WO2021109057A1

WO2021109057A1 - Long-afterglow luminescent organic microspheres, and method for preparation thereof and application thereof

Info

Publication number: WO2021109057A1
Application number: PCT/CN2019/123208
Authority: WO
Inventors: 李富友; 徐�明; 陈锐; 吴勇
Original assignee: 复旦大学; 上海泰辉生物科技有限公司
Priority date: 2019-12-05
Filing date: 2019-12-05
Publication date: 2021-06-10
Also published as: CN114761517A

Abstract

The present invention relates to long-afterglow luminescent organic microspheres, containing (A) at least one light-absorbing agent, and (B) at least one luminescent agent, said luminescent agent being a monomeric non-polymeric compound and its molecular weight being less than 10000 g mol^-1, (C) at least one photochemical buffering agent having the formula (I), and (D) a carrier medium used for adsorbing components (A) through (C); light-absorbing agent and the luminescent agent are compounds having different structures, and calculating according to the total mass of the four components (A) through (D), the content of the carrier medium is 30% to 99%, more preferably 35% to 95%, and most preferably 40% to 90%. The organic microspheres of the present invention are particularly suitable for immunochromatographic assay technology. In addition, the present invention also relates to a reagent strip, probe, and detection method used for immunochromatographic assays.

Description

Long afterglow light-emitting organic microspheres, preparation method and application thereof

Technical field

The present invention relates to a long-lasting light-emitting organic microsphere, and a preparation method and application thereof, in particular to the application of the long-lasting light-emitting organic microsphere in immunochromatographic detection technology.

Background technique

Immunochromatography (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 medicine. Inspection, environmental monitoring and food safety and other fields. Immunochromatographic detection technology effectively combines chromatographic technology and antigen-antibody immunoreaction technology. An immunochromatographic test strip is often used in immunochromatographic detection technology. Its main structure is supported by a polyvinyl chloride base plate, on which a sample pad (such as glass cellulose membrane) and a binding pad (such as glass Cellulose membrane), nitrocellulose membrane (NC membrane) and absorbent pad. When the sample flows under capillary action, the antigen is first combined with the probe on the binding pad to form an immune complex. As the liquid continues to flow, the immune complex will be enriched and trapped on the detection line (T line) of the NC membrane , The probes that have not formed immune complexes will be intercepted by the quality control line (line C), and finally interpreted by naked eyes or instruments.

Currently widely used immunochromatographic test strips mainly include colloidal gold immunochromatographic test strips and fluorescence immunochromatographic test strips. The 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 the antibody or antibody-coated microspheres, or embedded in the microspheres to modify the antibody, and use a light source such as ultraviolet light to excite the dye to emit light. However, during the detection of the luminescence signal, samples such as blood have spontaneous fluorescence signal interference, and the illumination of the excitation light source will also cause the interference of the optical signal. These unfavorable factors will seriously affect the accuracy and stability of the detection signal.

Long afterglow luminescent materials are a kind of special luminescent materials, which can continue to emit light for a long time after the excitation light source is removed. In the prior art, the long-lasting luminescent material generally has a luminescence lifetime of more than one hundred milliseconds, and it has important application value in the fields of biomedicine, life science and the like. At present, commercial long-lasting luminescent materials are generally inorganic long-lasting materials such as aluminates, silicates, titanates or sulfides doped with rare earths or transition metals. The use of long-lasting luminescent materials as signal indicator probes in immunochromatographic detection can avoid the interference of excitation light and background fluorescence. For example, CN105929155A discloses an immunochromatographic test paper based on long afterglow and its detection method, which uses inorganic long afterglow luminescent materials, which effectively eliminates interfering signals and improves the detection sensitivity and quantitative accuracy of the object to be detected.

These inorganic long-lasting 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-lasting luminescence properties. The luminescence properties of inorganic long-lasting materials synthesized by other non-high-temperature methods are significantly reduced and difficult to obtain widely used. However, the high-temperature solid-phase reaction conditions are harsh and energy consumption is high, 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 means of 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 1000 nm). For example, if the commercial micron-sized inorganic long afterglow powder is ground to the order of 100nm, the luminous brightness of the microspheres may be reduced by two orders of magnitude.

Therefore, it is currently difficult to prepare inorganic long-lasting luminescent microspheres, and the luminescence of inorganic long-lasting luminescent microspheres is weak, and it is difficult to obtain a long-lasting luminescence signal that is clearly visible to the naked eye. The detection of weak signals requires the help of complex professional equipment, and the effect when applied to immunochromatographic detection is also relatively limited.

Summary of the invention

In view of the above-mentioned defects in the prior art, the present invention provides a long-lasting light-emitting organic microsphere with higher brightness without affecting the afterglow time and possibly even longer.

Different from the prior art inorganic long-lasting luminescent material based on photophysical process, the long-lasting luminescent material of the present invention is based on an organic system, which utilizes the characteristics of photochemical reaction to introduce a photochemical reaction between light energy input and light energy output , The organic integration of optical physics and chemistry. In the long afterglow luminescent material based on this organic system, the luminescence process can involve the photochemical interaction between a variety of chemical substances. After a series of photochemical energy conversion and metabolism processes, the input excitation light energy is finally released in the form of luminescence. , So as to achieve long afterglow emission. Energy conversion and metabolism processes include energy input, energy buffering, energy extraction, energy transfer and energy release. Through the photochemical reaction, the originally very rapid photon radiation transition process (nanosecond to microsecond) 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) Level to hour level), greatly improving the short-lived limitation of organic molecules.

Furthermore, the inventor found that long afterglow materials based on organic systems can not only obtain longer afterglow and higher brightness afterglow, but also some long afterglow light-emitting organic materials are particularly suitable for making long-lasting light-emitting microspheres without loss of afterglow brightness. And the duration, which is particularly suitable for immunochromatographic detection technology. Even in many cases, the afterglow brightness can be increased to the level visible to the naked eye and above, and the long afterglow luminous signal can be collected and analyzed by common electronic devices such as mobile phones, which greatly increases the practicability of the material.

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

In this application, unless otherwise specified, the terms "long-lasting light-emitting organic microspheres", "long-lasting materials" and "long-lasting microspheres" have the same meaning and can be used interchangeably.

Therefore, in the first aspect, the present invention provides a long-lasting light-emitting organic microsphere, which comprises

A) at least one light absorbing agent,

B) At least one luminescent agent, which is a monomeric non-polymeric compound and its molecular weight is less than 10000g mol ^-1 ,

C) at least one photochemical caching agent of formula (I),

Where formula (I) is described in detail below, and

D) The carrier medium used to adsorb components A) to C);

Wherein, the light absorbing agent 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% based on the total mass of the four components A) to D) And most preferably 40% to 90%.

Preferably, the long-lasting light-emitting organic nanoparticles are composed of components A) to D).

In a second aspect, the present invention provides a probe containing the above-mentioned long-lasting light-emitting organic microspheres.

In the third aspect, the present invention provides a method for preparing the above-mentioned long-lasting light-emitting organic microspheres.

In a fourth aspect, the present invention provides a method for preparing a probe containing the above-mentioned long-lasting light-emitting organic microspheres.

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

In the sixth aspect, the present invention provides a method for immunochromatographic detection using the above-mentioned long afterglow light-emitting organic microspheres.

Other aspects of the invention are embodied in other independent claims and dependent claims.

In addition to the above advantages, the long-lasting luminescent microspheres according to the present invention have flexible component formulations, can design the composition and properties of materials according to actual needs, and can obtain flexible and diverse nanostructures, and have tailorable luminescence properties. The wavelength of the charged excitation light and the wavelength of the long afterglow emission can be adjusted separately, and it is convenient to adjust and replace the combination scheme of the light absorber and the luminescent agent, so as to efficiently realize the colorful long afterglow emission.

Preferably, the long-lasting luminescent nanomaterial according to the present invention does not contain or contains a very small amount of inorganic long-lasting components such as SrAl ₂ O ₄ :Eu ²⁺ , Dy ³⁺ , for example, no more than 0.1% by weight based on the material mixture.

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 nanometer particle 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 images taken by an electron microscope, and the average diameter of the microspheres obtained by multiple measurements is recorded as the particle size. The characterization method of such microspheres is known to the skilled person and can be measured, for example, using scanning electron microscope (SEM) and transmission electron microscope (TEM) instruments.

Under the same test conditions, the luminescence intensity of the long-lasting light-emitting organic microspheres according to the present invention can far exceed the level of the nano-scale commercial inorganic long-lasting material SrAl ₂ O ₄ :Eu ²⁺ , Dy ³⁺ . In particular, the long afterglow light-emitting organic microspheres according to the present invention can continue to emit light after the excitation light is turned off, and the long afterglow emission time can reach 100ms-3600s, preferably 500ms-1200s, more preferably 1s-600s, 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 0.32mcd m ^-2 -8000mcd m ^-2 , more preferably 1mcd 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.

In addition, the long afterglow microspheres of the present invention can prepare immunochromatographic nanoprobes with high afterglow brightness, and can obtain test strips for immunochromatographic detection with high stability, good repeatability and high sensitivity. Detection objects include mycotoxins, pathogenic bacteria, viruses, inflammatory factors and tumor markers.

Light absorbers and luminescent agents

In this application, the light absorbing agent and the luminescent agent themselves are known in the prior art. Light absorbers generally refer to substances that can absorb and capture light energy from natural light sources or artificial light sources. The selection of light absorbing agents includes traditional photosensitive reagents and other energy donor materials. The luminescent agent usually refers to a substance that can finally emit energy in the form of light energy. The luminescent agent may be a luminescent substance capable of generating fluorescence or phosphorescence. Related luminescent molecular groups are known per se, and can refer to, for example, the review paper Nature Methods, 2005, 2,910-919 by Jeff W. Lichtman et al.

In order to achieve the beneficial effects of the long afterglow material of the present invention, in particular, for example, to improve 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 component is responsible for absorbing light. The function of energy and release of light energy, so as to realize the energy utilization path of energy input, energy buffer and energy output after being combined with the specific selected photochemical buffer agent. This also means that, in an advantageous embodiment, a compound that has both a light-absorbing group and a light-emitting group in structure so that it can perform two functions with the same molecule is not a luminescent agent or a light-absorbing agent according to the present invention, and neither The excellent technical effect of the present invention will 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 cannot separately adjust the excitation and luminescence properties of the long afterglow material, for example, when it is based on the actual excitation and charging requirements. After selecting a compound, the luminescent properties of the material are also fixed at the same time, and vice versa; on the other hand, such a compound is equivalent to fixing the ratio of light absorber to luminescent agent at 1:1, and cannot adjust the intensity of light absorption at the same time. The level of weak and luminous levels; moreover, there are relatively few materials that have both high-efficiency absorption and high-efficiency luminescence functions, which limits the variety of long-lasting materials.

In the long-lasting luminescent material according to the present invention, the selection of its light-absorbing agent and luminescent agent has certain rules and standards. Generally speaking, a compound with a larger molar absorption coefficient is selected as a light absorbing agent, such as a photosensitizer or an energy donor dye; and a compound with a higher luminous 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.

The inventor of the present application found that in the long-lasting light-emitting organic microspheres according to the present invention, especially in terms of immunochromatographic detection technology, from the perspective of improving the luminescence brightness or luminescence signal intensity, the light absorber and the luminescence agent should be Advantageously, it is at least one compound of different molecular formulas or different structures selected from the following: porphyrin and phthalocyanine dyes, metal complexes, acene compounds, fluoroboron dipyrrole compounds (BODIPY), quantum dots (QDs) ), graphenes, 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 10,000 g mol ^-1 . In the context of the present application, the molecular weight refers to the weight average molecular weight of the compound, which can be measured by the methods of mass spectrometry, gas chromatography, and liquid chromatography. The available instrument can be, for example, a mass spectrometer or a liquid phase-mass spectrometer. Here, the non-polymeric compound means that the structure of the compound does not contain more than 2 repeating units obtained by polymerization or oligomerization.

More advantageously, especially from the consideration of immunochromatographic detection technology, the light absorbing agent and luminescent agent preferably used in the long-lasting organic microspheres of the present invention are selected from the following respectively.

(1) Light absorber

Preferably, the light absorbing agent 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 absorbing agents are mentioned below.

As porphyrin dyes and their complexes, for example, the following compounds can be mentioned:

As phthalocyanine dyes and their complexes, for example, the following can be mentioned:

In the structural formulas of these light absorber compounds shown above,

X represents halogen such as F, Cl, Br, I; and

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

Each substituent R such as R _1-24 represents H, hydroxyl, carboxyl, amino, mercapto, ester, aldehyde, nitro, sulfonic acid, halogen, or has 1-50, preferably 1-24, such as 2-14 Alkyl, alkenyl, alkynyl, aryl, N, O, or S heteroaryl, alkoxy, alkylamino, or a combination of three carbon atoms. Preferably, the aforementioned groups R such as R _1-24 are each independently selected from methoxy, ethoxy, dimethylamino, diethylamino, methyl, ethyl, propyl, butyl, tert-butyl, benzene Base or a combination of them.

Transition metal complexes that can be used as light absorbing agents are known per se, and are preferably complexes of porphyrin and phthalocyanine dyes as shown above.

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

Heavy metal quantum dots include, for example, Ag ₂ S, CdS, CdSe, PbS, CuInS, CuInSe, CuInGaS, CuInGaSe, InP quantum dots. A shell layer can be wrapped around it to form a core-shell structure. The shell layer can be one or more of Ag ₂ S, CdS, CdSe, PbS, CuInS, CuInSe, CuInGaS, CuInGaSe, or a ZnS layer.

Preferably, the quantum dots are modified with surface ligands. The surface ligands may be, for example, oleic acid, oleylamine, octadecene, octadecylamine, n-dodecanethiol, and combinations thereof. In some more favorable cases, the ligands on the surface of the quantum dots are partially replaced with molecular structures containing triplet states through a ligand exchange strategy, such as carboxyanthracene, carboxytetracene, carboxypentacene, aminoanthracene, aminotetracene , Aminopentacene, mercaptoanthracene, mercaptotetracene, mercaptopentacene, etc.

In a more preferred embodiment, the light absorbing agent is preferably selected from complexes of porphyrin and phthalocyanine, quantum dots (QDs), and derivatives of these compounds. For example, these exemplary compounds or compounds are as follows:

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

(2) Luminescent agent

Preferably, the luminescent agent may be selected from iridium complexes, rare earth complexes, acene compounds, fluoroboron dipyrrole compounds (BODIPY), and derivatives and copolymers of these compounds.

As the fluoroboron dipyrrole compound (BODIPY), for example, the following compounds can be mentioned:

As acene compounds, for example, the following compounds can be mentioned:

In the structural formulas of these luminescent agent compounds shown above,

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

Each substituent R such as R _1-16 represents H, hydroxyl, carboxy, amino, mercapto, ester, aldehyde, nitro, sulfonic acid, halogen, or has 1-50, preferably 1-24, such as 2-14 Alkyl, alkenyl, alkynyl, aryl, N, O, or S heteroaryl, alkoxy, alkylamino, or a combination of three carbon atoms. Preferably the group R such as R _{1-16 is} selected from methoxy, ethoxy, dimethylamino, diethylamino, methyl, ethyl, propyl, butyl, tert-butyl, phenyl; or their combination.

In an iridium complex suitable as a luminescent agent reagent, the composition of the ligand can be a combination of one or more different ligands. The schematic structure and the types of CN, NN, OO and ON ligands are exemplified as follows (wherein The CN, NN, OO and ON ligands shown are their schematic structural diagrams and highlight the coordination sites and the C and N atoms, two N atoms, two O atoms, and O and N atoms in the ligands respectively. The iridium atom Ir performs coordination, and such a representation method is familiar and understood by those skilled in the art):

(Where DMSO is dimethyl sulfoxide)

The C-N ligand may have, for example, the following structure:

The O-N ligand may have, for example, the following structure:

The N-N ligand may have, for example, the following structure:

The rare earth complex as a luminescent agent can 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. Generally, the central atom is Eu, Tb, Sm, Yb, Nd, Dy, Er, Ho, Pr, etc. The coordination number of these rare earth complexes is about 3-12, preferably 6-10. In actual rare earth complexes, the type of ligand, the number of each ligand and the total coordination number can vary. For rare earth complexes and their ligands, please refer to the review paper Coord. Chem. Rev., 2015, 293-294, 19-47 by Jean-Claude G. Bünzli, for example.

In a more preferred embodiment, the luminescent agent is selected from the group consisting of iridium complexes, rare earth complexes, fluoroboron dipyrrole compounds (BODIPY), perylene and derivatives of these compounds. For example, these exemplary compounds or compounds are as follows:

Photochemical caching agent

In the long afterglow luminescent material according to the present invention, the photochemical buffer agent is important. The function of the photochemical buffer agent 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 very weakly, and its molecular structure generally does not contain directly luminescent groups or conjugated structures. . In particular, the photochemical buffer agent according to the present invention is distinguished from the luminescent agent or light absorbing agent in kind, especially those luminescent agent or light absorbing agent substances listed in the present invention. The photochemical buffer agent 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 absorbing agent. In the photochemical reaction, through the reaction steps of addition, rearrangement or bond breaking, the energy extraction process of transition between energy levels is activated.

The photochemical buffer agent according to the present invention is a non-polymeric small molecule compound, and the molecular weight is preferably less than 2000 g mol ^-1 , more preferably less than 1000 g mol ^-1 . Similarly, the non-polymeric compound means that the structure of the buffer compound does not contain more than 2 repeating units obtained by polymerization or oligomerization.

In particular, the inventors found that some specific buffer compounds are particularly suitable for preparing stable and long-lasting microspheres with good luminescence properties. The buffering agent in the long-lasting luminescent microspheres suitable for the present invention is selected from the following structural formula (I):

among them,

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

R ₁ ′ and R ₂ ′ and R ₄ ′ to R ₈ ′ are each independently selected from H, hydroxyl, carboxyl, amino, mercapto, ester, nitro, sulfonic, halogen, amide, or have 1-50 , Preferably 1-24, such as 2-14 carbon atoms alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, aryl, aralkyl, heteroaryl with N, O or S Group or heteroaralkyl group, or a combination thereof, wherein the aryl group, aralkyl group, heteroaryl group or heteroaralkyl group optionally has one or more substituents L; and

L is selected from hydroxyl group, carboxyl group, amino group, mercapto group, ester group, nitro group, sulfonic acid group, halogen, amide group, or having 1-50, preferably 1-24, such as 2-14 or 6-12 carbon atoms Alkyl, alkenyl, alkynyl, alkoxy, and alkylamino, or combinations thereof; and

R ₃ ′ is an electron withdrawing group or an aryl group containing an electron withdrawing group.

In the context of this application, "aryl" means a group or ring formed by an aromatic compound that is distinguished from aliphatic compounds, which is directly connected to another structural group or to another structural group through one or more single bonds. The ring structure is fused, so it is 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 group" or an "aryloxy group" or an "arylester group". Similarly, it also applies to "heteroaryl groups", which can be regarded as replacing ring carbon atoms on aryl groups with heteroatoms N, S, Se or O or replacing aliphatic rings such as cycloolefins with said heteroatoms. A group formed from a carbon atom. In addition, if there is no indication to the contrary, the "aryl" or "heteroaryl" also includes aryl or heteroaryl substituted or fused with aryl, heteroaryl, such as biphenyl, phenylthienyl Or benzothiazolyl. In addition, the “aryl group” or “heteroaryl group” may also include groups formed by aromatic or heteroaromatic compounds having functional groups such as ether groups or carbonyl groups, such as anthrone, diphenyl ether, or thiazolone Wait. Advantageously, the "aryl" or "heteroaryl" according to the present invention has 4-30, more preferably 5-24, for example 6-14 or 6-10 carbon atoms. The term "fused" means that two aromatic rings have a common edge.

In the context of this application, the term "alkyl", "alkoxy" or "alkylthio" refers to a linear, branched, or cyclic saturated aliphatic hydrocarbon group, which is connected through a single bond, an oxy group, or a sulfur group. The group is connected to other groups, and it preferably has 1-50, more preferably 1-24, such as 1-18 carbon atoms. The term "alkenyl" or "alkynyl" refers to a linear, branched or cyclic unsaturated aliphatic hydrocarbon group with one or more CC double bonds or triple bonds, preferably 2-50, more preferably 2 -24, such as 4-18 carbon atoms.

In the context of this application, the term "alkylamino" refers to one or more alkyl substituted amino groups, including monoalkylamino or dialkylamino groups, such as methylamino, dimethylamino, diethylamino, diethylamino, and dialkylamino groups. Butylamino and so on.

In the context of this application, the term "halogen" includes fluorine, chlorine, bromine and iodine, with fluorine being preferred.

In the context of the present application, the term "electron withdrawing group" is understood as 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.

In addition, in the context of this application, the optional groups in the definitions of the various substituents listed can be combined with each other to form a new substituent that conforms to the principle of valence bond, which means that, for example, an alkyl group, an ester group, and a vinyl group are combined to form a new substituent group. For example, C1-C6 alkyl ester vinyl group (C _1-6 alkyl-OC(=O)-C=C-) is also in the definition of related substituents.

In a preferred embodiment, the ring part

Can be selected from

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

In a preferred embodiment, R ₁ ′ and R ₂ ′ and R ₄ ′ to R ₈ ′ are each independently selected from alkyl groups having 1-18, preferably 1-12, more preferably 1-16 carbon atoms, Alkoxy, alkylamino or aryl or a combination thereof, wherein the aryl group may be substituted or unsubstituted by one or more groups L and is preferably a phenyl group substituted or unsubstituted by one or more L groups.

Preferably, L is selected from hydroxyl, sulfonic acid, halogen, nitro, linear or branched alkyl having 1-12, more preferably 1-6 carbon atoms, alkoxy, alkylamino, amino, Or a combination of them.

More preferably, the groups R ₁ ′ and R ₂ ′ and R ₄ ′ to R ₈ ′ are selected from methoxy, ethoxy, dimethylamino, diethylamino, dibutylamino, methyl, ethyl, propyl Group, butyl group, tert-butyl group, or a combination thereof.

More preferably, the group R ₃ ′ is selected from an electron withdrawing group or an aryl group containing an electron withdrawing group, and the electron withdrawing group is preferably selected from a nitro group, a cyano group, a halogen, a halogenated alkyl group, and/or a combination thereof. Correspondingly, the aryl group containing an electron withdrawing group preferably includes an aryl group having one or more substituents selected from nitro, cyano, halogen and/or haloalkyl on the ring, preferably phenyl, such as fluorophenyl Or perfluorophenyl.

In a particularly preferred embodiment, the photochemical caching agent is selected from, for example, the following compounds:

Carrier medium

In addition to the aforementioned component A) light-absorbing agent, component B) luminescent agent and component C) photochemical buffering agent, the long-lasting organic microspheres of the present invention must also contain component D) carrier medium. Optionally, in addition to these, other processing aids used for the preparation of microspheres, or components that further improve the long-lasting luminescence effect may be included.

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 supporting the components A) to C). The inventors of the present application have found that the long afterglow organic microspheres of the present invention 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 detection test paper. The carrier medium is selected from styrene polymer microspheres, protein One or more of nanomedia and silicon microspheres, more preferably nanomedia formed by protein and styrene polymer microspheres. Silicon microspheres refer to silica microspheres. Styrene polymer microspheres include homopolymers of styrene or copolymers formed with other copolymerizable monomers. Examples of the copolymerizable monomers include olefins, alkynes, or unsaturated carboxylic acids or their anhydrides or esters. Etc., such as butadiene, maleic anhydride or (meth)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 size can be synthesized by known methods. The protein used to form the protein nanomedia is not particularly limited in theory, but is preferably one or more of bovine serum albumin (BSA), human serum albumin (HSA), silk fibroin, and casein, and more preferably bovine serum albumin (HSA). Serum albumin. Methods of forming microspheres from these proteins are also known in the art. In addition, it may be preferable to make the surface of these carrier substrates contain amino, carboxyl, and/or aldehyde groups, so that the surface of the microspheres of the present invention can be coupled with antibodies or aptamers using these groups. The body can react with specific antigens.

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 microsphere-shaped carrier media such as silicon microspheres to be mixed with other components to form long afterglow microspheres, or to mix non-spherical carrier media such as protein nanocarrier media with other components, and then The long afterglow microspheres are formed by a known microsphere formation process (as shown in the examples). The microsphere-shaped 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, and the like. Generally speaking, as the particle size of the long afterglow organic microspheres increases, the quantity or quality of the components A), B) and C) contained in a single microsphere increases, so that the long afterglow luminescence of a single microsphere increases to The high-efficiency detection of the test signal in the immunochromatography process is advantageous; but too large particle size is not conducive to the lateral chromatography of the microspheres on the test strip. Therefore, in order to obtain an ideal immunochromatographic detection effect, the long-lasting light-emitting 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.

In an advantageous embodiment, the content of the carrier medium D) based on the total mass of the four components A) to D) is preferably 30% to 98%, more preferably 35% to 95%, most preferably 40% To 90%, such as 50% to 80%. When the content of 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 D component is too low, the dispersibility and stability of the formed microspheres are poor, and even the material cannot form nanostructures, which cannot meet the application requirements of immunoassay.

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 an appropriate 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 content of the photochemical buffer agent based on the total mass of the three components A) to C) of the material may be 0.1% to 80%, preferably 0.3% to 60%, more preferably 0.5% to 40% , Most preferably 1% to 20%.

When the proportion of light absorber is too high, the adverse effect of long afterglow luminescence is absorbed by the light absorber and weakened. When the proportion of light absorber is too low, the absorbed excitation light energy is relatively limited, which will also result in weaker long afterglow luminescence. In addition, when the photochemical buffering agent is too small, the energy buffering capacity is weak, resulting in adverse effects on the performance of long-lasting luminescence, such as affecting the stability and brightness of long-lasting luminescence. When too much buffer agent is added in the system, it will hinder the collision energy transfer between the components, and the buffer energy cannot be effectively transmitted out and is dissipated, which reduces the long afterglow luminescence performance.

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

The long afterglow luminescent material of the present invention can easily control the excitation and emission wavelengths of the system, and can cover the spectral regions of violet, blue, green, yellow, red and near-infrared. By selecting the type of light absorber or luminescent agent and appropriate structural modification when necessary, the operation 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 300 nm to 1000 nm. In addition, the long afterglow light emission can be light emission based on an up-conversion mechanism, light emission based on a down-conversion mechanism, or light emission with zero Stokes shift. When using light with a wavelength of λ1 for excitation, the emission wavelength of long afterglow luminescence λ2 is flexibly distributed, and long afterglow luminescence can cover all wavelengths from ultraviolet to visible to near-infrared. When λ1<λ2, shorter wavelength light is excited to achieve longer wavelength light emission, that is, the excitation light wavelength is red-shifted from the emitted light wavelength, which belongs to the conventional down-conversion luminescence mode; when λ1>λ2, the longer wavelength The excitation light is excited to achieve shorter wavelength light emission, that is, the excitation light wavelength is blue-shifted than the emission light wavelength, which belongs to the up-conversion luminescence mode; when λ1=λ2, that is, the excitation light wavelength and the emission light wavelength are in the same wavelength band, which belongs to Luminous mode with zero Stokes shift.

A variety of light sources can be used to excite and charge the long-lasting luminescent material of the present invention. Common light source lighting equipment, point light sources, ring light sources, indoor and outdoor natural light can all excite and charge the long afterglow luminescent agent system based on photochemical mechanism. In a preferred solution, these light sources include solid-state 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, and incandescent lamps. Lamps, fluorescent lamps and natural sunlight, and combinations of these light sources. In a more preferred solution, lasers and light-emitting diodes are used as excitation light sources. These light sources have good monochromaticity and high luminous brightness, and can selectively and quickly excite and charge. In practical applications, the light source emits The outgoing light can be a focused, divergent, circular, or collimated beam. The light output intensity of the excitation light source can have a wide range of power density (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 continuous light, pulsed light or a combined output mode, where the pulsed light is modulated and has a wide modulation frequency range (0.001Hz-100KHz). In an advantageous solution, the ultra-bright long-lasting luminescent material according to the present invention requires a short excitation time, and the irradiation time of the excitation light is 0.1s-100s, preferably 0.5s-60s, more preferably 1s-30s, most preferably 2s–10s.

In the second aspect, the present invention relates to a probe containing the above-mentioned long-lasting light-emitting organic microspheres. The probe includes the long-lasting light-emitting organic microsphere as described above and an antibody or aptamer loaded or coupled to it.

In an advantageous embodiment, the content of the antibody or aptamer in the probe is preferably 1%-20% based on the mass of the entire probe, more preferably 2%-15%, most preferably 5%-12 %.

Theoretically, there are no particular restrictions on suitable antibodies or aptamers. Preferably they are capable of specific immunological binding to target antigens including mycotoxins, pathogenic bacteria, viruses, inflammatory factors or tumor markers waiting to be detected, preferably from C-reactive protein (CRP) antibodies, serum amyloid (SAA) antibodies , Procalcitonin (PCT) antibody, alpha-fetoprotein (AFP) antibody, carcinoembryonic antigen (CEA) antibody, prostate specific antigen (PSA) antibody, cardiac troponin (CTn-I) antibody and/or oligomeric nucleus Nucleotide fragments.

In the third aspect, the present invention relates to a method for preparing long-lasting light-emitting organic microspheres as described above, which includes the following steps:

(1) Provide components A) to C); and

(2) Disperse and adsorb components A) to C) on the carrier medium component D) in a dispersion or solution.

Here, it can be advantageous to first mix components A) to C) with each other, and then disperse or dissolve them in a suitable solvent, or disperse or dissolve components A) to C) in a suitable solvent in sequence to Form a solution. The suitable solvent is not particularly limited, as long as it can form a stable solution or dispersion. The solvent can be, for example, liquid paraffin, a mixture of phenylethyl alcohol-ethylene glycol and water, a mixture of mesitylene and ethanol, tetrahydrofuran, two Methyl chloride and so on.

After the solution or dispersion containing the components A) to C) is prepared, the carrier medium microspheres or the solution or dispersion thereof can be added thereto. Alternatively, the solution or dispersion containing the components A) to C) can also 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), and the like.

In the process of preparing the solution or dispersion, auxiliary equipment such as ultrasonic and high-pressure homogenizer can be used when necessary, or appropriate heating and stirring can be carried out.

After step (2) obtains a stable dispersion of long-lasting light-emitting organic microspheres containing components A) to D), they can be directly used for subsequent use without treatment as needed, such as for preparing suitable for immunoassays. Test paper. Alternatively, antibodies or aptamers can be further adsorbed or modified on the obtained microspheres, thereby obtaining the probe according to the present invention.

The preparation method of the probe itself is known or can be obtained by a person skilled in the art with a slight improvement based on the known technology. The preparation method mainly includes biological coupling of the above-mentioned long-lasting luminescent microspheres with antibodies or aptamers through functional reactive groups such as carboxyl groups, amino groups, and aldehyde groups. For example, a carboxy-amino group can react to form a coupling or an aldehyde group-amino group can react to form a coupling. Generally, the corresponding coupling method is selected according to the situation of the functional groups on the surface of the microspheres.

Therefore, the fourth aspect of the present invention relates to the preparation method of the probe as described above.

In a fifth aspect, the present invention provides a test paper for immunochromatographic detection comprising the long-lasting light-emitting organic microspheres as described above or the probes as described above. The test paper includes a sample pad, a bonding pad, a test line and a quality control line, wherein the bonding pad is provided with the above-mentioned long afterglow light-emitting organic microspheres or the above-mentioned probe.

The structure of the test paper itself used in immunochromatographic detection technology is known. The bonding pad, test line and quality control line can be attached to the bottom plate, such as a PVC bottom plate. For the structure of such a test paper, you can refer to, for example, the published patent document CN105929155A, the entire content of which is hereby incorporated into this application.

In an exemplary structure shown in FIG. 3, the test paper includes a PVC bottom plate 1, on which a sample pad 2, a binding pad 3, a nitrocellulose membrane 4, and a water-absorbing pad 5 are sequentially arranged, wherein the nitrocellulose membrane Along the direction from the sample pad 2 to the water-absorbent pad 5, a test line 6 and a quality control line 7 are also arranged on the 4 in turn.

Immunochromatographic techniques mainly include double antibody sandwich and competition methods. The double-antibody sandwich method is mainly used to detect proteins and other macromolecular substances, such as tumor markers, viruses and inflammatory factors. These detection methods are known per se. In an exemplary embodiment, the method uses a pair of paired antibodies against different epitopes of the antigen, the capture antibody is fixed on the T line of the NC membrane, and the detection antibody coupled and modified nanoprobe is fixed on the binding pad. Goat anti-mouse (or donkey anti-mouse, goat anti-rabbit, rabbit anti-mouse, etc.) secondary antibodies are fixed on the C line of the NC membrane as a quality control line. During the detection process, the sample is dropped on the sample pad, moves from left to right by capillary action, passes 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 molecule substances. In this method, for example, the whole antigen (the coupling product of small molecules and macromolecules) can be immobilized on the NC membrane to form a T line, and the nanoprobe modified by antibody coupling can be immobilized on the binding pad. Mouse, goat anti-rabbit, rabbit anti-mouse, etc.) secondary antibodies are used as the C-line. During the detection process, the sample is dripped onto the sample pad and passed through the binding pad, T-line and C-line sequentially through capillary action. The antigen immobilized on the T-line will competitively bind with the free antigen and antibody in the sample.

Finally, the present invention also relates to a method of immunochromatographic detection, which includes the following steps:

(1) Provide organic microspheres or probes or test papers with long afterglow emission as described above;

(2) Irradiating the organic microspheres or probes or test paper with excitation light; and

(3) Stop the irradiation and read the luminous signal.

Compared with similar technologies using inorganic long afterglow materials, such as CN105929155A, the immunochromatographic detection method of the present invention has more advantages. First, the excitation wavelength can be selected in a wider range, including the wavelength range of ultraviolet light, visible light and near-infrared light. Secondly, the absorption cross-section of the long-lasting light-emitting microspheres of the present invention is several orders of magnitude larger, which allows the light irradiation and charging time to be shorter, for example, 2s-10s is the most preferred. In addition, the long afterglow luminescence brightness of the long afterglow luminescent microspheres of the present invention is higher, which exceeds the brightness level visible to the naked eye, and the available detection equipment is more common. Preferably, the instrument used to read the luminescence signal in the detection is a mobile phone, a luminescence imaging system, a professional long afterglow luminescence detection device, and the like. More preferably, the detection device is a common commercial mobile phone, which is equipped with signal reading software, and can perform signal strength data analysis on pictures taken by the mobile phone.

Description of the drawings

Fig. 1 is a schematic diagram of the structure of a probe containing long-lasting 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.

Fig. 2 is a schematic diagram of the luminescence mechanism of the long-lasting luminescent nanomaterial according to the present invention.

Figure 3 is a schematic diagram of the immunochromatographic test strip of the present invention, including a PVC bottom plate 1, on which a sample pad 2, a binding pad 3, a nitrocellulose membrane 4, and a water-absorbing pad 5 are sequentially arranged; the upper edge of the nitrocellulose membrane 4 In the direction from the sample pad 2 to the absorbent pad 5, there are also a test line 6 and a quality control line 7 in sequence. The direction indicated by the arrow in the figure is the lateral chromatography direction.

FIG. 4 is a transmission electron microscope image of the long-lasting luminescent nanoparticles of Example 1. FIG.

Figure 5 Bright field (a) and long afterglow luminescence picture (b) of long afterglow material. The bright field is taken under indoor lighting, and the long afterglow picture is taken in the dark after the excitation light of 365nm is turned off. The sample on the left is the long afterglow luminescent nanomaterial of Example 1 of the present invention, and the sample on the right is the inorganic long afterglow SrAl ₂ O ₄ :Eu ²⁺ , Dy ³⁺ nanomaterial of Comparative Example 1.

6 is based on the standard curve of C-reactive protein (CRP) detection of the long-lasting luminescent nanomaterial of Example 31 of the present invention.

Figure 7 The effect of long afterglow immunochromatographic test strips for detecting C-reactive protein (CRP), taken with the same mobile phone. The long afterglow signal indicator probes used in immunochromatographic test strips are different. The left picture (a) is based on the CRP detection effect of the long afterglow luminescent nanomaterial of Example 31 of the present invention, and the right picture (b) is based on the comparison implementation. The CRP detection effect diagram of the inorganic long afterglow SrAl ₂ O ₄ :Eu ²⁺ ,Dy ^{3+ nanomaterial of Example 11.}

Fig. 8 is a standard curve of serum amyloid (SAA) detection based on the long-lasting luminescent nanomaterial of Example 32 of the present invention.

Fig. 9 is based on the standard curve of procalcitonin (PCT) detection of the long-lasting luminescent nanomaterial of Example 33 of the present invention.

Fig. 10 is based on the standard curve of alpha-fetoprotein (AFP) detection of the long-lasting luminescent nanomaterial of Example 34 of the present invention.

Figure 11 is based on the standard curve of carcinoembryonic antigen (CEA) detection of the long-lasting luminescent nanomaterial of Example 35 of the present invention.

Fig. 12 is based on the standard curve of prostate-specific antigen (PSA) detection of the long-lasting luminescent nanomaterial of Example 36 of the present invention.

Fig. 13 is 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

1. Performance test method

In the long afterglow luminescence test of the present invention, when a laser is used as an excitation light source, a wavelength tunable laser (Opolette 355) from Opotek, Inc. of the United States is used. Under certain circumstances, light-emitting diodes (LEDs) are also used as excitation light sources, and the power density of the excitation light remains the same. Excitation light of a specific wavelength irradiates the sample for charging, and the charging time is 3s. After charging, turn off the laser and start to test the luminous performance. A fluorescence spectrometer (Edinburgh FS-5) from Edinburgh Instruments, UK was used to test the long afterglow luminous intensity. Use the long afterglow test system (OPT-2003) of Beijing Aobodi Optoelectronics Technology Co., Ltd. to test the long afterglow luminous brightness. The invention uses a commercial smart phone or a common digital camera to take pictures, and records brightfield and long afterglow glow pictures.

The phrase "visible to the naked eye" used herein is a professional term in the field of long afterglow luminescent materials, which means that the luminous brightness of the material is greater than or equal to 0.32mcd·m ^-2 , and visible light can usually be seen by the naked eye when the brightness is at the radiation level and above. . The phrase "light-emitting time" used herein is a technical term in the field of long-lasting luminescent materials, which refers to the time elapsed when the luminous brightness of the material decays to a level visible to the naked eye. The phrase "blue long-lasting luminescence" used herein is the expression of the long-lasting luminescence color of the material, which means that there is obvious long-lasting luminescence in the blue wavelength range; similarly, the description is also applicable to this article. The description of other colors used. 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.

2. List of raw materials used

3. Preparation of long afterglow luminescent nanomaterials

Example 1

The long afterglow light-emitting organic microspheres were prepared, in which the content of the carrier medium was 75% based on the total mass of the four components A) to D). First, the light absorbing agent PdOEP, the luminescent agent Eu-1 and the photochemical buffer CA-1 are mixed in the methylene chloride solvent, and ultrasonic waves are used to assist the dissolution of the components. In this solution, the concentration of light absorbing agent PdOEP is 0.1 mmol L ^-1 , the molar concentration of photochemical buffer CA-1 is 3 mmol L ^-1 , the concentration of luminescent agent Eu-1 is 10 mmol L ^-1 , light absorbing agent, photochemical buffer The molar ratio of the three components of the luminescent agent and the luminescent agent is 1:30:100. Take more than 1 mL of the 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. The mixture was pre-emulsified with ultrasonic wave (Sonics VC750, Sonics & Materials, Inc) at room temperature in the dark for 5 minutes, and then the methylene chloride was removed using a rotary evaporator. 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. The emulsion was heated at 90 degrees Celsius in the dark for 1 hour. After the emulsion is cooled to room temperature, the long afterglow luminescent microspheres uniformly dispersed in water are obtained by gradient centrifugation and filtration. The above-mentioned microspheres were dyed with sodium phosphotungstate, and the morphology under the transmission electron microscope is shown in Figure 4. The afterglow performance test of the prepared long-lasting luminescent microspheres was carried out, and the long-lasting luminescent microspheres were prepared into an aqueous solution with a concentration of ^{1 mg mL -1.} First, use a 365nm wavelength LED light source to irradiate for 3s to charge. After the charge is completed, the light source is turned off, and the red long afterglow light visible to the naked eye is obtained. The test results are shown in Table 1.

Comparative Example 1 (C1)

The long-lasting light-emitting 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-lasting light-emitting nanoparticles. Among the commercially available inorganic long-lasting materials, SrAl ₂ O ₄ :Eu ²⁺ and Dy ³⁺ are currently the brightest green long-lasting luminescent materials and are widely used. The commercial SrAl ₂ O ₄ :Eu ²⁺ ,Dy ³⁺ material is a long afterglow powder obtained after high-temperature sintering and grinding. _{SrAl 2} O ₄ :Eu ²⁺ , Dy ³⁺ inorganic long afterglow nanoparticles with a particle size of about 50 nm were obtained by centrifugal separation. 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 phacoemulsification and other treatments. Finally, BSA-encapsulated inorganic long-lasting microspheres with a particle size of about 300nm were obtained. According to the method of Example 1, the BSA-coated inorganic long-lasting microspheres were prepared into ^{an aqueous solution with a concentration of 1 mg mL -1} , and the afterglow performance of the microspheres was tested. As a result, no afterglow light was seen by naked eye observation. The long afterglow luminous intensity is shown in Table 1.

Example 2-8

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

Example 9

The long afterglow light-emitting organic microspheres are prepared, in which the amount of the carrier medium is 50% based on the total mass of the four components A) to D). Add the light absorbing agent PdOEP, the luminescent agent Eu-1 and the photochemical buffer CA-1 to 5 mL of benzyl alcohol-ethylene glycol-water (v:v:v, 1:8:1) solution, and the concentration of the light absorbing agent PdOEP Is 0.1mmol L ^-1 , the concentration of photochemical buffer CA-1 is 3mmol L ^-1 , the concentration of luminescent agent Eu-1 is 10mmol L ^-1 , the molar ratio of the three components of light absorber, photochemical buffer and luminescent agent is 1:30:100. After the components were ultrasonically dispersed, 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 with ethanol and water centrifugation 3 times, and finally the microspheres were dispersed in water for storage. The afterglow performance of the prepared long-lasting luminescent microspheres was tested, and the long-lasting luminescent microspheres were prepared into an aqueous solution with a concentration of ^{1 mg mL -1.} First, irradiate the excitation light with a wavelength of 540nm for 3s for charging. After the charging is completed, the light source is turned off, and the red long afterglow luminescence visible to the naked eye is obtained. The test results are shown in Table 1.

Example 10

The long afterglow light-emitting organic microspheres are prepared, in which the amount of the carrier medium is 50% based on the total mass of the four components A) to D). Add light absorbing agent PdOEP, luminescent agent Eu-1 and photochemical buffer CA-1 to 10 mL of mesitylene-ethanol (v:v, 1:1) solution, where the concentration of light absorbing agent PdOEP is 0.1 mmol L ^-1 , The concentration of the photochemical buffer CA-1 is 3 mmol L ^-1 , the concentration of the luminescent agent Eu-1 is 10 mmol L ^-1 , and the molar ratio of the three components of the light absorber, the photochemical buffer and the luminescent agent is 1:30:100. After the components were dispersed ultrasonically, 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 with ethanol and water centrifugation 3 times, and finally the microspheres were dispersed in water for storage. The afterglow performance test of the prepared long-lasting luminescent microspheres was carried out, and the long-lasting luminescent microspheres were prepared into an aqueous solution with a concentration of ^{1 mg mL -1.} First, irradiate the excitation light with a wavelength of 540nm for 3s for charging. After the charging is completed, the light source is turned off, and the red long afterglow luminescence visible to the naked eye is obtained. The test results are shown in Table 1.

Examples 11-13

Comparative Example 2-3 (C2 and C3)

Comparative Example 4 (C4)

Using NCBS as a light-absorbing agent, PFVA as a luminescent agent, and DO as a photochemical buffer agent, long afterglow microspheres were prepared. The components are mixed in the dichloromethane solvent, and ultrasonic is used to assist the dissolution of the components, and finally a uniform and transparent solution is formed. In this solution, the concentration of the light absorbing agent NCBS is 0.1 mmol L ^-1 , the molar concentration of the photochemical buffering agent DO is 2 mmol L ^-1 , and the concentration of the luminescent agent PFVA is 10 mg mL ^-1 . Subsequently, take more than 1 mL of the 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. The mixture was pre-emulsified with ultrasonic wave (Sonics VC750, Sonics & Materials, Inc) at room temperature in the dark for 5 minutes, and then the methylene chloride was removed using a rotary evaporator. 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. The emulsion was heated at 90 degrees Celsius in the dark for 1 hour. After the emulsion is cooled to room temperature, long afterglow microspheres uniformly dispersed in water are obtained by gradient centrifugation and filtration. The afterglow performance test of the prepared long-lasting luminescent microspheres was carried out according to the method of Example 1, and the long-lasting luminescent microspheres were formulated into an aqueous solution with a concentration of ^{1 mg mL -1.} First, use 808nm wavelength excitation light for 3s to charge, and turn off the light source after the charge is completed. As a result, no afterglow is seen by naked eyes. The long afterglow luminous intensity measured by the instrument is shown in Table 1.

Comparative Example 5 (C5)

The operation of Comparative Example 6 was repeated, and the differences are shown in Table 1.

Comparative Example 6 (C6)

PtTPBP was used as a light-absorbing agent, Eu-1 was used as a luminescent agent, and CA-1 was used as a photochemical buffer agent to prepare long afterglow microspheres. First, dissolve the components in 2 mL of tetrahydrofuran (THF), where the concentration of the light-absorbing agent PdOEP is 0.1 mmol L ^-1 , the concentration of the photochemical caching agent CA-1 is 3 mmol L ^-1 , and the concentration of the luminescent agent Eu-1 is 10 mmol L ^-1 , The molar ratio of the three components: light absorbing agent, photochemical buffer agent, and luminescent agent is 1:30:100. Add 20 mg of F127 to the above solution and stir to dissolve, then remove the tetrahydrofuran, use ultrasound to disperse the obtained composition in 2 mL of water, and obtain long afterglow microspheres uniformly dispersed in water by centrifugation and filtration. The afterglow performance test of the prepared long-lasting luminescent microspheres was carried out according to the method of Example 1, and the long-lasting luminescent microspheres were formulated into an aqueous solution with a concentration of ^{1 mg mL -1.} First, use excitation light with a wavelength of 635nm for 3s to charge. After the charge is completed, the light source is turned off. No visible long afterglow luminescence is observed. The test results are shown in Table 1.

Table 1

Example 14

The light absorbing agent PdPc, the luminescent agent Eu-2 and the photochemical buffer agent CA-1 are mixed in the dichloromethane solvent, and ultrasonic waves are used to assist the dissolution of the components, and finally a uniform and transparent solution is formed. In this solution, the molar concentration of the photochemical caching agent CA-1 is 2 mmol L ^-1 , the concentration of the luminescent agent Eu-2 is 5 mmol L ^-1 , and the concentration of the light absorbing agent PdPc is 50 μmol L ^-1 . Subsequently, take more than 1 mL of the 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. The mixture was pre-emulsified with ultrasound (Sonics VC750, Sonics & Materials, Inc) at room temperature in the dark for 5 minutes, and then the methylene chloride was removed using a rotary evaporator. 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. The emulsion was heated at 90 degrees Celsius in the dark for 1 hour. After the emulsion is cooled to room temperature, long afterglow microspheres uniformly dispersed in water are obtained by gradient centrifugation and filtration. The afterglow performance test of the prepared long-lasting luminescent microspheres was carried out, and the long-lasting luminescent microspheres were prepared into an aqueous solution with a concentration of ^{1 mg mL -1.} First, irradiate the excitation light with a wavelength of 730nm for 3s for charging. After the charging is completed, turn off the light source. The test results are shown in Table 2.

Examples 15-19

The operation of Example 14 is repeated, and the difference is shown in Table 2, where the concentration of the luminescent agent Eu-2 is 5 mmol L ^-1 .

Comparative Examples 7-8 (C7 and C8)

Table 2

Example 20

The light absorbing agent PdPc, the luminescent agent Eu-1 and the photochemical buffer CA-1 are mixed in dichloromethane, and ultrasonic waves are used to assist the dissolution of the components, and finally a uniform and transparent solution is formed. In this solution, the molar ratio of the three components: light absorbing agent PdPc, photochemical caching agent CA-1, and luminescent agent Eu-1 is 1:300:3000. Then, the dichloromethane solvent is removed to obtain an oily mixture of A)/B)/C) three components. Weigh 50 mg of the three-component mixture and add it to component D) containing 30 mg of liquid paraffin and 20 mg of bovine serum albumin, wherein component D) accounts for 50% by weight. Use ultrasound (Sonics VC750, Sonics&Materials, Inc) to pre-emulsify the mixture from light at room temperature for 5 minutes, and then immediately use a high-pressure nano-homogenizer (FB-110Q, LiTu Mechanical equipment Engineering Co., Ltd) to continue emulsification from light 10 minutes. The emulsion was heated at 90 degrees Celsius in the dark for 1 hour. After the emulsion is cooled to room temperature, long afterglow microspheres uniformly dispersed in water are obtained by gradient centrifugation and filtration. The afterglow performance of the prepared long-lasting luminescent microspheres was tested, and the long-lasting luminescent microspheres were prepared into an aqueous solution with a concentration of ^{1 mg mL -1.} First, irradiate the excitation light with a wavelength of 540nm for 3s to charge. After the charge is completed, turn off the light source. The test results are shown in Table 3.

Examples 21-23

Repeat the operation of Example 20, the difference lies in the weight ratio of the D) component in the nanomaterial (as shown in Table 3). The test results are shown in Table 3.

Comparative Examples 9-10 (C9 and C10)

table 3

Example 24

Take 80nm styrene polymer (PS) microspheres with carboxyl group by centrifugation to remove the surfactant in the synthesis process, and then reconstitute 1g of PS microspheres solid into 100mL ultrapure water, and ultrasonically form a dispersed phase. Then, 1 mL each of 2% sodium dodecylbenzene sulfonate and 1% ethylenediamine polyoxyethylene polyoxypropylene block polyether were added to the PS microsphere aqueous solution, and stirred. Take the components A), B) and C) shown in Table 4 and disperse them in 10 mL of tetrahydrofuran solution to form a dispersed phase, where the concentrations of the three components A), B) and C) are 0.1 mmol L ^-1 , 2mmol L ^-1 and 10mmol L ^-1 . After the solution is prepared, the organic phase is quickly added to the water phase, and then the temperature is gradually increased to 50°C, and stirring is continued for 10 hours. Then, the obtained long afterglow PS microspheres with a particle size of 80nm were centrifuged to remove excess dye, and then washed twice with ultrapure water and ethanol, and stored in ultrapure water, protected from light at room temperature for later use.

Examples 25-28

The operation of Example 24 was repeated, except for the particle size of the carboxyl-containing styrene polymer (PS) microspheres as the nanocarrier medium (as shown in Table 4). The test results are shown in Table 4.

Table 4

Example 29

The probe is prepared by coupling the alpha-fetoprotein (AFP) antibody AFP-Ab _{1 with fluorescent long afterglow microspheres:}

1) Centrifuge 100 mg of the long afterglow luminescent microspheres prepared according to Example 26, reconstitute them in 18 mL of BBS buffer with pH 7.4, and fully sonicate them to make them uniformly dispersed; 2) Add 2 mg of 1-(3) to them. -Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and 2mg of N-hydroxysuccinimide sulfonate sodium salt (NHSS), react for 2 hours at room temperature; 3) the end of the reaction Afterwards, it was washed by centrifugation and reconstituted in 10 mL of BBS buffer with a pH of 7.4, 10 mg of AFP-Ab _{type 1} monoclonal antibody was added to it, and reacted for 4 hours at room temperature; 4) After the reaction, washed by centrifugation and reconstituted Add 100 mg of BSA to 10 mL of BBS buffer with pH 7.4, and react for 2 hours at room temperature; 5) After the reaction is over, centrifuge and wash, and reconstitute into 10 mL of BBS buffer with pH 7.4 at 4°C Save it for later use.

Example 30

The probe is prepared by coupling the prostatic specific antigen (PSA) aptamer with long afterglow microspheres:

1) Take 10 mg of the carboxyl-containing long afterglow microspheres prepared according to Example 26 by centrifugation, reconstitute it in 1.8 mL of BBS buffer (pH 7.4), and fully sonicate to make it evenly dispersed; 2) Add 0.6 mg of EDC and 0.2 mg of NHSS were shaken and reacted at room temperature for 2 hours; 3) After the reaction, washed by centrifugation, reconstituted in 2 mL of BBS buffer, and added 20 μL of PSA aptamer ^{containing 2 μmol mL -1 to it,} The sequence is (NH ₂ -ATTAAAGCTCGCCATCAAATAGCTGC), react for 4 hours at room temperature; 4) After the reaction, wash by centrifugation and reconstitute into 2mL BBS buffer, add 10mg of BSA to it, and react for 2 hours at room temperature; 5) Reaction After the end, centrifuge and wash twice, reconstitute in 4mL BBS buffer (pH 7.4), and store at 4°C for later use.

Example 31

Preparation of lateral chromatography immunoassay strip containing long afterglow luminescent microspheres of the present invention and its application in C-reactive protein (CRP) detection

(1) Preparation of immunochromatographic test strips for the detection of C-reactive protein (CRP):

1) Coupling the long afterglow microspheres in Example 1 to CRP-Ab _{1 as} follows: Use PEG with carboxyl groups at both ends to activate the carboxyl groups at both ends first, and then one end to react with the amino groups on the BSA nanospheres, and the other One end is reactively connected to the amino group on CRP-Ab _1. Among them, 10 mg of CRP-Ab _{type 1} monoclonal antibody was added to 100 mg. After the reaction, it was centrifuged and washed, reconstituted in 10 mL of BBS buffer with pH 7.4, and stored at 4°C for later use.

2) Preparation of CRP immunochromatographic test strip NC membrane: use PBS buffer (1% BSA, 1% sucrose, 50 mM NaCl and 0.5% TWEEN 20) to separate CRP-Ab _{type 2} monoclonal antibody and donkey The anti-mouse IgG ^{was streaked on the nitrocellulose membrane with a streaking device at a concentration of 1 mg mL -1} and 1 mg mL ^-1 at an interval of 8 mm, and dried at 37°C overnight.

3) Preparation of CRP immunochromatographic test strip sample pad: Take the fluorescent probe prepared in step 1), centrifuge it, and reconstitute it to 20 mg mL ^-1 with a membrane spray buffer, and use a membrane sprayer to detect the fluorescence probe. The needle solution was poured into the instrument, and the fluorescent probe was sprayed on the glass fiber at a speed of ^{1.2 μL cm -1 and baked overnight at 37°C.}

4) The assembly of CRP immunochromatographic test strips: On the white PVC bottom plate, _{the glass fibers of fluorescent microspheres labeled with CRP-Ab type 1} monoclonal antibody are affixed with CRP-Ab type 1 monoclonal antibody and marked with CRP-Ab. ₂ T line and donkey anti-mouse IgG C line NC film, and finally paste absorbent paper. Next, the assembled chromatography plate is cut into a 3.8mm wide test strip by a high-speed chopper, and then the test strip is fixed with two matching upper and lower plastic cassettes to obtain an immunochromatographic test strip.

(2) Establishment of CRP standard curve in the sample:

1) The antigen stock was diluted CRP is a whole blood sample for different concentrations of CRP antigen solutions at concentrations of ^{^{0μg mL -1, 5μg mL -1,}} 20μg mL -1, 40μg mL -1, 160μg mL -1 and 320μg mL ^-1 .

2) Take 1 μL of the 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 well.

3) Add 100 μL of the mixed solution to the sample hole of the immunochromatographic test strip, and the liquid will pass through the sample area, the detection area and the water absorption area 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 moves to the test line (T line) with the liquid to form a sandwich immune complex _{with CRP-Ab 2.} The long afterglow luminescent probe swims to the control line (line C) and binds to the donkey anti-mouse secondary antibody. When there is no antigen in the test sample, it will drive the long afterglow luminescent probe to swim directly to the C line and bind to the donkey anti-mouse secondary antibody.

4) After the reaction has proceeded for 5 minutes, the immunochromatographic test strip is tested with a long afterglow luminescence detector. By measuring the long afterglow luminescence intensity of the T line and the C line, the peak area is integrated, and then the ratio of the peak area is calculated, and the standard curve is established by the logarithm of the ratio and the antigen concentration (Figure 6). The long afterglow luminescence detector is a commercial smart phone that is used daily, and is equipped with signal reading software, which can perform data analysis of signal strength on pictures taken by the phone.

(3) Immunochromatographic detection of CRP in the sample:

It is only necessary to use the excitation light to excite the prepared probe containing the long-lasting luminescent microspheres of the present invention before the reading, and the excitation light is turned off during the subsequent reading process. This method eliminates the interference of the background fluorescence signal and can realize the waiting Highly sensitive quantitative detection of analytes. 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) had a detection sensitivity improvement of 100% compared with the detection system based on inorganic long afterglow (Figure 7 right). However, there is no obvious change in the processing requirements of the test sample, and the test time part only needs 3s in the excitation time. According to the detection result of the long afterglow luminescence immunochromatographic test strip of the present invention, the sample contains 100 ng mL ^-1 CRP antigen, which is far lower than the normal physiological level.

Comparative Example 11 (C11)

The operation of Example 31 was repeated, except that: in the first step, the inorganic long-lasting microspheres in Comparative Example 1 were used to couple CRP-Ab ₁ . According to the prepared inorganic long afterglow nano immunochromatographic test strip, the long afterglow luminescence signal is weak, invisible to the naked eye and no signal can be captured by the mobile phone, as shown in the right picture in Figure 7.

Example 32-37

Repeat the operation of Example 31 to obtain an antigen detection standard curve based on long afterglow lateral chromatography immunoassay strips. The difference is that the detected target antigens were replaced with SAA (Example 32, Figure 8), PCT ( Example 33, Figure 9), AFP (Example 34, Figure 10), CEA (Example 35, Figure 11), PSA (Example 36, Figure 12), CTn-I (Example 37, Figure 13).

Claims

A long-lasting light-emitting organic microsphere, which comprises

A) at least one light absorbing agent,

B) At least one luminescent agent, which is a monomeric non-polymeric compound and its molecular weight is less than 10000g mol -1 ,

C) at least one photochemical caching agent of formula (I),

among them,

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

R 1 ′ and R 2 ′ and R 4 ′ to R 8 ′ are each independently selected from H, hydroxyl, carboxyl, amino, mercapto, ester, nitro, sulfonic, halogen, amide, or have 1-50 , Preferably 1-24, such as 2-14 carbon atoms alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, aryl, aralkyl, heteroaryl with N, O or S Group or heteroaralkyl group, or a combination thereof, wherein the aryl group, aralkyl group, heteroaryl group or heteroaralkyl group optionally has one or more substituents L; and

L is selected from hydroxyl group, carboxyl group, amino group, mercapto group, ester group, nitro group, sulfonic acid group, halogen, amide group, or having 1-50, preferably 1-24, such as 2-14 or 6-12 carbon atoms Alkyl, alkenyl, alkynyl, alkoxy, and alkylamino, or combinations thereof; and

R 3 ′ is an electron withdrawing group or an aryl group containing an electron withdrawing group, preferably the electron withdrawing group is selected from the group consisting of nitro, halogen, halogenated alkyl, sulfonic acid, cyano, acyl, carboxyl and/or them Combination of; and

D) The carrier medium used to adsorb components A) to C);

Wherein, the light absorbing agent 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), more preferably 35% to 95% And most preferably 40% to 90%.
The long-lasting light-emitting organic microspheres according to claim 1, wherein the ring part
Selected from
More preferably, G and T are selected from S and O, most preferably one of G and T is S and the other is O.
The long-lasting light-emitting organic microspheres according to claim 1 or 2, wherein the groups R 1 ′ and R 2 ′ and R 4 ′ to R 8 ′ are each independently selected from those having 1-18, preferably 1- 12. More preferably alkyl, alkoxy, alkylamino or aryl groups or combinations of 1-16 carbon atoms, wherein the aryl group may be substituted or unsubstituted by one or more groups L and is preferably One or more L-substituted or unsubstituted phenyl groups.
The long-lasting light-emitting organic microspheres according to any one of the preceding claims, wherein L is selected from the group consisting of hydroxyl, sulfonic acid, halogen, nitro, and has 1-12, more preferably 1-6 carbon atoms. Linear or branched alkyl, alkoxy, alkylamino, amino, or a combination thereof;

It is more preferably selected from halogens, linear or branched alkyl groups having 1-12, more preferably 1-6 carbon atoms, alkoxy groups, alkylamino groups, or combinations thereof.
The long-lasting light-emitting organic microspheres according to any one of the preceding claims, wherein the groups R 1 ′ and R 2 ′ and R 4 ′ to R 8 ′ are selected from the group consisting of methoxy, ethoxy, dimethyl Amino, diethylamino, dibutylamino, methyl, ethyl, propyl, butyl, tert-butyl, or a combination thereof.
The long-lasting light-emitting organic microspheres according to any one of the preceding claims, wherein the electron withdrawing group is selected from the group consisting of nitro, cyano, halogen, halogenated alkyl and/or combinations thereof, and the inclusion The aryl group of the electron withdrawing group is selected from aryl groups having one or more substituents selected from nitro, cyano, halogen and/or haloalkyl on the ring, preferably phenyl, wherein said halogen and haloalkyl are preferably Fluorine and fluoroalkyl.
The long-lasting light-emitting organic microspheres according to any one of the preceding claims, wherein the photochemical buffer agent is selected from one or more of the following compounds:
The long-lasting light-emitting organic microspheres according to any one of the preceding claims, wherein the carrier medium is selected from one or more of styrene polymer microspheres, protein nanomedium media, and silicon microspheres, more preferably Protein nanomedium and/or styrene polymer microspheres; preferably, the surface of the carrier matrix contains amino groups, carboxyl groups and/or aldehyde groups.
The long-lasting light-emitting organic microspheres according to claim 8, wherein the protein used to form the protein nanomedium is selected from one or more of bovine serum albumin, human serum albumin, silk fibroin, and casein. Furthermore, bovine serum albumin is more preferable.
The long-lasting light-emitting organic microspheres according to any one of the preceding claims, wherein the long-lasting light-emitting organic microspheres have a particle size in the range of 5nm-1000nm, more preferably 50nm-800nm, most preferably 100nm-500nm .
The long-lasting light-emitting organic microspheres according to any one of the preceding claims, wherein 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.
The long-lasting light-emitting organic microspheres according to any one of the preceding claims, wherein the photochemical buffer agent is based on the total mass of components A) to C), and its content is 0.1% to 80%, preferably 0.3% to 60%. %, more preferably 0.5% to 40%, most preferably 1% to 20%.
The long-lasting light-emitting organic microspheres according to any one of the preceding claims, wherein the long-lasting light-emitting organic nanoparticles are composed of components A) to D).
The long-lasting light-emitting organic microspheres according to any one of the preceding claims, wherein the luminescent agent can be selected from the group consisting of iridium complexes, rare earth complexes, acene compounds, fluoroboron dipyrrole compounds (BODIPY) , And derivatives and copolymers of these compounds.
A probe, which comprises the long-lasting light-emitting organic microsphere according to any one of claims 1 to 14 and an antibody or aptamer loaded or coupled thereto.
The probe according to claim 15, wherein the content of the antibody or aptamer in the probe is preferably 1%-20%, more preferably 2%-15%, based on the mass of the entire probe, Most preferably 5%-12%.
The probe according to claim 15 or 16, wherein the antibody or aptamer is selected from C-reactive protein (CRP) antibody, serum amyloid (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 fragments.
A method for preparing long-lasting light-emitting organic microspheres according to any one of claims 1 to 14, which comprises the following steps:

(1) Provide components A) to C); and

(2) Disperse and adsorb components A) to C) on the carrier medium component D) in a dispersion or solution.
The method according to claim 18, characterized in that one or more solvents selected from the group consisting of liquid paraffin, a mixture of phenethyl alcohol-ethylene glycol and water, a mixture of mesitylene and ethanol, tetrahydrofuran and dichloromethane are used for dispersion Or dissolve components A) to C).
The method for preparing the probe according to claim 15, wherein the antibody or aptamer is adsorbed or modified on the long-lasting light-emitting organic microsphere according to any one of claims 1 to 14.
The method according to claim 20, which comprises biologically coupling the long-lasting light-emitting organic microspheres and antibodies or aptamers through functional reactive groups such as carboxyl groups, amino groups, and aldehyde groups.
A test paper for immunochromatographic detection, which comprises the long-lasting light-emitting organic microsphere according to any one of claims 1 to 14 or the probe according to any one of claims 15 to 17.
The test paper according to claim 22, which comprises a sample pad, a bonding pad, a test line and a quality control line, wherein the long afterglow light-emitting organic microspheres or the probe are arranged on the bonding pad.
The method of immunochromatographic detection includes the following steps:

(1) Provide the long-lasting light-emitting organic microspheres according to any one of claims 1 to 14, the probes according to any one of claims 15 to 17, or the probes according to any one of claims 20 to 21 Test paper

(2) Irradiating the organic microspheres or probes or test paper with excitation light; and

(3) Stop the irradiation and read the luminous signal.
The detection method according to claim 24, wherein the adjustable range of the excitation wavelength is 300nm-1000nm.
The detection method according to claim 24, wherein the light irradiation time is 2s-10s.
The detection method according to claim 24, wherein the instrument for reading the luminescence signal is selected from the group consisting of a mobile phone, a luminescence imaging system and/or a professional long afterglow luminescence detection device, more preferably a mobile phone.