more preferably 1s-600s, and most preferably 2s-60s. The long afterglow luminescence brightness of the long afterglow material according to the present invention can reach 0.1mcd m -2 –10000mcd m -2 , preferably at 0.32 mcd m -2 –8000mcd m -2 , more preferably at 1 mcd m -2 –5000mcd m -2 Based on the above properties, the long afterglow microspheres of the present invention can provide a complete material basis for immunochromatographic detection technology.

[0031] In addition, the long afterglow microspheres of the present invention can be used to prepare immunochromatographic nanoprobes with high afterglow brightness, and test strips for immunochromatographic detection with high stability, good repeatability and high sensitivity can be obtained. The detection objects include fungal toxins, pathogenic bacteria, viruses, inflammatory factors and tumor markers.

[0032] Light absorbers and luminescent agents

[0033] In the present application, light absorbers and luminescent agents are known in the prior art. Light absorbers generally refer to substances that can absorb and capture light energy from natural or artificial light sources. The selection range of light absorbers includes traditional photosensitizers and other energy donor materials. Luminescent agents generally refer to substances that can ultimately emit energy in the form of light energy. Luminescent agents can be luminescent substances that can produce fluorescence or phosphorescence. The relevant luminescent molecular groups are known in themselves, and reference can be made to, for example, the review paper Nature Methods, 2005, 2, 910-919 by Jeff W. Lichtman et al.

[0034] In order to achieve the beneficial effects of the long afterglow material of the present invention, especially, for example, improving the afterglow intensity and time, the two components of the luminescent agent and the light absorber are clearly distinguished in the composition of the present invention, so that each of them respectively takes on the role of absorbing light energy and releasing light energy, thereby realizing the energy utilization path of energy input, energy caching and energy output after being combined with a specifically selected photochemical buffer. This also means that, in an advantageous embodiment, a compound that has both a light absorbing group and a luminescent group in its structure so that the same molecule can perform two functions is not a luminescent agent or a light absorber according to the present invention, and the excellent technical effect of the present invention will not be obtained. On the one hand, such a compound is equivalent to packaging and binding the light absorber and the luminescent agent together with their properties, and it is impossible to adjust the excitation and luminescence properties of the long afterglow material separately. For example, when a compound is selected according to the actual excitation and charging needs, the luminescence properties of the material are also fixed, and vice versa. On the other hand, such a compound is equivalent to fixing the ratio of the light absorber to the luminescent agent to, for example, 1:1, and it is impossible to adjust the intensity of the absorption and the level of luminescence at the same time. Moreover, there are relatively few materials that have both high-efficiency light absorption and high-efficiency luminescence functions, which limits the richness of long afterglow materials.

[0035] In the long afterglow luminescent material according to the present invention, the selection of the light absorber and the luminescent agent has certain rules and standards. Generally speaking, a compound with a larger molar absorption coefficient is selected as a light absorber, such as a photosensitizer or an energy donor dye; and a compound with a higher luminescence quantum efficiency is selected as a luminescent agent, such as a luminescent dye. In addition, the absorption peak of the light absorber should overlap as little as possible with the emission peak of the luminescent agent to avoid the adverse effect of the long afterglow luminescence being absorbed by the absorber and weakened.

[0036] The inventors of the present application have found that in the long afterglow luminescent organic microspheres according to the present invention, especially in the immunochromatographic detection technology, from the perspective of improving the luminescent brightness or the luminescent signal intensity, the light absorber and the luminescent agent should be advantageously selected from at least one compound of different molecular formulas or different structures as follows: porphyrin and phthalocyanine dyes, metal complexes, acene compounds, fluoroboron dipyrrole compounds (BODIPY), quantum dots (QDs), graphene, and derivatives or copolymers of these compounds. However, advantageously, the luminescent agent used in the present invention is a monomeric non-polymeric compound and its molecular weight is less than 10000 g mol -1In the context of this application, the molecular weight refers to the weight average molecular weight of the compound, which can be measured by mass spectrometry, gas chromatography, or liquid chromatography. The instrument that can be used can be, for example, a mass spectrometer or a liquid chromatography-mass spectrometer. Here, the non-polymeric compound refers to a compound structure that does not contain more than two repeating units obtained by polymerization or oligomerization.

[0037] More advantageously, particularly from the perspective of immunochromatographic detection technology, the light absorber and the light emitting agent preferably used in the long afterglow organic microspheres of the present invention are selected from the following.

[0038] (1) Light absorber

[0039] Preferably, the light absorber can be selected from porphyrin and phthalocyanine dyes, transition metal complexes, quantum dots (QDs), and derivatives or copolymers of these compounds. These compounds themselves are known to those skilled in the art, and some non-limiting examples of light absorbers are mentioned below.

[0040] As porphyrin dyes and their complexes, for example, the following compounds may be mentioned:

[0041]

[0042]

[0043] As phthalocyanine dyes and their complexes, for example, the following may be mentioned:

[0044]

[0045]

[0046] In the structural formulas of the light absorber compounds shown above,

[0047] X represents a halogen such as F, Cl, Br, I; and

[0048] M = metal elements, such as Al, Pd, Pt, Zn, Ga, Ge, Cu, Fe, Co, Ru, Re, Os, etc.

[0049] Each substituent R such as R 1-24 represents H, hydroxyl, carboxyl, amino, thiol, ester, aldehyde, nitro, sulfonic acid, halogen, or an alkyl, alkenyl, alkynyl, aryl, heteroaryl with N, O or S, alkoxy, alkylamino, or a combination thereof having 1 to 50, preferably 1 to 24, such as 2 to 14 carbon atoms. 1-24 Each is independently selected from methoxy, ethoxy, dimethylamino, diethylamino, methyl, ethyl, propyl, butyl, tert-butyl, phenyl or a combination thereof.

[0050] Transition metal complexes which can be used as light absorbers are known per se and are preferably complexes of porphyrin and phthalocyanine dyes such as those indicated above.

[0051] Suitable quantum dot materials include, for example, graphene quantum dots, carbon quantum dots, and heavy metal quantum dots.

[0052] Heavy metal quantum dots include, for example, Ag2S, CdS, CdSe, PbS, CuInS, CuInSe, CuInGaS, CuInGaSe, and InP quantum dots. They may be wrapped with a shell layer to form a core-shell structure, and the shell layer may be one or more of Ag2S, CdS, CdSe, PbS, CuInS, CuInSe, CuInGaS, and CuInGaSe, or may be a ZnS layer.

[0053] Preferably, the quantum dots are modified with surface ligands, which may be, for example, oleic acid, oleylamine, octadecene, octadecylamine, n-dodecyl mercaptan, and combinations thereof. In some more favorable cases, the ligands on the surface of the quantum dots are partially replaced with triplet-containing molecular structures, such as carboxyanthracene, carboxytetracene, carboxypentacene, aminoanthracene, aminotetracene, aminopentacene, mercaptoanthracene, mercaptotetracene, mercaptopentacene, and the like, by a ligand exchange strategy.

[0054] In a more preferred embodiment, the light absorber is preferably selected from porphyrin and phthalocyanine complexes, quantum dots (QDs), and derivatives of these compounds, such as one or more of the following exemplary compounds:

[0055]

[0056]

[0057] There are also quantum dot materials such as graphene quantum dots, CdSe quantum dots and PbS quantum dots.

[0058] (2) Luminescent agent

[0059] Preferably, the luminescent agent can be selected from iridium complexes, rare earth complexes, acene compounds, boron dipyrrolidone compounds (BODIPY), and derivatives and copolymers of these compounds.

[0060] As the boron dipyrrole compounds (BODIPY), for example, the following compounds may be mentioned:

[0061]

[0062]

[0063] As the acene compounds, there may be mentioned, for example, the following compounds:

[0064]

[0065] In the structural formulas of the luminescent compounds shown above,

[0066] n = an integer greater than or equal to 0, such as 0, 1, 2, and 3;

[0067] Each substituent R such as R 1-16 represents H, hydroxyl, carboxyl, amino, thiol, ester, aldehyde, nitro, sulfonic acid, halogen, or an alkyl, alkenyl, alkynyl, aryl, heteroaryl with N, O or S, alkoxy, alkylamino group having 1 to 50, preferably 1 to 24, such as 2 to 14 carbon atoms, or a combination thereof. Preferably, the group R is such as R 1-16 Selected from methoxy, ethoxy, dimethylamino, diethylamino, methyl, ethyl, propyl, butyl, tert-butyl, phenyl; or combinations thereof.

[0068] In the iridium complex suitable as a luminescent agent, the composition of the ligand can be a combination of one or more different ligands, and its structural schematic diagram and the types of some CN, NN, OO and ON ligands are exemplarily shown as follows (the CN, NN, OO and ON ligands shown therein are simplified structural schematic diagrams and highlight the coordination of the iridium atom Ir with the C and N atoms, two N atoms, two O atoms and O and N atoms in the ligand as coordination sites, respectively. Such a representation method is familiar and understood by those skilled in the art):

[0069]

[0070]

[0071] (DMSO is dimethyl sulfoxide)

[0072] The CN ligand may have, for example, the following structure:

[0073]

[0074] The ON ligand may have, for example, the following structure:

[0075]

[0076] The NN ligand may have, for example, the following structure:

[0077]

[0078] The rare earth complex used as a luminescent agent may be, for example, a structure in which the central atom is a lanthanide element, and the ligand is coordinated with the central atom by O or N, and the central atom is generally Eu, Tb, Sm, Yb, Nd, Dy, Er, Ho, Pr, etc. The coordination number of these rare earth complexes is about 3 to 12, preferably 6 to 10. In actual rare earth complexes, the type of ligand, the number of each ligand, and the total coordination number may vary. For rare earth complexes and their ligands, reference may be made to, for example, Jean-Claude G. Bünzli's review paper Coord. Chem. Rev., 2015, 293-294, 19-47.

[0079] In a more preferred embodiment, the luminescent agent is selected from iridium complexes, rare earth complexes, boron dipyrrole compounds (BODIPY), perylene and derivatives of these compounds, such as one or more of the following exemplary compounds:

[0080]

[0081]

[0082] Photochemical Buffer

[0083] In the long afterglow luminescent material according to the present invention, the photochemical buffer is important. The function of the photochemical buffer is mainly the conversion of photochemical energy. Unlike the luminescent agent whose main function is to emit light, the buffer agent molecule itself does not emit light or emits light very weakly, and its molecular structure generally does not contain groups or conjugated structures that can directly emit light. In particular, the photochemical buffer according to the present invention is different in type from the luminescent agent or light absorber, especially those luminescent agents or light absorber substances listed in the present invention. The photochemical buffer according to the present invention can assist in participating in the photochemical reaction and build a bridge for energy exchange and storage between the luminescent agent and the light absorber. In the photochemical reaction, the energy extraction process of the transition between energy levels is activated through the reaction steps of addition, rearrangement or bond breaking.

[0084] The photochemical buffer according to the present invention is a non-polymeric small molecule compound, and the molecular weight is preferably less than 2000 gmol -1 , more preferably less than 1000 g mol -1 Likewise, the non-polymeric compound refers to a compound structure of the cache agent that does not contain more than two repeating units obtained by polymerization or oligomerization.

[0085] In particular, the inventors have found that some specific buffer compounds are particularly suitable for preparing stable microspheres with good long afterglow luminescence performance. The buffer used in the long afterglow luminescent microspheres of the present invention is selected from the following structural formula (I):

[0086]

[0087] in,

[0088] G and T are heteroatoms selected from O, S, Se and N;

[0089] R1′ and R2′ and R4′ to R8′ are each independently selected from H, hydroxyl, carboxyl, amino, thiol, ester, nitro, sulfonic acid, halogen, amide, or an alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, aryl, aralkyl, heteroaryl or heteroaralkyl having 1 to 50, preferably 1 to 24, such as 2 to 14 carbon atoms, or a combination thereof, wherein the aryl, aralkyl, heteroaryl or heteroaralkyl optionally has one or more substituents L; and

[0090] L is selected from hydroxy, carboxyl, amino, thiol, ester, nitro, sulfonic acid, halogen, amide, or alkyl, alkenyl, alkynyl, alkoxy and alkylamino groups having 1 to 50, preferably 1 to 24, such as 2 to 14 or 6 to 12 carbon atoms, or a combination thereof; and

[0091] R3' is an electron withdrawing group or an aryl group containing an electron withdrawing group.

[0092] In the context of this application, "aryl" means a group or ring formed by an aromatic compound that is different from an aliphatic compound, which is directly connected to another structural group or fused to another ring structure through one or more single bonds, and is therefore distinguished from a group connected to another structural group through a spacer such as an alkylene group or an ester group, such as an "aralkyl" or "aryloxy" or "arylate". Similarly, it also applies to "heteroaryl", which can be regarded as a group formed by replacing a ring carbon atom on an aryl with a heteroatom N, S, Se or O, or replacing a carbon atom on an aliphatic ring such as a cycloolefin with the heteroatom. In addition, unless otherwise indicated, the "aryl" or "heteroaryl" also includes an aryl or heteroaryl substituted or fused with an aryl or heteroaryl, such as a biphenyl, a phenylthienyl or a benzothiazolyl. In addition, the "aryl" or "heteroaryl" can also include a group formed by an aromatic or heteroaromatic compound having a functional group such as an ether group or a carbonyl group, such as an anthrone, a diphenyl ether or a thiazolone. Advantageously, an "aryl" or "heteroaryl" according to the invention has 4 to 30, more preferably 5 to 24, for example 6 to 14 or 6 to 10 carbon atoms. The term "fused" means that the two aromatic rings have a common edge.

[0093] In the context of the present application, the term "alkyl", "alkoxy" or "alkylthio" refers to a straight-chain, branched or cyclic saturated aliphatic hydrocarbon group, which is connected to other groups via a single bond, an oxy group or a thiol group, and preferably has 1-50, more preferably 1-24, such as 1-18 carbon atoms. The term "alkenyl" or "alkynyl" refers to a straight-chain, branched or cyclic unsaturated aliphatic hydrocarbon group having one or more C-C double or triple bonds, and preferably has 2-50, more preferably 2-24, such as 4-18 carbon atoms.

[0094] In the context of the present application, the term "alkylamino" refers to an amino group substituted with one or more alkyl groups, including monoalkylamino or dialkylamino, such as methylamino, dimethylamino, diethylamino, dibutylamino and the like.

[0095] In the context of this application, the term "halogen" includes fluorine, chlorine, bromine and iodine, preferably fluorine.

[0096] In the context of this application, the term "electron withdrawing group" is understood to be a group that reduces the electron cloud density on the ring when the group replaces the hydrogen on the aromatic or heteroaromatic ring. Such groups are widely known in the chemical field. Preferably, in the present invention, the electron withdrawing group is selected from nitro, halogen, haloalkyl, sulfonic acid, cyano, acyl, carboxyl and / or combinations thereof.

[0097] In addition, in the context of this application, the selected groups in the definitions of the various substituents listed can be combined with each other to form new substituents that conform to the valence principle, which means that, for example, a C1-C6 alkyl ester vinyl (C1-C6 alkyl ester vinyl) formed by combining an alkyl group, an ester group and a vinyl group 1-6 Alkyl-OC(=O)-C=C-) is also included in the definition of the relevant substituents.

[0098] In a preferred embodiment, the ring portion Can be selected from

[0099] More preferably, G and T are selected from S and O, and most preferably one of G and T is S and the other is O.

[0100] In a preferred embodiment, R1′ and R2′ and R4′ to R8′ are each independently selected from an alkyl group, an alkoxy group, an alkylamino group or an aryl group or a combination thereof having 1 to 18, preferably 1 to 12, more preferably 1 to 16 carbon atoms, wherein the aryl group may be substituted or unsubstituted by one or more groups L and is preferably a phenyl group which is substituted or unsubstituted by one or more L groups.

[0101] Preferably, L is selected from hydroxyl, sulfonic acid, halogen, nitro, linear or branched alkyl, alkoxy, alkylamino, amino, or combinations thereof having 1 to 12, more preferably 1 to 6 carbon atoms.

[0102] More preferably, the groups R1′ and R2′ and R4′ to R8′ are selected from methoxy, ethoxy, dimethylamino, diethylamino, dibutylamino, methyl, ethyl, propyl, butyl, tert-butyl, or combinations thereof.

[0103] More preferably, radical R3' is selected from electron withdrawing group or the aryl comprising electron withdrawing group, and the electron withdrawing group is preferably selected from nitro, cyano, halogen, haloalkyl and / or their combination. Correspondingly, the aryl comprising electron withdrawing group preferably comprises the aryl having one or more substituents selected from nitro, cyano, halogen and / or haloalkyl on the ring, preferably phenyl, such as fluorophenyl or perfluorophenyl.

[0104] In a particularly preferred embodiment, the photochemical buffer is selected from compounds such as:

[0105]

[0106]

[0107]

[0108] Carrier medium

[0109] The long afterglow organic microspheres of the present invention must contain component D) a carrier medium in addition to the above-mentioned component A) light absorber, component B) luminescent agent and component C) photochemical buffer. Optionally, other processing aids for microsphere preparation or components that further improve the long afterglow luminescence effect may also be included.

[0110] According to the present invention, the carrier medium is used to adsorb the specific components A) to C) as described above, and helps to form stable microspheres loaded with components A) to C). The inventors of the present application have found that the long afterglow organic microspheres that are particularly suitable for the present invention and can meet the above requirements, and are particularly suitable for use in immunochromatographic detection technology to prepare test strips, are selected from one or more of styrene polymer microspheres, protein nano-mediums and silicon microspheres, and more preferably protein-formed nano-mediums and styrene polymer microspheres. Silicon microspheres refer to silica microspheres. Styrene polymer microspheres include homopolymers of styrene or copolymers thereof formed with other copolymerizable monomers, examples of which include olefins, alkynes or unsaturated carboxylic acids or their anhydrides or esters, such as butadiene, maleic anhydride or (methyl) acrylic acid. Silicon microspheres and styrene polymer microspheres are known and commercialized types of microspheres in the field, and large quantities of microspheres with uniform particle sizes can be synthesized by known methods. The protein used to form the protein nanometer medium is not particularly limited in theory, but is preferably selected from one or more of bovine serum albumin (BSA), human serum albumin (HSA), fibroin, casein, more preferably bovine serum albumin. The method of forming microspheres by these proteins is also known in the art. In addition, it is preferred that these carrier matrix surfaces contain groups such as amino, carboxyl, and / or aldehyde groups, so that the surface of the microspheres of the present invention can utilize these groups to couple antibodies or aptamers, and the antibodies or aptamers can react immunologically with specific antigens.

[0111] Within the scope of the present invention, the skilled person can understand that the morphology of the long afterglow microspheres of the present invention actually depends on the structural morphology or processing technology of the carrier medium. Therefore, it is possible to directly and advantageously use a microspherical carrier medium such as a silicon microsphere mixed with other components to form a long afterglow microsphere, or to mix a non-spherical carrier medium such as a protein nanocarrier medium with other components, and then form the long afterglow microsphere by a known microsphere formation process (as shown in the embodiment). The microspherical carrier medium may include the following microsphere structures: core-shell structure, oil-in-water structure, water-in-oil structure, mesoporous structure, hollow structure, swellable structure, etc. In general, as the particle size of the long afterglow organic microsphere increases, the number or mass of components A), B) and C) contained in a single microsphere increases, so that the long afterglow luminescence enhancement of a single microsphere is beneficial to the efficient detection of the test signal in the immunochromatography process; but if the particle size is too large, it is not conducive to the lateral chromatography of the microsphere on the test strip. Therefore, in order to obtain an ideal immunochromatographic detection effect, the long afterglow luminescent organic microspheres of the present invention advantageously have a particle size in the range of 5nm-1000nm, more preferably 50nm-800nm, and most preferably 100nm-500nm.

[0112] In an advantageous embodiment, the carrier medium D) is preferably present in an amount of 30% to 98%, more preferably 35% to 95%, and most preferably 40% to 90%, such as 50% to 80%, based on the total mass of the four components A) to D). When the content of the component D) is too high, the brightness of the long afterglow luminescence is reduced, making it impossible to perform effective immunoassay based on the long afterglow luminescence signal. When the content of the component D is too low, the dispersion and stability of the formed microspheres are poor, and the material may even fail to form a nanostructure, which cannot meet the application requirements of immunoassay.

[0113] In addition, in the long afterglow material composition according to the present invention, adjusting the molar ratio of the light absorber to the luminescent agent within a suitable range can further improve the effect of the long afterglow. In an advantageous embodiment, the molar ratio of the light absorber to the luminescent agent is 1:2 to 1:10000, preferably 1:10 to 1:8000 or 1:50 to 1:6000, more preferably 1:100 to 1:4000 or 1:200 to 1:2000. In an advantageous embodiment, the photochemical buffering agent may be present in an amount of 0.1% to 80%, preferably 0.3% to 60%, more preferably 0.5% to 40%, and most preferably 1% to 20%, based on the total mass of the three components A) to C) of the material.

[0114] When the ratio of the light absorber is too high, the long afterglow luminescence will be absorbed by the light absorber and weakened. When the ratio of the light absorber is too low, the absorbed excitation light energy is relatively limited, which will also lead to weak long afterglow luminescence. In addition, when the photochemical buffer is too little, the energy buffering capacity is weak, resulting in the performance of the long afterglow luminescence being adversely affected, such as affecting the stability and brightness of the long afterglow luminescence. When too much buffer is added to the system, it will hinder the collision energy transfer between the components, and the buffered energy cannot be effectively transmitted and is dissipated, which reduces the performance of the long afterglow luminescence.

[0115] The long afterglow material of the present invention can be directly processed from a solution to prepare long afterglow luminescent organic microspheres, thereby being conveniently applied to the field of immunochromatographic test strip detection.

[0116] The long afterglow luminescent material of the present invention has an easily adjustable excitation and emission wavelength, which can cover the spectral regions of purple, blue, green, yellow, red and near-infrared. By selecting the type of light absorber or luminescent agent and making appropriate structural modifications when necessary, the operable range of excitation and emission is very wide, so the actual combination of excitation and emission properties is very rich. Preferably, the adjustable range of the wavelength of the excitation light is 300nm to 1000nm. In addition, the long afterglow luminescence can be luminescence based on an up-conversion mechanism, luminescence based on a down-conversion mechanism, or luminescence with zero Stokes shift. When light with a wavelength band of λ1 is used for excitation, the emission light wavelength band λ2 of the long afterglow luminescence is flexibly distributed, and the long afterglow luminescence can cover all wavelength bands from ultraviolet visible to near-infrared. When λ1<λ2, shorter wavelength light excites and realizes emission of longer wavelength light, that is, the wavelength of excitation light is red-shifted compared to the wavelength of emission light, which belongs to the conventional down-conversion luminescence mode; when λ1>λ2, longer wavelength light excites and realizes emission of shorter wavelength light, that is, the wavelength of excitation light is blue-shifted compared to the wavelength of emission light, which belongs to the up-conversion luminescence mode; when λ1=λ2, the wavelength of excitation light and the wavelength of emission light are in the same band, which belongs to the zero Stokes shift luminescence mode.

[0117] A variety of light sources can be used to excite and charge the long afterglow luminescent material of the present invention. Common light source lighting equipment, point light sources, annular light sources, indoor and outdoor natural lighting can all be used to excite and charge the long afterglow luminescent agent system based on the photochemical mechanism. In a preferred embodiment, these light sources include solid lasers, gas lasers, semiconductor lasers, photodiodes, D65 standard light sources, organic light emitting diodes, ultraviolet lamps, flashlights, flashlights, xenon lamps, sodium lamps, mercury lamps, tungsten filament lamps, incandescent lamps, fluorescent lamps and natural sunlight, as well as combinations of these light sources. In a more preferred embodiment, lasers and light emitting diodes are used as excitation light sources. The output light of these light sources has good monochromaticity and high luminous brightness, and can be selectively and quickly excited and charged. In practical applications, the light emitted by the light source can be a focused, divergent, annular, or collimated beam. The light output intensity of the excitation light source can have a wide power density range (1μW cm -2 –1000W cm -2 ), and the excitation time also has a wide dynamic range (1μs-1h). In addition, the excitation light output by the light source can be a continuous light, a pulsed light, or a combination mode output mode, wherein the pulsed light is modulated and has a wide modulation frequency range (0.001Hz-100KHz). In an advantageous scheme, the excitation time required for the ultra-bright long afterglow luminescent material according to the present invention is short, and the irradiation time of the excitation light is 0.1s-100s, preferably 0.5s-60s, more preferably 1s-30s, and most preferably 2s-10s.

[0118] In a second aspect, the present invention relates to a probe comprising the long afterglow luminescent organic microspheres as described above, and an antibody or aptamer loaded or coupled thereto.

[0119] In an advantageous embodiment, the content of the antibody or aptamer in the probe is preferably 1%-20% by weight, more preferably 2%-15%, and most preferably 5%-12% by weight of the entire probe.

[0120] Theoretically, there is no particular limitation on suitable antibodies or aptamers. Preferably, they can specifically immunobind with target antigens to be detected, including mycotoxins, pathogens, viruses, inflammatory factors or tumor markers, and are preferably selected from C-reactive protein (CRP) antibodies, serum amyloid protein (SAA) antibodies, procalcitonin (PCT) antibodies, alpha-fetoprotein (AFP) antibodies, carcinoembryonic antigen (CEA) antibodies, prostate-specific antigen (PSA) antibodies, cardiac troponin (CTn-I) antibodies and / or oligonucleotide fragments.

[0121] In a third aspect, the present invention relates to a method for preparing the long afterglow luminescent organic microspheres as described above, which comprises the following steps:

[0122] (1) providing components A) to C); and

[0123] (2) Dispersing and adsorbing components A) to C) on a carrier medium component D) in a dispersion or solution.

[0124] Here, components A) to C) may be advantageously first mixed with one another and then dispersed or dissolved in a suitable solvent, or components A) to C) may be dispersed or dissolved in a suitable solvent in sequence to form a solution. There are no particular restrictions on the suitable solvent as long as it can form a stable solution or dispersion, and the solvent may be, for example, liquid paraffin, a mixture of phenylethanol-ethylene glycol and water, a mixture of mesitylene and ethanol, tetrahydrofuran, dichloromethane, and the like.

[0125] After the solution or dispersion containing components A) to C) is prepared, the carrier medium microspheres or their solution or dispersion can be added thereto. Alternatively, the solution or dispersion containing components A) to C) can be added to the solution or dispersion containing the carrier medium microspheres. Water or other suitable solvents can be used to disperse the carrier medium, such as deionized water, phosphate buffered saline (PBS), borate buffered saline (BBS), etc.

[0126] In the process of preparing the solution or dispersion, auxiliary equipment such as ultrasound, a high-pressure homogenizer, etc. may be used if necessary, or appropriate heating may be performed accompanied by stirring.

[0127] After obtaining a stable dispersion of long-lasting luminescent organic microspheres containing components A) to D) in step (2), it can be directly used for subsequent use without treatment as needed, such as for preparing test paper suitable for immunoassay. Alternatively, antibodies or aptamers can be further adsorbed or modified on the obtained microspheres to obtain the probe according to the present invention.

[0128] The preparation method of the probe is known per se or can be obtained by those skilled in the art with slight improvements based on known techniques. The preparation method mainly comprises biocoupling the long afterglow luminescent microspheres as described above with antibodies or aptamers through functional reactive groups such as carboxyl, amino, and aldehyde groups. For example, coupling can be formed by carboxyl-amino reaction or aldehyde-amino reaction. Usually, the corresponding coupling method is selected according to the situation of the functional groups on the surface of the microspheres.

[0129] Therefore, the fourth aspect of the present invention relates to a method for preparing the probe as described above.

[0130] In a fifth aspect, the present invention provides a test paper for immunochromatographic detection comprising the long afterglow luminescent organic microspheres or the probes as described above. The test paper comprises a sample pad, a conjugate pad, a test line and a quality control line, wherein the conjugate pad is provided with the long afterglow luminescent organic microspheres or the probes as described above.

[0131] The structure of the test paper itself for immunochromatographic detection technology is known. The conjugate pad, the test line and the quality control line can be attached to a base plate, such as a PVC base plate. For the structure of such a test paper, reference can be made to, for example, the published patent document CN105929155A, the entire contents of which are hereby introduced into this application.

[0132] In such Figure 3 In an exemplary structure shown, the test paper includes a PVC base plate 1, on which a sample pad 2, a binding pad 3, a nitrocellulose membrane 4 and a water absorbent pad 5 are sequentially arranged, wherein the nitrocellulose membrane 4 is also sequentially provided with a test line 6 and a quality control line 7 along the direction from the sample pad 2 to the water absorbent pad 5.

[0133] Immunochromatography mainly includes double antibody sandwich and competition method. Double antibody sandwich method is mainly used to detect macromolecular substances such as proteins, such as tumor markers, viruses and inflammatory factors. These detection methods are known in themselves. In an exemplary embodiment, the method uses a pair of paired antibodies for different epitopes of the antigen, fixes the capture antibody on the T line of the NC membrane, and the detection antibody coupled modified nanoprobe is fixed on the binding pad, and the goat anti-mouse (or donkey anti-mouse, goat anti-rabbit, rabbit anti-mouse, etc.) secondary antibody is fixed on the C line of the NC membrane as a quality control line. During the detection process, the sample is dripped on the sample pad, and it swims from left to right through the capillary action, passing through the binding pad in turn, and the T line and C line undergo specific immune reactions. The competition method is mainly used for the detection of small molecules. In this method, for example, the whole antigen (the coupling product of small molecules and macromolecules) can be fixed on the NC membrane to form a T line, and the antibody coupled modified nanoprobe is fixed on the binding pad, and the goat anti-mouse (or donkey anti-mouse, goat anti-rabbit, rabbit anti-mouse, etc.) secondary antibody is used as the C line. During the detection process, the sample is dropped on the sample pad and passes through the binding pad, T line and C line in turn through capillary action. The antigen fixed on the T line will competitively bind with the free antigen and antibody in the sample.

[0134] Finally, the present invention also relates to a method for immunochromatographic detection, which comprises the following steps:

[0135] (1) providing the long afterglow luminescent organic microspheres, probes or test papers as described above;

[0136] (2) irradiating the organic microspheres, probes or test papers with excitation light; and

[0137] (3) Stop irradiation and read the luminescence signal.

[0138] Compared with similar technologies such as CN105929155A that use inorganic long afterglow materials, the immunochromatographic detection method of the present invention has more advantages. First, the selectable range of excitation wavelengths is wider, including the wavelength ranges of ultraviolet light, visible light and near-infrared light. Secondly, the absorption cross-section of the long afterglow luminescent microspheres of the present invention is several orders of magnitude larger, which allows the light irradiation charging time to be shorter, for example, 2s-10s is most preferred. In addition, the long afterglow luminescent microspheres of the present invention have a higher brightness, exceeding the brightness level visible to the naked eye, and the optional detection equipment is more common. Preferably, the instrument used to read the luminescent signal in the detection is a mobile phone, a luminescent imaging system, a professional long afterglow luminescent detection device, and the like. More preferably, the detection equipment is a common commercial mobile phone, which is equipped with signal reading software that can perform data analysis of the signal intensity of pictures taken by the mobile phone. BRIEF DESCRIPTION OF THE DRAWINGS

[0139] Figure 1Schematic diagram of the structure of the probe comprising the long afterglow luminescent microspheres according to the present invention. It can be seen that components A) to C) are all adsorbed on the carrier medium microspheres, and the carrier medium is also coupled with antibodies or aptamers.

[0140] Figure 2 Schematic diagram of the luminescence mechanism of the long afterglow luminescent nanomaterial according to the present invention.

[0141] Figure 3 The schematic diagram of the immunochromatographic test strip of the present invention comprises a PVC base plate 1, on which a sample pad 2, a binding pad 3, a nitrocellulose membrane 4 and a water absorbent pad 5 are sequentially arranged; a test line 6 and a quality control line 7 are also sequentially arranged on the nitrocellulose membrane 4 along the direction from the sample pad 2 to the water absorbent pad 5, and the direction indicated by the arrow in the figure is the lateral chromatography direction.

[0142] Figure 4 This is a transmission electron microscope image of the long afterglow luminescent nanoparticles of Example 1.

[0143] Figure 5 The bright field (a) and long afterglow luminescence image (b) of the long afterglow material. The bright field was taken under indoor lighting, and the long afterglow luminescence image was taken in the dark after the 365nm excitation light was turned off. The left sample is the long afterglow luminescent nanomaterial of Example 1 of the present invention, and the right sample is the inorganic long afterglow SrAl2O4:Eu 2+ ,Dy 3+ Nanomaterials.

[0144] Figure 6 A standard curve for detecting C-reactive protein (CRP) based on the long afterglow luminescent nanomaterial of Example 31 of the present invention.

[0145] Figure 7 The effect diagram of long afterglow immunochromatographic test strips detecting C-reactive protein (CRP), taken with the same mobile phone. The long afterglow signal indicator probes used in the immunochromatographic test strips are different. The left figure (a) is a CRP detection effect diagram based on the long afterglow luminescent nanomaterial of Example 31 of the present invention, and the right figure (b) is a CRP detection effect diagram based on the inorganic long afterglow SrAl2O4:Eu based on Comparative Example 11. 2 + ,Dy 3+ CRP detection effect diagram of nanomaterials.

[0146] Figure 8 A standard curve for detecting serum amyloid protein (SAA) based on the long afterglow luminescent nanomaterial of Example 32 of the present invention.

[0147] Fig. 9 A standard curve for the detection of procalcitonin (PCT) based on the long afterglow luminescent nanomaterial of Example 33 of the present invention.

[0148] Fig.10 A standard curve for the detection of alpha-fetoprotein (AFP) based on the long afterglow luminescent nanomaterial of Example 34 of the present invention.

[0149] Fig.11 A standard curve for detection of carcinoembryonic antigen (CEA) based on the long afterglow luminescent nanomaterial of Example 35 of the present invention.

[0150] Fig.12 A standard curve for prostate-specific antigen (PSA) detection based on the long afterglow luminescent nanomaterial of Example 36 of the present invention.

[0151] Fig.13 A standard curve for the detection of cardiac troponin (CTn-I) based on the long afterglow luminescent nanomaterial of Example 37 of the present invention. Example

[0152] 1. Performance testing method

[0153] In the long afterglow luminescence test of the present invention, when the laser is used as the excitation light source, a wavelength tunable laser (Opolette 355) from Opotek, Inc. of the United States is used. In certain cases, a light emitting diode (LED) is also used as an excitation light source, and the power density of the excitation light remains consistent. The excitation light of a specific wavelength irradiates the sample for charging, and the irradiation and charging time is 3s. After the charging is completed, the laser is turned off and the luminescence performance is tested. The fluorescence spectrometer (Edinburgh FS-5) of Edinburgh Instruments of the United Kingdom is used to test the long afterglow luminescence intensity. The long afterglow test system (OPT-2003) of Beijing Aobodi Optoelectronic Technology Co., Ltd. is used to test the long afterglow luminescence brightness. The present invention uses a commercial smart phone or a common digital camera to take pictures and record bright field and long afterglow luminescence pictures.

[0154] The phrase "visible to the naked eye" used in this article is a professional term in the field of long-lasting luminescent materials, meaning that the luminous brightness of the material is greater than or equal to 0.32 mcd·m -2 , visible light can usually be seen by the naked eye when it is at a radiation level of this brightness or above. The phrase "luminescence time" used in this article is a professional term in the field of long afterglow luminescent materials, which means the time it takes for the luminescence brightness of the material to decay to a level visible to the naked eye. The phrase "blue long afterglow luminescence" used in this article is a description of the long afterglow luminescence color of the material, meaning that there is obvious long afterglow luminescence in the blue wavelength range; similarly, this description is also applicable to the description of other colors used in this article. In actual situations, due to differences in observation methods or individual differences, there may be errors in observation results such as luminescence color or luminescence time.

[0155] 2. List of raw materials used

[0156]

[0157]

[0158]

[0159]

[0160]

[0161] 3. Preparation of long afterglow luminescent nanomaterials

[0162] Example 1

[0163] Long afterglow luminescent organic microspheres were prepared, wherein the content of the carrier medium was 75% based on the total mass of the four components A) to D). First, the light absorber PdOEP, the luminescent agent Eu-1 and the photochemical buffer CA-1 were mixed in a dichloromethane solvent, and ultrasound was used to assist the dissolution of each component. In the solution, the concentration of the light absorber PdOEP was 0.1 mmol L -1 The molar concentration of photochemical buffer CA-1 was 3 mmol L -1 The concentration of luminescent agent Eu-1 is 10 mmol L -1 , the molar ratio of the three components of the light absorber, photochemical buffer and luminescent agent is 1:30:100. Take 1 mL of the above solution, add 10 mg of liquid paraffin and 20 mg of bovine serum albumin (BSA) to it, and then add 10 mL of deionized water. Use ultrasound (Sonics VC750, Sonics & Materials, Inc) to pre-emulsify the mixture in the dark for 5 minutes at room temperature, and then use a rotary evaporator to remove dichloromethane. Then immediately use a high-pressure nano homogenizer (FB-110Q, LiTu Mechanical equipment Engineering Co., Ltd) to continue emulsification in the dark for 10 minutes. Heat the emulsion in the dark at 90 degrees Celsius for 1 hour. After the emulsion is cooled to room temperature, gradient centrifugation and filtration are used to obtain long afterglow luminescent microspheres uniformly dispersed in water. The above microspheres are stained with sodium phosphotungstate, and the morphology under the transmission electron microscope is as follows Figure 4 The prepared long afterglow luminescent microspheres were tested for their afterglow performance. The long afterglow luminescent microspheres were prepared into 1 mg mL -1 First, a 365nm wavelength LED light source was used to irradiate for 3s for charging. After the charging was completed, the light source was turned off, and a long red afterglow visible to the naked eye was obtained. The test results are shown in Table 1.

[0164] Comparative Example 1 (C1)

[0165] Long afterglow luminescent inorganic microspheres are prepared, wherein the content of the carrier medium is 67% based on the total mass of the carrier medium and the inorganic long afterglow luminescent nanoparticles. 2+ ,Dy 3+ It is the brightest green long afterglow luminescent material and is widely used. 2+ ,Dy 3+ The material is a long afterglow powder obtained by high temperature sintering and then grinding. SrAl2O4:Eu with a particle size of about 50nm is obtained by centrifugal separation. 2+ ,Dy 3+ Inorganic long afterglow nanoparticles. Then, as described in Example 1, 10 mg of the inorganic nanoparticles were modified with 20 mg of BSA in an aqueous solution and subjected to ultrasonic emulsification and other treatments. Finally, BSA-coated inorganic long afterglow microspheres were obtained, with a particle size of about 300 nm. According to the method of Example 1, the BSA-coated inorganic long afterglow microspheres were prepared into 1 mg mL -1 The concentration of aqueous solution was used to test the afterglow performance of the microspheres. The results showed that no afterglow was observed by naked eyes. The long afterglow luminescence intensity tested by the instrument is shown in Table 1.

[0166] Embodiment 2-8

[0167] The operation of Example 1 was repeated, wherein the molar ratio of the three components of the light absorber, the photochemical buffer and the luminescent agent was maintained at 1:30:100. The differences are shown in Table 1.

[0168] Example 9

[0169] Long afterglow luminescent organic microspheres were prepared, wherein the amount of the carrier medium was 50% of the total mass of the four components A) to D). The light absorber PdOEP, the luminescent agent Eu-1 and the photochemical buffer CA-1 were added to 5 mL of benzyl alcohol-ethylene glycol-water (v:v:v, 1:8:1) solution, wherein the concentration of the light absorber PdOEP was 0.1 mmol L -1 The concentration of photochemical buffer CA-1 was 3 mmol L -1 The concentration of luminescent agent Eu-1 is 10 mmol L -1 The molar ratio of the three components of the absorbent, photochemical buffer and luminescent agent is 1:30:100. After ultrasonic dispersion of each component, 50 mg of styrene polymer (PS) microspheres with carboxyl groups on the surface were added and heated at 110 ° C for 30 minutes. Then, it was cooled to room temperature, washed by centrifugation with ethanol and water for 3 times, and finally the microspheres were dispersed in water for storage. The afterglow performance of the prepared long afterglow luminescent microspheres was tested, and the long afterglow luminescent microspheres were prepared into 1 mg mL-1 First, the excitation light with a wavelength of 540 nm was used for 3 seconds for charging. After the charging was completed, the light source was turned off, and a long red afterglow visible to the naked eye was obtained. The test results are shown in Table 1.

[0170] Example 10

[0171] Long afterglow luminescent organic microspheres were prepared, wherein the amount of the carrier medium was 50% of the total mass of the four components A) to D). The light absorber PdOEP, the luminescent agent Eu-1 and the photochemical buffer CA-1 were added to 10 mL of mesitylene-ethanol (v:v, 1:1) solution, wherein the concentration of the light absorber PdOEP was 0.1 mmol L -1 The concentration of photochemical buffer CA-1 was 3 mmol L -1 The concentration of luminescent agent Eu-1 is 10 mmol L -1 The molar ratio of the three components of the absorbent, photochemical buffer and luminescent agent is 1:30:100. After ultrasonic dispersion of each component, 100 mg of silicon microspheres with amino groups on the surface were added and heated at 80 ° C for 2 hours. Then, it was cooled to room temperature, washed by centrifugation with ethanol and water for 3 times, and finally the microspheres were dispersed in water for storage. The afterglow performance of the prepared long afterglow luminescent microspheres was tested, and the long afterglow luminescent microspheres were prepared into 1 mg mL -1 First, the excitation light with a wavelength of 540 nm was used for 3 seconds for charging. After the charging was completed, the light source was turned off, and a long red afterglow visible to the naked eye was obtained. The test results are shown in Table 1.

[0172] Examples 11-13

[0173] The operation of Example 1 was repeated, wherein the molar ratio of the three components of the light absorber, the photochemical buffer and the luminescent agent was maintained at 1:30:100. The differences are shown in Table 1.

[0174] Comparative Examples 2-3 (C2 and C3)

[0175] The operation of Example 1 was repeated, wherein the molar ratio of the three components of the light absorber, the photochemical buffer and the luminescent agent was maintained at 1:30:100. The differences are shown in Table 1.

[0176] Comparative Example 4 (C4)

[0177] Long afterglow microspheres were prepared using NCBS as a light absorber, PFVA as a luminescent agent, and DO as a photochemical buffer. The components were mixed in a dichloromethane solvent, and ultrasonic waves were used to assist the dissolution of the components to form a uniform and transparent solution. In the solution, the concentration of the light absorber NCBS was 0.1 mmol L -1The molar concentration of the photochemical buffer DO is 2 mmol L -1 The concentration of luminescent agent PFVA is 10 mg mL -1 . Subsequently, take 1 mL of the above solution, add 10 mg of liquid paraffin and 20 mg of bovine serum albumin (BSA) thereto, and then add 10 mL of deionized water. Use ultrasound (Sonics VC750, Sonics & Materials, Inc) to pre-emulsify the mixture in the dark at room temperature for 5 minutes, and then use a rotary evaporator to remove dichloromethane. Then immediately use a high-pressure nano homogenizer (FB-110Q, LiTu Mechanical equipment Engineering Co., Ltd) to continue emulsifying in the dark for 10 minutes. Heat the emulsion in the dark at 90 degrees Celsius for 1 hour. After the emulsion is cooled to room temperature, gradient centrifugation and filtration are performed to obtain long afterglow microspheres uniformly dispersed in water. The afterglow performance of the prepared long afterglow luminescent microspheres was tested according to the method of Example 1, and the long afterglow luminescent microspheres were formulated into 1 mg mL -1 First, the excitation light with a wavelength of 808 nm was used for 3 seconds for charging. After the charging was completed, the light source was turned off. As a result, no afterglow light was observed by naked eyes. The long afterglow luminescence intensity tested by the instrument is shown in Table 1.

[0178] Comparative Example 5 (C5)

[0179] The procedure of Comparative Example 6 was repeated, with the differences shown in Table 1.

[0180] Comparative Example 6 (C6)

[0181] Long afterglow microspheres were prepared using PtTPBP as a light absorber, Eu-1 as a luminescent agent, and CA-1 as a photochemical buffer. First, each component was dissolved in 2 mL of tetrahydrofuran (THF), where the concentration of the light absorber PdOEP was 0.1 mmol L -1 The concentration of photochemical buffer CA-1 was 3 mmol L -1 The concentration of luminescent agent Eu-1 is 10 mmol L -1 , the molar ratio of the three components of the light absorber, photochemical buffer, and luminescent agent is 1:30:100. 20 mg of F127 was added to the above solution and stirred to dissolve, and then tetrahydrofuran was removed. The obtained composition was dispersed in 2 mL of water using ultrasound, and long afterglow microspheres uniformly dispersed in water were obtained by centrifugation and filtration. The afterglow performance of the prepared long afterglow luminescent microspheres was tested according to the method of Example 1, and the long afterglow luminescent microspheres were prepared into 1 mg mL -1First, the excitation light with a wavelength of 635nm was used for 3s to charge the battery. After the charging was completed, the light source was turned off, and no long afterglow was observed that was obviously visible to the naked eye. The test results are shown in Table 1.

[0182] Table 1

[0183]

[0184]

[0185] Embodiment 14

[0186] The absorbent PdPc, luminescent agent Eu-2 and photochemical buffer CA-1 were mixed in dichloromethane solvent, and ultrasonic waves were used to assist the dissolution of each component to form a uniform and transparent solution. In this solution, the molar concentration of the photochemical buffer CA-1 was 2 mmol L -1 The concentration of luminescent agent Eu-2 is 5 mmol L -1 The concentration of the light absorber PdPc is 50 μmol L -1 . Subsequently, take 1 mL of the above solution, add 10 mg of liquid paraffin and 20 mg of bovine serum albumin (BSA) to it, and then add 10 mL of deionized water. Use ultrasound (Sonics VC750, Sonics & Materials, Inc) to pre-emulsify the mixture in the dark at room temperature for 5 minutes, and then use a rotary evaporator to remove dichloromethane. Then immediately use a high-pressure nano homogenizer (FB-110Q, LiTu Mechanical equipment Engineering Co., Ltd) to continue emulsification in the dark for 10 minutes. Heat the emulsion in the dark at 90 degrees Celsius for 1 hour. After the emulsion is cooled to room temperature, gradient centrifugation and filtration are used to obtain long afterglow microspheres uniformly dispersed in water. The afterglow performance of the prepared long afterglow luminescent microspheres was tested, and the long afterglow luminescent microspheres were formulated into 1 mgmL -1 First, the excitation light with a wavelength of 730 nm was used for 3 seconds for charging. After the charging was completed, the light source was turned off. The test results are shown in Table 2.

[0187] Examples 15-19

[0188] The procedure of Example 14 was repeated, except that the concentration of the luminescent agent Eu-2 was 5 mmol L -1 .

[0189] Comparative Examples 7-8 (C7 and C8)

[0190] The procedure of Example 14 was repeated, except that the concentration of the luminescent agent Eu-2 was 5 mmol L -1.

[0191] Table 2

[0192]

[0193] Embodiment 20

[0194] The light absorber PdPc, the luminescent agent Eu-1 and the photochemical buffer CA-1 were mixed in dichloromethane, and ultrasound was used to assist the dissolution of each component to finally form a uniform and transparent solution. In the solution, the molar ratio of the three components of the light absorber PdPc, the photochemical buffer CA-1 and the luminescent agent Eu-1 was 1:300:3000. Then, the dichloromethane solvent was removed to obtain an oily mixture of the three components A) / B) / C). 50 mg of the three-component mixture was weighed and added to the D) component containing 30 mg of liquid paraffin and 20 mg of bovine serum albumin, wherein the weight proportion of component D) was 50%. The aqueous solution of the mixture was pre-emulsified in the dark for 5 minutes at room temperature using ultrasound (SonicsVC750, Sonics&Materials, Inc), and then immediately emulsified in the dark for 10 minutes using a high-pressure nano homogenizer (FB-110Q, LiTu Mechanical equipment Engineering Co., Ltd). The emulsion was heated at 90 degrees Celsius in the dark for 1 hour. After the emulsion cooled to room temperature, gradient centrifugation and filtration were performed to obtain long afterglow microspheres uniformly dispersed in water. The afterglow performance of the prepared long afterglow luminescent microspheres was tested. The long afterglow luminescent microspheres were prepared into 1 mg mL -1 First, the excitation light with a wavelength of 540 nm was used for 3 seconds for charging. After the charging was completed, the light source was turned off. The test results are shown in Table 3.

[0195] Examples 21-23

[0196] The operation of Example 20 was repeated, except that the weight proportion of the D) component in the nanomaterial was (as shown in Table 3). The test results are shown in Table 3.

[0197] Comparative Examples 9-10 (C9 and C10)

[0198] The operation of Example 20 was repeated, except that the weight proportion of the D) component in the nanomaterial was (as shown in Table 3). The test results are shown in Table 3.

[0199] Table 3

[0200]

[0201] Embodiment 24

[0202] Take 80nm carboxyl-containing styrene polymer (PS) microspheres and centrifuge to remove the surfactant in the synthesis process, then dissolve 1g of PS microsphere solid in 100mL ultrapure water and form a dispersed phase by ultrasound. Then add 1mL of 2% sodium dodecylbenzene sulfonate and 1% ethylenediamine polyoxyethylene polyoxypropylene block polyether to the PS microsphere aqueous solution and stir. Take components A), B) and C) shown in Table 4 and disperse them in 10mL tetrahydrofuran solution to form a dispersed phase, where the concentrations of A), B) and C) are 0.1mmol L -1 , 2mmol L -1 and 10mmol L -1 After the solution is prepared, the organic phase is quickly added to the aqueous phase, and then the temperature is gradually raised to 50°C and stirred for 10 hours. The obtained long afterglow PS microspheres with a particle size of 80nm are then centrifuged to remove excess dye, and then washed twice with ultrapure water and ethanol, and stored in ultrapure water, protected from light and placed at room temperature for use.

[0203] Examples 25-28

[0204] The operation of Example 24 was repeated, except that the particle size of the styrene polymer (PS) microspheres having carboxyl groups as the nanocarrier medium was changed (as shown in Table 4). The test results are shown in Table 4.

[0205] Table 4

[0206]

[0207] Embodiment 29

[0208] The probe was prepared by coupling the alpha-fetoprotein (AFP) antibody AFP-Ab1 with fluorescent long-lasting microspheres:

[0209] 1) Take 100 mg of the long afterglow luminescent microspheres prepared according to Example 26, centrifuge, redissolve in 18 mL of BBS buffer with a pH of 7.4, and fully sonicate to make them uniformly dispersed; 2) Add 2 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 2 mg of N-hydroxysuccinimide sulfonic acid sodium salt (NHSS) thereto, respectively, and react at room temperature for 2 hours; 3) After the reaction is completed, centrifuge and wash, redissolve in 10 mL of BBS buffer with a pH of 7.4, add 10 mg of AFP-Ab1 type monoclonal antibody thereto, and react at room temperature for 4 hours; 4) After the reaction is completed, centrifuge and wash, redissolve in 10 mL of BBS buffer with a pH of 7.4, add 100 mg of BSA thereto, and react at room temperature for 2 hours; 5) After the reaction is completed, centrifuge and wash, redissolve in 10 mL of BBS buffer with a pH of 7.4, and store at 4°C for use.

[0210] Embodiment 30

[0211] Preparation of probes by coupling long-lasting microspheres with prostate-specific antigen (PSA) aptamers:

[0212] 1) 10 mg of the long afterglow microspheres containing carboxyl groups prepared according to Example 26 were centrifuged and redissolved in 1.8 mL of BBS buffer (pH 7.4), and fully ultrasonicated to make them uniformly dispersed; 2) 0.6 mg of EDC and 0.2 mg of NHSS were added thereto respectively, and the reaction was shaken at room temperature for 2 hours; 3) After the reaction was completed, the microspheres were washed by centrifugation, redissolved in 2 mL of BBS buffer, and 20 μL of 2 μmol mL -1 The PSA aptamer, whose sequence is (NH2-ATTAAAGCTCGCCATCAAATAGCTGC), was reacted at room temperature for 4 hours; 4) after the reaction, the mixture was centrifuged and washed, redissolved in 2 mL of BBS buffer, 10 mg of BSA was added thereto, and reacted at room temperature for 2 hours; 5) after the reaction, the mixture was centrifuged and washed twice, redissolved in 4 mL of BBS buffer (pH 7.4), and stored at 4°C for later use.

[0213] Embodiment 31

[0214] Preparation of lateral flow immunoassay strips containing long afterglow luminescent microspheres of the present invention and application thereof in C-reactive protein (CRP) detection

[0215] (1) Preparation of immunochromatographic test strips for detecting C-reactive protein (CRP):

[0216] 1) The long afterglow microspheres in Example 1 were coupled to CRP-Ab1 as follows: PEG with carboxyl groups at both ends was used, and the carboxyl groups at both ends were first activated, and then one end was reacted and connected with the amino group on the BSA nanosphere, and the other end was reacted and connected with the amino group on CRP-Ab1. 10 mg of CRP-Ab1 type monoclonal antibody was added to 100 mg. After the reaction was completed, the mixture was centrifuged and washed, and then redissolved in 10 mL of BBS buffer with a pH of 7.4 and stored at 4°C for later use.

[0217] 2) Preparation of CRP immunochromatographic test strip NC membrane: CRP-Ab2 monoclonal antibody and donkey anti-mouse IgG were respectively diluted in PBS buffer (1% BSA, 1% sucrose, 50 mM NaCl and 0.5% TWEEN 20) at 1 mg mL -1 and 1mgmL -1 The concentration was 8 mm and the membrane was streaked onto a nitrocellulose membrane using a membrane streaker and dried at 37°C overnight.

[0218] 3) Preparation of CRP immunochromatographic test strip sample pad: Take the fluorescent probe prepared in step 1), centrifuge it, and reconstitute it with spray buffer to 20 mg mL -1 The fluorescent probe solution was poured into the instrument through the film sprayer at a rate of 1.2 μL cm -1 The fluorescent probe was sprayed onto the glass fiber at a speed of 100 μl and baked at 37°C overnight.

[0219] 4) Assembly of CRP immunochromatographic test strips: Glass fibers labeled with fluorescent microspheres of CRP-Ab1 monoclonal antibodies, NC membranes marked with T lines of CRP-Ab2 and C lines of donkey anti-mouse IgG are attached to the white PVC base plate in a staggered manner of 3 mm, and finally absorbent paper is attached. The assembled chromatography plate is then cut into 3.8 mm wide test strips by a high-speed chopper, and then the test strips are fixed with the matching upper and lower plastic card shells to obtain immunochromatographic test strips.

[0220] (2) Establishment of CRP standard curve in samples:

[0221] 1) Use the sample to dilute the CRP antigen stock solution into whole blood CRP antigen solutions of different concentrations, with concentrations of 0 μg mL -1 , 5 μg mL -1 , 20 μg mL -1 , 40 μg mL -1 , 160 μg mL -1 and 320 μg mL -1 .

[0222] 2) Take 1 μL of sample CRP antigen solution and add it to 99 μL of PBS buffer (containing 1% BSA, 0.1% SDS and 0.1% B66), and mix thoroughly.

[0223] 3) Add 100 μL of the evenly mixed solution to the sample well of the immunochromatographic test strip. The liquid will pass through the sample area, the detection area and the water absorption area in sequence through capillary action. When the test sample contains an antigen solution, the antigen first combines with the long afterglow luminescent probe in the sample area to form an immune complex, and then swims with the liquid to the test line (T line) to form a sandwich immune complex with CRP-Ab2, and the excess long afterglow luminescent probe swims to the control line (C line) to combine with the donkey anti-mouse secondary antibody. When there is no antigen in the test sample, the long afterglow luminescent probe will be driven to swim directly to the C line to combine with the donkey anti-mouse secondary antibody.

[0224] 4) After the reaction has been carried out for 5 minutes, the immunochromatographic test strip is tested using a long afterglow luminescence detector. The long afterglow luminescence intensity of the T line and the C line is measured, the peak area is integrated, and then the ratio of the peak areas is calculated. The standard curve is established by the ratio and the logarithm of the antigen concentration ( Figure 6The long afterglow luminescence detector is a commercial smart phone for daily use, which is equipped with signal reading software and can perform data analysis on the signal strength of pictures taken by the mobile phone.

[0225] (3) Immunochromatographic detection of CRP in samples:

[0226] The probe containing the long afterglow luminescent microspheres of the present invention only needs to be excited with excitation light before reading, and the excitation light is turned off during the subsequent reading process. This method eliminates the interference of background fluorescence signals and can achieve highly sensitive quantitative detection of the object to be tested. In the detection of samples containing CRP, it was found that the immunochromatographic test strip based on the long afterglow luminescent microspheres of the present invention ( Figure 7 Left) is more sensitive than the detection system based on inorganic long afterglow ( Figure 7 The detection time of the long afterglow luminescent immunochromatographic test strip of the present invention is only 3s. -1 The CRP antigen is far below the normal physiological level.

[0227] Comparative Example 11 (C11)

[0228] The operation of Example 31 was repeated, except that in the first step, the inorganic long afterglow microspheres in Comparative Example 1 were used to couple CRP-Ab1. The inorganic long afterglow nano-immunochromatographic test strip prepared in this way had a weak long afterglow luminescence signal, which was invisible to the naked eye and could not be photographed by a mobile phone. Figure 7 As shown in the middle right picture.

[0229] Examples 32-37

[0230] The operation of Example 31 was repeated to obtain a standard curve for antigen detection based on a long afterglow lateral flow immunoassay strip, except that the target antigens detected were replaced with: SAA (Example 32, Figure 8 )、PCT (Example 33, Fig. 9 )、AFP (Example 34, Fig.10 )、CEA (Example 35, Fig.11 )、PSA (Example 36, Fig.12 )、CTn-I (Example 37, Fig.13 ).

Claims

1. A long afterglow luminescent organic microsphere comprising A) at least one light absorber, wherein the light absorber is selected from the group consisting of porphyrin and phthalocyanine complexes, quantum dots, and derivatives of these compounds, and the quantum dots are selected from the group consisting of graphene quantum dots, carbon quantum dots, and heavy metal quantum dots; B) at least one luminescent agent, said luminescent agent being a monomeric, non-polymeric compound and having a molecular weight of less than 10,000 gmol -1 , the luminescent agent is selected from iridium complexes, rare earth complexes, fluoroboron dipyrrole compounds, perylene and derivatives of these compounds; C) at least one photochemical buffering agent of formula (I), （I） in, Ring part" "Selected from , , ; R1' and R2' are selected from H or an alkyl group having 1 to 14 carbon atoms; R4' to R8' are each independently selected from H, an alkoxy group having 1 to 14 carbon atoms, an alkylamino group having 1 to 14 carbon atoms, or a combination thereof; R3' is a phenyl group comprising an electron withdrawing group, and the electron withdrawing group is selected from a nitro group, a halogen group, a cyano group and / or a combination thereof; and D) a carrier medium for adsorbing components A) to C), wherein the carrier medium is selected from one or more of styrene polymer microspheres and protein nano-medium; wherein the light absorber and the luminescent agent are compounds with different structures, and the content of the carrier medium is 30% to 99% based on the total mass of the four components A) to D); The photochemical buffering agent is contained in an amount of 0.1% to 80% by weight based on the total weight of components A) to C); and The molar ratio of the light absorber to the light emitting agent is between 1:2 and 1:10000.

2. The long afterglow luminescent organic microsphere according to claim 1, characterized in that: The content of the carrier medium is 35% to 95% based on the total mass of the four components A) to D).

3. The long afterglow luminescent organic microsphere according to claim 1, characterized in that: The content of the carrier medium is 40% to 90% based on the total mass of the four components A) to D).

4. The long afterglow luminescent organic microsphere according to any one of claims 1 to 3, characterized in that: The radicals R1' and R2' are selected from H, methyl, ethyl, propyl, butyl or tert-butyl.

5. The long afterglow luminescent organic microsphere according to any one of claims 1 to 3, characterized in that: The groups R4' to R8' are each independently selected from methoxy, ethoxy, butoxy, dimethylamino, diethylamino, dibutylamino, or a combination thereof.

6. The long afterglow luminescent organic microsphere according to any one of claims 1 to 3, characterized in that: The photochemical buffer is selected from one or more of the following compounds: 。 7. The long afterglow luminescent organic microsphere according to any one of claims 1 to 3, characterized in that: The surface of the carrier medium contains amino groups, carboxyl groups and / or aldehyde groups.

8. The long afterglow luminescent organic microsphere according to any one of claims 1 to 3, characterized in that: The protein used to form the protein nano-medium is selected from one or more of bovine serum albumin, human serum albumin, silk fibroin and casein.

9. The long afterglow luminescent organic microsphere according to claim 8, characterized in that: The protein used to form the protein nano-medium is selected from bovine serum albumin.

10. The long afterglow luminescent organic microsphere according to any one of claims 1 to 3, characterized in that: The long afterglow luminescent organic microspheres have a particle size in the range of 5 nm-1000 nm.

11. The long afterglow luminescent organic microsphere according to any one of claims 1 to 3, characterized in that: The long afterglow luminescent organic microspheres have a particle size in the range of 50 nm-800 nm.

12. The long afterglow luminescent organic microsphere according to any one of claims 1 to 3, characterized in that: The long afterglow luminescent organic microspheres have a particle size in the range of 100 nm-500 nm.

13. The long afterglow luminescent organic microsphere according to any one of claims 1 to 3, characterized in that: The molar ratio of the light absorber to the light emitting agent is 1:10 to 1:8000.

14. The long afterglow luminescent organic microsphere according to any one of claims 1 to 3, characterized in that: The molar ratio of the light absorber to the light emitting agent is 1:50 to 1:6000.

15. The long afterglow luminescent organic microsphere according to any one of claims 1 to 3, characterized in that: The molar ratio of the light absorber to the light emitting agent is between 1:100 and 1:4000.

16. The long afterglow luminescent organic microsphere according to any one of claims 1 to 3, characterized in that: The molar ratio of the light absorber to the light emitting agent is 1:200 to 1:2000.

17. The long afterglow luminescent organic microsphere according to any one of claims 1 to 3, characterized in that: The photochemical buffering agent is contained in an amount of 0.3% to 60% by weight based on the total weight of components A) to C).

18. The long afterglow luminescent organic microsphere according to any one of claims 1 to 3, characterized in that: The photochemical buffering agent is contained in an amount of 0.5% to 40% by weight based on the total weight of components A) to C).

19. The long afterglow luminescent organic microsphere according to any one of claims 1 to 3, characterized in that: The photochemical buffering agent is contained in an amount of 1% to 20% by weight based on the total weight of components A) to C).

20. The long afterglow luminescent organic microsphere according to any one of claims 1 to 3, characterized in that: The long afterglow luminescent organic nanoparticles consist of components A) to D).

21. A probe comprising the long afterglow luminescent organic microsphere according to any one of claims 1 to 20 and an antibody or aptamer loaded or coupled thereto.

22. The probe according to claim 21, characterized in that The content of the antibody or aptamer in the probe is 1%-20% based on the mass of the entire probe.

23. The probe according to claim 21, characterized in that The content of the antibody or aptamer in the probe is 2%-15% based on the mass of the entire probe.

24. The probe according to claim 21, characterized in that The content of the antibody or aptamer in the probe is 5%-12% based on the mass of the entire probe.

25. The probe according to any one of claims 21 to 24, characterized in that The antibody or aptamer is selected from C-reactive protein (CRP) antibody, serum amyloid protein (SAA) antibody, procalcitonin (PCT) antibody, alpha-fetoprotein (AFP) antibody, carcinoembryonic antigen (CEA) antibody, prostate specific antigen (PSA) antibody, cardiac troponin (CTn-I) antibody and / or oligonucleotide fragment.

26. A method for preparing the long afterglow luminescent organic microspheres according to any one of claims 1 to 20, comprising the following steps: (1) providing components A) to C); and (2) Dispersing and adsorbing components A) to C) on a carrier medium component D) in a dispersion or solution.

27. The method according to claim 26, characterized in that Components A) to C) are dispersed or dissolved using one or more solvents selected from liquid paraffin, a mixture of phenylethanol-ethylene glycol and water, a mixture of mesitylene and ethanol, tetrahydrofuran and dichloromethane.

28. A method for preparing the probe according to claim 21, wherein an antibody or an aptamer is adsorbed or modified on the long afterglow luminescent organic microsphere according to any one of claims 1 to 20.

29. The method according to claim 28, comprising bio-coupling the long afterglow luminescent organic microspheres with antibodies or aptamers via functional reactive groups.

30. The method according to claim 29, wherein the functional reactive group is selected from a carboxyl group, an amino group or an aldehyde group.

31. A test paper for immunochromatographic detection, comprising the long afterglow luminescent organic microspheres according to any one of claims 1 to 20 or the probes according to any one of claims 21 to 25.

32. The test paper according to claim 31, comprising a sample pad, a conjugation pad, a test line and a quality control line, wherein the conjugation pad is provided with the long afterglow luminescent organic microsphere or the probe.

33. An immunochromatographic detection method, which does not involve the diagnosis and treatment of a disease, comprising the following steps: (1) providing the long afterglow luminescent organic microsphere according to any one of claims 1 to 20, the probe according to any one of claims 21 to 25, or the test paper according to any one of claims 31 to 32; (2) irradiating the organic microspheres, probes or test papers with excitation light; and (3) Stop irradiation and read the luminescence signal.

34. The detection method according to claim 33, characterized in that: The excitation wavelength can be adjusted in the range of 300 nm–1000 nm.

35. The detection method according to claim 33, characterized in that: The light exposure time was 2s–10s.

36. The detection method according to claim 33, characterized in that: The instrument used to read the luminescence signal is selected from a mobile phone, a luminescence imaging system and / or a professional long afterglow luminescence detection device.

37. The detection method according to claim 33, characterized in that: The instrument for reading the luminescent signal is selected from a mobile phone.

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