WO2021109059A1 - 长余辉发光的苯乙烯聚合物微球、其制备方法和应用 - Google Patents

长余辉发光的苯乙烯聚合物微球、其制备方法和应用 Download PDF

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WO2021109059A1
WO2021109059A1 PCT/CN2019/123220 CN2019123220W WO2021109059A1 WO 2021109059 A1 WO2021109059 A1 WO 2021109059A1 CN 2019123220 W CN2019123220 W CN 2019123220W WO 2021109059 A1 WO2021109059 A1 WO 2021109059A1
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long
styrene polymer
group
emitting
light
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PCT/CN2019/123220
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English (en)
French (fr)
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李富友
徐�明
陈锐
吴勇
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复旦大学
上海泰辉生物科技有限公司
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Priority to CN201980103292.2A priority Critical patent/CN114846329B/zh
Priority to PCT/CN2019/123220 priority patent/WO2021109059A1/zh
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
    • C08K5/00—Use of organic ingredients
    • C08K5/36—Sulfur-, selenium-, or tellurium-containing compounds
    • C08K5/45—Heterocyclic compounds having sulfur in the ring
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08K—Use of inorganic or non-macromolecular organic substances as compounding ingredients
    • C08K5/00—Use of organic ingredients
    • C08K5/36—Sulfur-, selenium-, or tellurium-containing compounds
    • C08K5/45—Heterocyclic compounds having sulfur in the ring
    • C08K5/46—Heterocyclic compounds having sulfur in the ring with oxygen or nitrogen in the ring
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic

Definitions

  • the invention relates to a long-lasting light-emitting styrene polymer microsphere, and a preparation method and application thereof, in particular to the application of the long-lasting light-emitting styrene polymer microsphere in immunochromatographic detection technology.
  • 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.
  • sample pad such as glass cellulose membrane
  • binding pad such as glass Cellulose membrane
  • NC membrane nitrocellulose membrane
  • the antigen is first combined with the probe on the binding pad to form an immune complex.
  • 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.
  • 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.
  • 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 antibody-coated microspheres, or embedded in nano-microspheres to modify the antibody, and use a light source such as ultraviolet light to excite the dye to emit light.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • the luminous brightness of the nano-microspheres may be reduced by two orders of magnitude.
  • the high uniformity of the luminescent probes used is of great significance to ensure the repeatability and reliability of the detection results.
  • the inorganic long afterglow luminescent material used as the luminescent core in CN105929155A is inorganic particles (particle size greater than 15nm, even up to 250nm), and its processability in the nanometer scale is poor, and the surface of the material is not easy to modify for use. With functional groups for biological coupling, it is difficult to obtain large quantities and uniform long-lasting luminescence nanoprobes according to the technical solution disclosed in CN105929155A.
  • the present invention provides a styrene polymer-based organic long-lasting light-emitting microsphere with higher brightness without affecting the afterglow time and possibly even longer.
  • 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.
  • 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.
  • organic long-lasting light-emitting materials can be particularly suitably combined with styrene polymer microspheres to produce long-lasting light-emitting styrene polymer microspheres with higher afterglow brightness and possibly longer duration. It is particularly suitable for immunochromatographic detection.
  • the light-emitting core of the organic long afterglow system is composed of molecular components and has good processability.
  • the molecular components in the solution are easily dispersed and absorbed into uniform styrene polymer microspheres, and prepared into large quantities And with high uniformity, long afterglow light-emitting styrene polymer microspheres and probes.
  • 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.
  • photochemical reaction is a series of chain reactions, including reaction processes of photochemical addition, photooxidation, photochemical dissociation, and bond-breaking recombination.
  • the present invention provides a long-lasting light-emitting styrene polymer microsphere, which comprises
  • At least one luminescent agent which is a monomeric non-polymeric compound and its molecular weight is less than 10000g mol -1 ,
  • a carrier medium for adsorbing components A) to C), the carrier medium is styrene polymer microspheres;
  • the total content of components A) to C) is 0.1% to 30%, preferably 0.2% to 25%, more preferably 0.5% to 20%, and most preferably 1% based on the total mass of components A) to D). To 15%.
  • the long-lasting light-emitting styrene polymer microspheres are composed of components A) to D).
  • the present invention provides a probe containing the above-mentioned long-lasting light-emitting styrene polymer microspheres.
  • the present invention provides a method for preparing the above-mentioned long-lasting light-emitting styrene polymer microspheres.
  • the present invention provides a method for preparing a probe containing the above-mentioned long-lasting light-emitting styrene polymer microspheres.
  • the present invention provides a test strip for immunochromatographic detection.
  • the present invention provides a method for immunochromatographic detection using the long-lasting light-emitting styrene polymer microspheres as described above.
  • the long-lasting light-emitting styrene polymer microspheres according to the present invention have flexible formulations, the composition and properties of the materials can be designed according to actual needs, and flexible and diverse nanostructures can be obtained, and they can be tailored.
  • the luminous performance 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.
  • 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.
  • 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 light-emitting styrene polymer microspheres of the present invention can reach 5nm-1000nm, more preferably 50nm-800nm, and most preferably the nanometer particle size is 100nm-500nm.
  • the morphology and particle size of all particles of the nano-microspheres can be characterized by images taken by an electron microscope, and the average diameter of the nano-microspheres obtained by multiple measurements is recorded as the particle size.
  • the characterization method of such nano-microspheres is known to the skilled person and can be measured, for example, using scanning electron microscope (SEM) and transmission electron microscope (TEM) instruments.
  • the luminescence intensity of the long-lasting light-emitting styrene polymer 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+ .
  • the long afterglow light-emitting styrene polymer 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 .
  • the long afterglow nanospheres of the present invention can provide a complete material basis for immunochromatographic detection technology.
  • the long afterglow nanospheres of the present invention can be used to prepare immunochromatographic nanoprobes with high afterglow brightness, and can obtain test strips for immunochromatographic detection with high stability, good repeatability, and high sensitivity. Including mycotoxins, pathogenic bacteria, viruses, inflammatory factors and tumor markers.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the selection of its light-absorbing agent and luminescent agent has certain rules and standards.
  • 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.
  • 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 inventors of the present application found that in the long-lasting light-emitting styrene polymer 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 luminescent agent should advantageously be at least one compound selected from the following different molecular formulas or different structures: porphyrin and phthalocyanine dyes, metal complexes, acene compounds, fluoroboron dipyrrole compounds (BODIPY), quantum Dots (QDs), graphenes, and derivatives or copolymers of these compounds.
  • the luminescent agent used in the present invention is a monomeric non-polymeric compound and its molecular weight is less than 10000 g mol -1 .
  • 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.
  • the non-polymeric compound means that the structure of the compound does not contain more than 2 repeating units obtained by polymerization or oligomerization.
  • the light-absorbing agent and luminescent agent preferably used in the long-lasting styrene polymer microspheres of the present invention are selected from the following.
  • 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.
  • porphyrin dyes and their complexes for example, the following compounds can be mentioned:
  • phthalocyanine dyes and their complexes for example, the following can be mentioned:
  • X represents halogen such as F, Cl, Br, I;
  • 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.
  • 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.
  • 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.
  • 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.
  • a ligand exchange strategy such as carboxyanthracene, carboxytetracene, carboxypentacene, aminoanthracene, aminotetracene , Aminopentacene, mercaptoanthracene, mercaptotetracene, mercaptopentacene, etc.
  • the light absorbing agent is preferably selected from complexes of porphyrin and phthalocyanine, quantum dots (QDs), and derivatives of these compounds.
  • QDs quantum dots
  • these exemplary compounds or compounds are as follows:
  • quantum dot materials such as graphene quantum dots, CdSe quantum dots and PbS quantum dots.
  • 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.
  • fluoroboron dipyrrole compound for example, the following compounds can be mentioned:
  • acene compounds for example, the following compounds can be mentioned:
  • 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.
  • 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.
  • 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):
  • 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.
  • 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.
  • 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.
  • 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.
  • these exemplary compounds or compounds are as follows:
  • the photochemical buffer agent is important.
  • the function of the photochemical buffer agent is mainly the conversion of photochemical energy.
  • 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.
  • 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 caching agent according to the present invention is preferably a non-polymer small molecule compound, and the molecular weight is preferably less than 2000 g mol -1 , more preferably less than 1000 g mol -1 .
  • the compound that is not a polymer means that the compound is not obtained by a conventional polymerization reaction, and it is preferable that the compound does not contain or contains no more than 2 repeating units.
  • the buffering agent in the long-lasting light-emitting styrene polymer microspheres suitable for the present invention is selected from the following structural formula (I):
  • 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.
  • 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”.
  • aryl groups 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.
  • the "aryl” or “heteroaryl” also includes aryl or heteroaryl substituted or fused with aryl, heteroaryl, such as biphenyl, phenylthienyl Or benzothiazolyl.
  • 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.
  • 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.
  • alkyl 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.
  • 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.
  • 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.
  • halogen includes fluorine, chlorine, bromine and iodine.
  • 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.
  • the electron withdrawing group is selected from nitro, halogen, haloalkyl, sulfonic acid, cyano, acyl, carboxyl and/or combinations thereof.
  • 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.
  • the ring part Can be selected from
  • G and T are selected from S and O, most preferably one of G and T is S and the other is O.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the photochemical caching agent is selected from, for example, the following compounds:
  • styrene polymer microspheres of the present invention in addition to the above-mentioned component A) light-absorbing agent, component B) luminescent agent and component C) photochemical buffering agent, styrene polymer microspheres must also be contained as a component.
  • Sub D) carrier medium in addition to these, other processing aids for the preparation of nano-microspheres, or components that further improve the long-lasting luminescence effect may be included.
  • the styrene polymer microspheres can particularly well adsorb the specific components A) to C) as described above, thereby forming stable nanomicrospheres supporting the components A) to C).
  • the "styrene polymer microspheres” are formed of styrene polymer.
  • the "styrene polymer” refers to a homopolymer of styrene or a polymer copolymer formed with other copolymerizable monomers. Examples of such copolymerizable monomers include alkenes, alkynes, ethylenically unsaturated carboxylic acids or their anhydrides or amides or esters, etc., and their derivatives with one or more substituents, such as butadiene, horse Acid anhydride, (meth)acrylic acid or (meth)acrylamide, etc.
  • alkene or “alkyne” refers to a linear, branched or cyclic unsaturated aliphatic hydrocarbon with one or more CC double bonds or triple bonds, preferably 2-50, More preferably 2-24, such as 4-18 carbon atoms.
  • the "unsaturated carboxylic acid” especially refers to aliphatic ethylenically unsaturated carboxylic acids, that is, having the formula Y-COOH, where Y is C2-C18, having one or more CC double bonds instead of CC single bonds, Such as C3-C8 linear, branched or cyclic aliphatic alkyl, more preferably (meth)acrylic acid.
  • the "substituent” includes halogen, amino, amide, aldehyde, and/or carboxy, and the like.
  • the surface of the styrene polymer microspheres may contain coupling groups selected from amino groups, amide groups, carboxyl groups, and/or aldehyde groups, so that the surface of the nanomicrospheres of the present invention can utilize these groups for better coupling.
  • the antibodies or aptamers can react immunologically with specific antigens. Therefore, preferred comonomers have one or more groups selected from amino groups, amide groups, carboxyl groups and/or aldehyde groups, such as (meth)acrylic acid, (meth)acrylamide or amino-substituted olefins.
  • the styrene polymer forming the microspheres contains 1%-15%, more preferably 2%-10% of the aforementioned comonomers based on the total weight of all monomers.
  • the styrene polymer or microspheres thereof according to the present invention contains 0.05% to 5%, more preferably 0.1% to 2%, of the coupling group as described above based on the total weight of the polymer.
  • Such preferred styrene polymers are readily available and are particularly suitable for immunodetection applications.
  • the styrene polymer is a copolymer formed from styrene, (meth)acrylic acid or its ester or (meth)acrylamide and optionally other comonomers.
  • the coefficient of variation of the particle diameter of the styrene polymer microspheres according to the present invention is less than 10%, preferably less than 5%, and more preferably less than 3%.
  • the coefficient of variation (CV) represents the ratio of the standard deviation of the data to the average value. It is a statistical measure of the dispersion of data points around the average in a series of data.
  • the smaller the coefficient of variation of the particle size of the styrene polymer microspheres the more favorable it is to obtain stable and repeatable detection effects in practical applications.
  • Those skilled in the art are familiar with the measurement method of the coefficient of variation and the required measuring instruments.
  • styrene polymer carrier medium a styrene polymer synthesized by itself in the form of microspheres can be directly used.
  • Styrene polymers can be prepared using suitable free radical polymerization reactions known to those skilled in the art.
  • the microsphere-like 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.
  • the structure of the styrene polymer microspheres is selected from a hollow structure, a mesoporous structure and a core-shell structure.
  • the hollow structure of styrene polymer microspheres can adsorb more A), B) and C three components.
  • A), B) and C three components.
  • the particle size of styrene polymer microspheres increases, so that the long afterglow luminescence of a single microsphere is enhanced. It is beneficial for the high-efficiency detection of the test signal in the immunochromatographic process; but too large particle size is not conducive to the lateral chromatography of the microspheres on the test strip.
  • the long-lasting light-emitting styrene polymer 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.
  • the total content of the components A) to C) is 0.1% to 30%, preferably 0.2% to 25%, more preferably 0.5% based on the total mass of the components A) to D) To 20% and most preferably 1% to 15%.
  • the total content of components A) to C) is too low, it is equivalent to that the content of component D) is too high, resulting in a decrease in the brightness of the long afterglow luminescence, making it impossible to perform effective immunoassay based on the long afterglow luminescence signal.
  • the total content of components A) to C) is too high, it means that the content of component D) is too low, resulting in poor dispersion and stability of the formed nano-microspheres, such as particle agglomeration and sedimentation. Even the materials cannot form monodisperse nanostructures, which cannot meet the application requirements of immunoassays.
  • 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 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%.
  • the proportion of light absorber 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 styrene polymer 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.
  • the operation range of excitation and emission is very wide, so the actual combination of excitation and emission properties is very rich.
  • the adjustable range of the wavelength of the excitation light is 300 nm to 1000 nm.
  • 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.
  • the emission wavelength of long afterglow luminescence ⁇ 2 is flexibly distributed, and long afterglow luminescence can cover all wavelengths from ultraviolet to visible to near-infrared.
  • 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;
  • ⁇ 1> ⁇ 2 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;
  • 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.
  • these light sources include solid-state lasers, gas lasers, semiconductor lasers, photodiodes, D65 standard light sources, styrene polymer light-emitting diodes, ultraviolet lamps, flashlights, flashlights, xenon lamps, sodium lamps, mercury lamps, tungsten filaments Lamps, incandescent lamps, fluorescent lamps and natural sunlight, and combinations of these light sources.
  • 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 (1 ⁇ W cm -2 -1000W cm -2 ), and the excitation time also has a wide dynamic range (1 ⁇ s-1h).
  • 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).
  • 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.
  • the present invention relates to a probe containing the above-mentioned long-lasting light-emitting styrene polymer microspheres.
  • the probe includes the long-lasting light-emitting styrene polymer microsphere as described above and the antibody or aptamer loaded or coupled to it.
  • 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 %.
  • suitable antibodies or aptamers 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, human chorionic gonadotropin Hormone (HCG), antistreptolysin O (ASO), rheumatoid factor (RF) and/or oligonucleotide fragments.
  • CRP C-reactive protein
  • SAA serum amyloid
  • PCT Procalcitonin
  • AFP alpha-fetoprotein
  • CEA carcinoembryonic antigen
  • PSA prostate specific antigen
  • CCG human chorionic gonadotropin Hormone
  • the present invention relates to a method for preparing the aforementioned long-lasting light-emitting styrene polymer microspheres, which includes the following steps:
  • 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.
  • the carrier medium microspheres or the solution or dispersion thereof can be added thereto.
  • 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.
  • auxiliary equipment such as ultrasonic waves and high-pressure homogenizers can be used when necessary, or appropriate heating and stirring can be carried out.
  • step (2) After step (2) obtains a stable dispersion of long-lasting light-emitting styrene polymer microspheres containing components A) to D), it can be directly used for subsequent use without treatment as needed, such as for preparing suitable Test paper for immunoassay.
  • antibodies or aptamers can be further adsorbed or modified on the obtained nanospheres to obtain 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 biologically coupling the above-mentioned long-lasting light-emitting styrene polymer microspheres with antibodies or aptamers through functional reactive groups such as carboxyl groups, amino groups, and aldehyde groups.
  • a carboxy-amino group can react to form a coupling or an aldehyde group-amino group can react to form a coupling.
  • the corresponding coupling method is selected according to the functional groups on the surface of the nanospheres.
  • the fourth aspect of the present invention relates to the preparation method of the probe as described above.
  • the present invention provides a test paper for immunochromatographic detection comprising the long-lasting light-emitting styrene polymer 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 styrene polymer microspheres or the above-mentioned probe.
  • test paper itself used in immunochromatographic detection technology
  • the sample pad, bonding pad, test line and quality control line can be attached to the bottom plate, such as a PVC bottom plate.
  • the bottom plate such as a PVC bottom plate.
  • 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.
  • 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.
  • 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.
  • 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.
  • the sample is dropped on the sample pad, and passes through the binding pad, T-line and C-line sequentially through capillary action.
  • the antigen fixed on the T-line will competitively bind with the free antigen and antibody in the sample.
  • the general procedure of the immunochromatographic detection method is known to those skilled in the art, and the embodiment of the method is exemplified in the examples of this application.
  • the present invention also relates to a method of immunochromatographic detection, which includes the following steps:
  • the immunochromatographic detection method of the present invention has more advantages.
  • the excitation wavelength can be selected in a wider range, including the wavelength range of ultraviolet light, visible light and near-infrared light.
  • the long-lasting light-emitting styrene polymer microspheres of the present invention have a larger absorption cross-section by several orders of magnitude, which makes the light irradiation charging time shorter, for example, 2s-10s is the most preferred.
  • the long afterglow luminescence brightness of the long afterglow light-emitting styrene polymer 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.
  • the instrument used to read the luminescence signal in the detection is a smart phone, a luminescence imaging system, a professional long afterglow luminescence detection device, and the like.
  • 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.
  • Fig. 1 is a schematic diagram of the structure of a probe containing long-lasting light-emitting styrene polymer microspheres according to the present invention. It can be seen that components A) to C) are all adsorbed on the carrier medium nanospheres, and antibodies or aptamers are also coupled to the carrier medium.
  • 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 arranged in sequence; the upper edge of the nitrocellulose membrane 4 Along 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.
  • Figure 4 is a transmission electron microscope image of 300nm carboxyl-containing styrene polymer nanospheres synthesized in Example 1
  • Example 5 is a transmission electron microscope image of the long-lasting styrene polymer nanospheres of Example 2.
  • Figure 6 is based on the storage stability of the long-lasting styrene polymer nanospheres tested in Example 29 of the present invention.
  • Fig. 7 is based on the standard curve of C-reactive protein (CRP) detection of the long-lasting luminescent nanomaterial of Example 33 of the present invention.
  • CRP C-reactive protein
  • Figure 8 The effect of long afterglow immunochromatographic test strips for the detection of C-reactive protein (CRP).
  • the pictures were all taken with a mobile phone.
  • the long afterglow signal indicator probes used in the immunochromatographic test strips are different.
  • the left picture (a) is the CRP detection effect picture based on the long-lasting luminescent nanomaterial of Example 33 of the present invention, and the right picture (b) is based on the comparative implementation.
  • Figure 9 is based on the standard curve of serum amyloid (SAA) detection of the long-lasting luminescent nanomaterial of Example 34 of the present invention.
  • Fig. 10 is based on the standard curve of procalcitonin (PCT) detection of the long-lasting luminescent nanomaterial of Example 35 of the present invention.
  • Figure 11 is based on the standard curve of alpha-fetoprotein (AFP) detection of the long-lasting luminescent nanomaterial of Example 36 of the present invention.
  • AFP alpha-fetoprotein
  • Figure 12 is based on the standard curve of carcinoembryonic antigen (CEA) detection of the long-lasting luminescent nanomaterial of Example 37 of the present invention.
  • CEA carcinoembryonic antigen
  • Fig. 13 is based on the standard curve of prostate-specific antigen (PSA) detection of the long-lasting luminescent nanomaterial of Example 38 of the present invention.
  • PSA prostate-specific antigen
  • Fig. 14 is a standard curve of cardiac troponin (cTn-I) detection based on the long afterglow luminescent nanomaterial of Example 39 of the present invention.
  • Fig. 15 is a standard curve of human chorionic gonadotropin (HCG) detection based on the long-lasting luminescent nanomaterial of Example 40 of the present invention.
  • Figure 16 is based on the standard curve of anti-streptolysin O (ASO) detection of the long afterglow luminescent nanomaterial of Example 41 of the present invention.
  • ASO anti-streptolysin O
  • Figure 17 is based on the standard curve of the rheumatoid factor (RF) detection of the long-lasting luminescent nanomaterial of Example 42 of the present invention.
  • a wavelength tunable laser (Opolette 355) from Opotek, Inc. of the United States is used.
  • LEDs light-emitting diodes
  • 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.
  • Fluorescence spectrometer (Edinburgh FS-5) of Edinburgh Instrument Company, UK was used to test the long afterglow luminous intensity.
  • the invention uses a commercial smart phone or a common digital camera to take pictures, and records brightfield and long afterglow glow pictures.
  • 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.
  • 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.
  • 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.
  • Synthesize styrene polymer nanospheres as a carrier medium for long-lasting luminescent materials The styrene polymer nanospheres were synthesized by emulsion polymerization: firstly, 39.3 g styrene, 2.1 g methacrylic acid and 0.5 g sodium dodecylbenzene sulfonate were dispersed in 100 mL ultrapure water, and the The solution was added to a 500 mL three-necked flask, and nitrogen gas was introduced. The temperature was maintained at 25° C., and stirring was continued for 30 minutes.
  • the surface of the styrene polymer nanospheres prepared according to the method contains carboxyl groups, and the content of the carboxyl groups measured by the conductivity titration method accounts for 0.2% of the total weight of the styrene polymer microspheres.
  • the scanning electron microscope image of the prepared styrene polymer nanospheres is shown in Fig. 4, and the particle size is 300 nm and very uniform.
  • the coefficient of variation between the nanoparticles is less than 5%.
  • the synthesis and control methods of the above-mentioned styrene polymer nanospheres are easy to understand, and other styrene polymer nanospheres with different particle diameters can be obtained by adjusting the amount of synthetic raw materials added. .
  • Inorganic long-lasting styrene polymer microspheres are prepared from commercialized inorganic long-lasting materials. In the preparation process, a dispersion polymerization method known to those skilled in the professional field is used. 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 nanospheres with a particle size of about 250 nm are obtained by centrifugal separation, and cetyltrimethyl amine bromide (CTAB) is used to treat inorganic particles with the aid of ultrasound.
  • CTAB cetyltrimethyl amine bromide
  • the long afterglow nanospheres are subjected to lipophilic treatment.
  • disperse 15g styrene and 1g methacrylic acid into 80mL ethanol-water mixed solution (v:v, 1:3), put the solution into a 250mL three-necked flask, and add the inorganic long afterglow nanometer after CTAB treatment.
  • Example 2 the inorganic long-lasting styrene polymer microspheres were prepared into an aqueous solution with a concentration of 1 mg mL -1 , and the afterglow performance of the nano-microspheres was tested. As a result, no afterglow light was seen by naked eye observation. The measured long afterglow luminous intensity is shown in Table 1.
  • Example 2 The operation of Example 2 was repeated, wherein the molar ratio of the three components of the light absorbing agent, the photochemical buffering agent, and the luminescent agent was maintained at 1:600:1000, and the differences are shown in Table 1.
  • Example 1 The operation of Example 1 is repeated, wherein the molar ratio of the three components of the light absorber, the photochemical buffering agent, and the luminescent agent is maintained at 1:600:1000, and the differences are shown in Table 1.
  • NCBS benzyl alcohol-ethylene glycol-water
  • concentration of PdOEP is 5 ⁇ mol L -1
  • CA- The concentration of 1 is 3 mmol L -1
  • concentration of Eu-1 is 5 mmol L -1 .
  • Example 2 After the components were ultrasonically dispersed, 50 mg of the 300 nm styrene polymer nanospheres with carboxyl groups on the surface of Example 1 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 nanoparticles were dispersed in water for storage.
  • the afterglow performance test of the prepared long-lasting light-emitting styrene polymer microspheres was carried out according to the method of Example 2, and the long-lasting light-emitting styrene polymer microspheres were prepared into an aqueous solution with a concentration of 1 mg mL -1. First, use 808nm wavelength excitation light for 3s to charge. After the charge is completed, the light source is turned off. As a result, no afterglow is seen by naked eyes. The long afterglow luminous intensity measured by the instrument is shown in Table 1.
  • Example 13 The operation of Example 13 is repeated, and the difference is shown in Table 2, where the concentration of the luminescent agent Eu-2 is 10 mmol L -1 .
  • Example 13 The operation of Example 13 was repeated, and the difference is shown in Table 2, where the concentration of the light absorbing agent PdPc was 10 mmol L -1 .
  • the light absorbing agent PdPc, the luminescent agent Eu-2 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.
  • the molar ratio of the three components of the light absorbing agent PdPc, the photochemical caching agent CA-1, and the luminescent agent Eu-2 is 1:600:2000.
  • the dichloromethane solvent is removed to obtain an oily mixture of A), B), and C).
  • the mass fraction of the three components ABC in the total weight of the microspheres is measured according to the following method.
  • the prepared long afterglow styrene polymer microspheres are fully dissolved in tetrahydrofuran, and then the three components A), B) and C) are separated, and the mass fraction of the three components in the nano microspheres is calculated .
  • Example 20 Repeat the operation of Example 20, the difference lies in the carrier medium and component content in the nanomaterial (as shown in Table 3), by adjusting the mass of the oily three-component mixture to obtain the ABC three components in the total weight of the microspheres Different quality scores.
  • Table 3 The test results are shown in Table 3.
  • Example 20 Repeat the operation of Example 20, the difference lies in the carrier medium and component content in the nanomaterial (as shown in Table 3), by adjusting the mass of the oily three-component mixture to obtain the ABC three components in the total weight of the microspheres Different quality scores.
  • Table 3 The test results are shown in Table 3.
  • the light absorbing agent PdPc, the luminescent agent Eu-2 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.
  • the molar ratio of the three components of the light absorbing agent PdPc, the photochemical caching agent CA-1, and the luminescent agent Eu-2 is 1:600:2000.
  • the dichloromethane solvent is removed to obtain an oily mixture of A), B), and C). Weigh 10 mg of the three-component mixture and add it to 10 mL of mesitylene-ethanol (v:v, 1:1) solution.
  • the mass fraction of the three components ABC in the total weight of the microspheres is measured according to the following method. Dissolve the prepared long-lasting silicon nanospheres in toluene, and fully ultrasonically dissolve the adsorbed ABC three components, and then separate the three components A), B) and C), and calculate the three components in the nano The mass fraction of the microspheres.
  • the light absorbing agent PdPc, the luminescent agent Eu-2 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.
  • the molar ratio of the three components of the light absorbing agent PdPc, the photochemical caching agent CA-1, and the luminescent agent Eu-2 is 1:600:2000.
  • the dichloromethane solvent is removed to obtain an oily mixture of A), B), and C).
  • 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.
  • Example 24 The operation of Example 24 was repeated, except for the particle size of the carboxyl-bearing styrene polymer microspheres as the nanocarrier medium (as shown in Table 4). The test results are shown in Table 4.
  • Example 3 we repeated the same operation 10 times to prepare 10 batches of long-lasting light-emitting styrene polymer microspheres.
  • the prepared long-lasting luminescent nanoparticles were prepared into aqueous solutions with a concentration of 1 mg mL -1 respectively, and the afterglow performance of the 10 batches of long-lasting luminescent styrene polymer microspheres was tested.
  • the test results are shown in Table 5. Analyzing the intensity of the obtained 10 times long afterglow luminescence, the result shows that the deviation is less than 5%, indicating that the long afterglow luminescence system has good stability and repeatability.
  • the probe is prepared by coupling the alpha-fetoprotein (AFP) antibody AFP-Ab 1 with fluorescent long afterglow nanospheres:
  • the probe is prepared by coupling the prostatic specific antigen (PSA) aptamer with long afterglow nanospheres:
  • Example 1 The long afterglow nanospheres in Example 1 were coupled to CRP-Ab 1 as follows: 100 mg of the long afterglow luminescent styrene polymer microspheres prepared in Example 3 were centrifuged and reconstituted to 18 mL with a pH of 7.4 In the BBS buffer solution, fully sonicate to make the dispersion uniform; add 10mg of EDC and 2.5mg of NHSS to it, respectively, and react at room temperature for 2 hours; after the reaction is over, centrifuge and wash, and reconstitute into 10mL of BBS buffer with a pH of 7.4 To the solution, add 10mg of CRP-Ab type 1 monoclonal antibody and react for 4 hours at room temperature; after the reaction is over, wash by centrifugation, reconstitute it in 10mL of BBS buffer with pH 7.4, and add 100mg of BSA to it , React for 2 hours at room temperature; after the reaction, centrifuge and wash, reconstitute in 10 mL of BBS buffer
  • CRP immunochromatographic test strip NC membrane uses PBS buffer (1% BSA, 1% sucrose, 50 mM NaCl and 0.5% TWEEN 20) to separate CRP-Ab type 2 monoclonal antibody and donkey
  • PBS buffer 1% BSA, 1% sucrose, 50 mM NaCl and 0.5% TWEEN 20
  • 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.
  • the sample pad of the CRP immunochromatographic test strip is made of glass fiber.
  • the CRP immunochromatographic test strip binding pad is also made of glass fiber, the difference is that the glass fiber is sprayed with long-lasting light-emitting styrene polymer nano-probes.
  • Probe spraying process on glass fiber Take the fluorescent probe prepared in step 1), centrifuge it, reconstitute it with spray buffer to 20mg mL -1 , and pour the fluorescent probe solution into the instrument through the sprayer , Spray the fluorescent probe on the glass fiber at a speed of 1.2 ⁇ L cm -1 and bake it at 37°C overnight.
  • the concentrations are 0 ⁇ g mL -1 , 0.1 ⁇ g mL -1 , 0.5 ⁇ g mL -1 , 5 ⁇ g mL -1 , 20 ⁇ g mL -1 , 40 ⁇ g mL -1 , 160 ⁇ g mL -1 and 320 ⁇ g mL -1 .
  • the immunochromatographic test strip is tested with a long afterglow luminescence detector.
  • the excitation light irradiation time is 3s.
  • the long afterglow luminescence signal on the test strip is collected. Measure the long afterglow luminescence intensity of the T line and the C line, and then calculate the ratio of the intensity, and establish a standard curve through the corresponding relationship between the ratio and the antigen concentration ( Figure 7).
  • 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.
  • the prepared long-lasting luminescence nano-indicating probe only needs to use excitation light to irradiate and excite before reading, and the excitation light is turned off during the subsequent reading process.
  • This method eliminates the interference of background fluorescence signal and can realize the test object Highly sensitive quantitative detection.
  • the immunochromatographic test strips based on the long-lasting light-emitting styrene polymer microspheres of the present invention (Figure 8 left) are more sensitive than the detection system based on inorganic long-lasting ( Figure 8 right).
  • the sample contains 21 ⁇ g mL -1 CRP antigen.
  • Example 33 The operation of Example 33 was repeated, with the difference that: in the first step, the inorganic long afterglow nanospheres in Comparative Example 1 were used to couple CRP-Ab 1 .
  • 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 8.
  • Example 33 Repeat the operation of Example 33 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 34, Figure 9), PCT ( Example 35, Figure 10), AFP (Example 36, Figure 11), CEA (Example 37, Figure 12), PSA (Example 38, Figure 13), cTn-I (Example 39, Figure 14), HCG (Example 40, Figure 15), ASO (Example 41, Figure 16) and RF (Example 42, Figure 17).
  • SAA Example 34, Figure 9
  • PCT Example 35, Figure 10
  • AFP Example 36, Figure 11
  • CEA Example 37, Figure 12
  • PSA Example 38, Figure 13
  • cTn-I Example 39, Figure 14
  • HCG Example 40, Figure 15
  • ASO Example 41, Figure 16
  • RF Example 42, Figure 17
  • the CRP antigen standard purchased from Zhongjian Institute was diluted to 21 ⁇ g mL -1 , and the sample was tested in parallel for 30 times. The samples to be tested were divided into 30 evenly, and the operation of Example 33 was repeated to perform the tests respectively.
  • the 30 test results obtained are shown in Table 6. Analyzing the obtained 30 detection results, it is found that the coefficient of variation of the detection results is less than 5%, indicating that the detection method of the long afterglow luminescence probe and the immunochromatographic test strip of the present invention has good detection accuracy and repeatability.

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Abstract

一种长余辉发光苯乙烯聚合物微球，其包含 A)至少一种吸光剂， B)至少一种发光剂， C)至少一种式(I)的光化学缓存剂，和 (I) D)用于吸附组分A)到C)的载体介质，所述载体介质是苯乙烯聚合物微球；其中，所述吸光剂与发光剂是结构不同的化合物，并且所述组分A)到C)的总含量以组分A)到D)的总质量计为0.1％到30％、优选0.2％到25％、更优选0.5％到20％和最优选1％到15％。所述苯乙烯聚合物微球特别适用于免疫层析检测技术。此外，还涉及用于免疫层析检测的试纸、探针以及检测方法。

Description

长余辉发光的苯乙烯聚合物微球、其制备方法和应用 技术领域
本发明涉及一种长余辉发光苯乙烯聚合物微球，其制备方法和应用，特别是所述长余辉发光苯乙烯聚合物微球在免疫层析检测技术中的应用。
背景技术
免疫层析检测技术(Immunochromatography assay，ICA)或侧向层析免疫检测技术(Lateral flow assay，LFA)最早被用于检测人绒毛膜促性腺激素，随着标记技术的发展，也广泛应用于医学检验，环境监控和食品安全等领域。免疫层析检测技术有效地将色谱技术和抗原-抗体免疫反应技术结合在一起。在免疫层析检测技术中经常使用一种免疫层析检测试纸条，其主要结构是以聚氯乙烯底板作为支撑，其上设置有样品垫(如玻璃纤维素膜)、结合垫(如玻璃纤维素膜)、硝酸纤维素膜(NC膜)和吸水垫。当样品在毛细作用下流动时，抗原先与结合垫上的探针结合，形成免疫复合物，随着液体继续流动，免疫复合物将被富集并截留在NC膜的检测线(T线)上，没有形成免疫复合物的探针将会被质控线(C线)截留，最后通过肉眼或者仪器进行判读。
目前广泛使用的免疫层析检测试纸条主要包括胶体金免疫层析试纸条和荧光免疫层析试纸条。传统的免疫层析检测技术主要以胶体金作为输出信号，由于胶体金探针的光密度不足，导致检测灵敏度较低，且难以定量，无法满足临床诊断的要求。随后，荧光探针在免疫层析检测上得到发展，其中基于荧光染料的发光探针的应用最为广泛。荧光染料通常直接标记在抗体或包被抗体的微球上，或包埋于纳米微球内再修饰抗体，采用例如紫外光的光源激发使得染料发光。但是，在发光信号的检测过程中，例如血液等样本存在自发的荧光信号干扰，另外激发光源的照射也会产生光信号的干扰，这些不利因素将严重影响到检测信号的准确性和稳定性。
长余辉发光材料是一类特殊的发光材料，其在激发光源去除后还可持续长时间的发光。现有技术中，通常长余辉发光材料的发光寿命大于一百毫秒，其在生物医学、生命科学等领域具有重要的应用价值。目前，商品化的长余辉发光材料一般为稀土或过渡金属掺杂的铝酸盐、硅酸盐、钛酸盐或硫化物等无机长余辉材料。将长余辉发光材料作为信号指示探针应用于免疫层析检测中，可以避免激发光和背景荧光的干扰。 例如，在CN105929155A中公开了一种基于长余辉的免疫层析试纸及其检测方法，其使用无机长余辉发光材料，有效消除了干扰信号，提升了对待检测物的检测灵敏度和定量准确性。
这些基于稀土或过渡金属掺杂的无机长余辉发光材料通常通过高温固相煅烧而制得。高温固相合成是这类材料最普遍和有效的生产方法，主要是因为高温有利于获得较好的长余辉发光性质，采用其他非高温方法合成的无机长余辉材料的发光性能显著降低而难以获得广泛应用。但是，高温固相反应条件苛刻且能耗高，难以控制材料的形貌均匀，材料的粒径普遍较大。虽然通过研磨并筛选的手段，可以将高温固相法合成的材料进一步变小，但是粒径变小至纳米量级后(例如当颗粒直径小于1000nm时)发光亮度急剧下降。例如，如果将商业化的微米级无机长余辉粉体研磨到100nm量级时，则纳米微球的发光亮度可能会有两个数量级的降低。
另外，在侧向层析免疫检测应用中，所使用发光探针的高度均一性对保证检测结果的可重复性和可靠性具有重要的意义。但是，在CN105929155A的中作为发光核心的无机长余辉发光材料是无机的颗粒(粒径大于15nm，甚至达到250nm)，其在纳米尺度内的可加工性能较差，材料表面也不易于修饰用于生物偶联的功能基团，根据CN105929155A公示的技术方案很难获得大批量且均匀的长余辉发光纳米探针。
因此，目前无机长余辉发光微球及探针的制备比较困难，而且无机的长余辉发光微球及探针发光较弱，导致长余辉发光信号逐渐衰减到肉眼可见水平时所经历的余辉时间过短，甚至完全不能获得肉眼明显可见的长余辉发光信号。弱信号的检测需要借助于复杂的专业设备，而且在应用于免疫层析检测时的效果也比较受限。
发明内容
针对现有技术的上述这些缺陷，本发明提供了一种具有更高亮度而余辉时间不受影响甚至可能更长的基于苯乙烯聚合物的有机长余辉发光微球。
不同于现有技术的基于光物理过程的无机长余辉发光材料，本发明的长余辉发光材料是基于有机体系的，该体系利用光化学反应的特性，在光能输入和光能输出之间引入光化学反应，将光物理与化学有机融合。在基于该有机体系的长余辉发光材料中，发光过程可以涉及多种化学物质间的光化学相互作用，其中经过一系列的光化学能量转化与代谢过程，输入的激发光能量最终以发光的形式释放出来，从而实现长余辉发光。能量转化与代谢过程包括能量输入、能量缓存、能量提取、能量转移和能量释放。 通过光化学反应使原本非常迅速的光子辐射跃迁进程(纳秒量级至微秒量级)发生改变，能量缓慢释放并最终以光能的形式发射出来，由此获得超长的发光时间(毫秒量级至小时量级)，大大改善了有机分子发光寿命短的限制。
进一步地，发明人发现，某些有机长余辉发光材料可以特别合适地与苯乙烯聚合物微球组合而制成余辉亮度更高和持续时间可能更长的长余辉发光苯乙烯聚合物微球，其特别适合用于免疫层析技术检测。该有机长余辉体系的发光核心由分子组分构成，具有很好的可加工性能，例如在溶液中各分子组分很容易分散和吸附到均匀的苯乙烯聚合物微球中，制备成大批量且具有高度均一性的长余辉发光苯乙烯聚合物微球及探针。甚至在很多情况中，余辉亮度可以提高到肉眼可见及以上的水平，长余辉发光信号能够被手机等常见的电子设备采集分析，这极大地增加了材料的实用性。
在本申请中，术语光化学反应是一系列的连锁反应，包括光化学加成、光氧化、光化学解离和断键重组的反应过程。
在本申请中，如无特殊说明，术语“长余辉发光苯乙烯聚合物微球”、“长余辉材料”和“长余辉纳米微球”等均具有相同含义并可互换使用。
因此，第一个方面，本发明提供一种长余辉发光苯乙烯聚合物微球，其包含
A)至少一种吸光剂，
B)至少一种发光剂，所述发光剂是单体的非聚合的化合物且其分子量小于10000g mol -1，
C)至少一种式(I)的光化学缓存剂，
Figure PCTCN2019123220-appb-000001
其中式(I)如下文详细描述，和
D)用于吸附组分A)到C)的载体介质，所述载体介质是苯乙烯聚合物微球；
其中，组分A)到C)的总含量以组分A)到D)的总质量计为0.1％到30％、优选0.2％到25％、更优选0.5％到20％和最优选1％到15％。
优选地，所述长余辉发光苯乙烯聚合物微球由组分A)到D)组成。
第二个方面，本发明提供一种包含上述长余辉发光苯乙烯聚合物微球的探针。
第三个方面，本发明提供制备上述长余辉发光苯乙烯聚合物微球的方法。
第四个方面，本发明提供制备包含上述长余辉发光苯乙烯聚合物微球的探针的方法。
第五个方面，本发明提供一种用于免疫层析检测的试纸条。
第六个方面，本发明提供一种利用如上所述的长余辉发光苯乙烯聚合物微球进行免疫层析检测的方法。
本发明的其他方面体现在其他的独立权利要求和从属权利要求中。
除了上述优点之外，根据本发明的长余辉发光苯乙烯聚合物微球的组分配制灵活，可以根据实际需求来设计材料的组成与性质，并可获得灵活多样的纳米结构，且具有可剪裁的发光性能。充能的激发光波长与长余辉发光的波长可以分别地进行调节，很方便地对吸光剂和发光剂的组合方案进行调整更换，以此高效地实现色彩丰富的长余辉发光。
优选的，根据本发明的长余辉发光纳米材料中不包含或包含极少量的无机长余辉成分如SrAl 2O 4:Eu 2+,Dy 3+，例如基于材料混合物计不超过0.1重量％。
本发明的长余辉发光苯乙烯聚合物微球的粒径可以达到5nm–1000nm，更优选为50nm–800nm，最优选纳米粒径为100nm–500nm。在本发明的上下文中，纳米微球所有的颗粒的形貌和粒径可以通过电子显微镜拍摄图像表征，并且将多次测量得到的纳米微球的平均直径记录为粒径。这样的纳米微球的表征方法是技术人员已知的并且可以例如采用扫描电子显微镜(SEM)和透射电子显微镜(TEM)仪器测得。
在相同的测试条件下，根据本发明的长余辉发光苯乙烯聚合物微球的发光强度可以远超过纳米尺度的商业化无机长余辉材料SrAl 2O 4:Eu 2+,Dy 3+的水平。特别的，根据本发明的长余辉发光苯乙烯聚合物微球在激发光关闭之后还能持续发光，且长余辉发光时间可以达到100ms–3600s，优选地在500ms–1200s，更优选地在1s–600s，最优选2s–60s。根据本发明的长余辉材料的长余辉发光亮度可以达到0.1mcd m -2–10000mcd m -2，优选地在0.32mcd m -2–8000mcd m -2，更优选地在1mcd m -2–5000mcd m -2。基于上述性质，本发明的长余辉纳米微球能够为免疫层析检测技术提供完备的材料基础。
此外，利用本发明的长余辉纳米微球可以制备余辉亮度高的免疫层析纳米探针，可获得稳定性高、重复性好、灵敏度高的用于免疫层析检测的试纸条，检测对象包括真菌毒素、致病菌、病毒、炎性因子和肿瘤标志物等。
吸光剂和发光剂
在本申请中，吸光剂和发光剂本身是现有技术已知的。吸光剂通常指能吸收并捕获来自于自然光源或人工光源的光能的物质。吸光剂的选取范围包括传统的光敏试剂和其他能量给体材料等。而发光剂通常指能够将能量最终以光能的形式发射出来的物质。发光剂可以是能够产生荧光或磷光等的发光物质。有关的发光分子基团本身是已知的，并且可参考例如Jeff W.Lichtman等人的综述性论文Nature Methods,2005,2,910-919。
为了实现本发明的长余辉材料的有益效果，特别是例如改善余辉强度和时间，在本发明的组合物中对发光剂和吸光剂两个组分做了明确区分，使其各自分别承担吸收光能和释放光能的作用，从而在与经过特定筛选的光化学缓存剂组合之后实现能量输入、能量缓存和能量输出的能量利用路径。这也意味着，在有利的实施方式中，在结构上既具有吸光基团也具有发光基团从而可以以同一分子发挥两种功能的化合物不是根据本发明的发光剂或吸光剂，并且也不会得到本发明的优异技术效果。一方面，这样的化合物等同于把吸光剂与发光剂连同它们的性质一起打包绑定，就无法分别地对长余辉材料的激发和发光性能进行调节，例如当其根据实际的激发充能的需要选取了一个化合物后，材料的发光性质也同时固定了，反之亦然；另一方面，这样的化合物等同于把吸光剂与发光剂的比例固定为例如1:1，无法同时调节吸光程度的强弱和发光水平的高低；而且，同时具备高效吸光功能与高效发光功能的材料相对较少，这就限制了长余辉材料的种类丰富性。
在根据本发明的长余辉发光材料中，其吸光剂与发光剂的选取具有一定的规则标准。一般而言，将具有较大的摩尔吸光系数的化合物选取作为吸光剂，例如光敏剂或能量给体染料；而将具有更高发光量子效率的化合物选取作为发光剂，例如发光染料。另外，吸光剂的吸收峰应该与发光剂的发射峰尽可能少的重叠，避免长余辉发光被吸收剂吸收而减弱的不利影响。
本申请的发明人发现，在根据本发明的长余辉发光苯乙烯聚合物微球中，特别是在免疫层析检测技术方面，从提高发光亮度或发光信号强度的角度考虑，所述吸光剂和发光剂应当有利地分别是选自如下的不同分子式或不同结构的至少一种化合物：卟啉和酞菁类染料、金属配合物、并苯类化合物、氟硼二吡咯类化合物(BODIPY)、量子点(QDs)、石墨烯类，以及这些化合物的衍生物或共聚物。有利的，在本发明中所用的发光剂是单体的非聚合的化合物且其分子量小于10000g mol -1。在本申请上下文中，所述分子量指的是化合物的重均分子量，其可以通过质谱、气相色谱、液相色谱 的方法测得。可供选用的仪器可以是例如质谱分析仪、或者液相-质谱联用仪。在此，所述非聚合的化合物指的是该化合物结构中不包含通过聚合或低聚反应得到的超过2个的重复单元。
更有利地，特别从免疫层析检测技术考虑，优选用于本发明的长余辉苯乙烯聚合物微球的吸光剂和发光剂分别选自如下这些。
(1)吸光剂
优选的，所述吸光剂可选自卟啉类和酞菁类染料、过渡金属配合物、量子点(QDs)，以及这些化合物的衍生物或共聚物。这些化合物本身是本领域技术人员已知的，以下提及吸光剂的一些非限制性的实例。
作为卟啉类染料及其配合物可以提及例如以下这些化合物：
Figure PCTCN2019123220-appb-000002
Figure PCTCN2019123220-appb-000003
作为酞菁类染料及其配合物可以提及例如以下这些：
Figure PCTCN2019123220-appb-000004
在以上所示的这些吸光剂化合物的结构式中，
X表示卤素如F,Cl,Br,I；和
M＝金属元素，如Al,Pd,Pt,Zn,Ga,Ge,Cu,Fe,Co,Ru,Re,Os等。
各个取代基R如R 1-24表示H、羟基、羧基、氨基、巯基、酯基、醛基、硝基、磺酸基、卤素，或具有1-50、优选1-24、如2-14个碳原子的烷基、烯基、炔基、芳基、具有N、O或S的杂芳基、烷氧基、烷氨基，或者它们的组合。优选地，上述基团R如R 1-24各自独立地选自甲氧基、乙氧基、二甲氨基、二乙氨基、甲基、乙基、丙基、丁基、叔丁基、苯基或者它们的组合。
可以用作吸光剂的过渡金属配合物本身是已知的，并且优选是如上所示的那些卟啉类和酞菁类染料的配合物。
合适的量子点材料包括例如石墨烯量子点、碳量子点和重金属量子点。
重金属量子点包括例如Ag 2S、CdS、CdSe、PbS、CuInS、CuInSe、CuInGaS、CuInGaSe、InP量子点。其外可以包裹壳层，形成核壳结构，壳层可以为Ag 2S、CdS、CdSe、PbS、CuInS、CuInSe、CuInGaS、CuInGaSe中的一种或几种，也可以为ZnS层。
优选地，采用表面配体对量子点进行修饰，所述表面配体可以是例如油酸、油胺、十八烯、十八胺、正十二硫醇及其组合等。在一些更有利的情况下，量子点表面的配体通过配体交换策略部分更换为含有三线态的分子结构，例如羧基蒽，羧基并四苯、羧基并五苯、氨基蒽、氨基并四苯、氨基并五苯、巯基蒽、巯基并四苯、巯基并五苯等。
在一个更优选的实施方式中，所述吸光剂优选自卟啉和酞菁类的配合物、量子点(QDs)、以及这些化合物的衍生物。例如以下这些示例性的一种或多种化合物：
Figure PCTCN2019123220-appb-000005
Figure PCTCN2019123220-appb-000006
以及还有石墨烯量子点、CdSe量子点和PbS量子点等量子点材料。
(2)发光剂
优选的，所述发光剂可选自铱配合物、稀土配合物、并苯类化合物、氟硼二吡咯类化合物(BODIPY)、以及这些化合物的衍生物和共聚物。
作为氟硼二吡咯类化合物(BODIPY)，可以提及例如以下这些化合物：
Figure PCTCN2019123220-appb-000007
Figure PCTCN2019123220-appb-000008
作为并苯类化合物，可以提及例如以下这些化合物：
Figure PCTCN2019123220-appb-000009
在以上所示的这些发光剂化合物的结构式中，
n＝大于等于0的整数，例如0、1、2和3；
各个取代基R如R 1-16表示H、羟基、羧基、氨基、巯基、酯基、醛基、硝基、磺酸基、卤素，或具有1-50、优选1-24、如2-14个碳原子的烷基、烯基、炔基、芳基、具有N、O或S的杂芳基、烷氧基、烷氨基，或者它们的组合。优选地基团R如R 1-16选自甲氧基、乙氧基、二甲氨基、二乙氨基、甲基、乙基、丙基、丁基、叔丁基、苯基；或者它们的组合。
适合作为发光剂试剂的铱配合物中，配体的组成可以是一种或多种不同配体的组合，其结构示意及部分C-N，N-N，O-O和O-N配体的种类例示性展示如下(其中所示的C-N，N-N，O-O和O-N配体为其简略结构示意图且分别突出表示以配体中的C与N原子、两个N原子、两个O原子和O与N原子为配位点与铱原子Ir进行配位作用，这样的表示方法是本领域技术人员熟悉和理解的)：
Figure PCTCN2019123220-appb-000010
Figure PCTCN2019123220-appb-000011
(其中DMSO为二甲基亚砜)
Figure PCTCN2019123220-appb-000012
其中C-N配体可以具有例如以下结构：
Figure PCTCN2019123220-appb-000013
O-N配体可以具有例如以下结构：
Figure PCTCN2019123220-appb-000014
N-N配体可以具有例如以下结构：
Figure PCTCN2019123220-appb-000015
作为发光剂的稀土配合物可以例如是这样的结构，其中中心原子为镧系元素，配体以O或N与中心原子配位，一般中心原子为Eu、Tb、Sm、Yb、Nd、Dy、Er、Ho、Pr等。这些稀土配合物的配位数大约在3到12，优选6到10。在实际的稀土配合物中，配体种类、每个配体的个数和总的配位数可以发生变化。稀土配合物及其配体可参考例如Jean-Claude G.Bünzli的综述性论文Coord.Chem.Rev.,2015,293-294,19-47。
在一个更优选的实施方式中，所述发光剂选自铱配合物、稀土配合物、氟硼二吡咯类化合物(BODIPY)、苝以及这些化合物的衍生物。例如以下这些示例性的一种或多种化合物：
Figure PCTCN2019123220-appb-000016
Figure PCTCN2019123220-appb-000017
光化学缓存剂
在根据本发明的长余辉发光材料中，光化学缓存剂是重要的。光化学缓存剂的功能主要是光化学能量的转化，与主要功能为发光的发光剂不同，缓存剂分子本身不发光或发光很弱，其分子结构中一般不包含直接能发光的基团或共轭结构。特别的，根据本发明的光化学缓存剂在种类上区别于发光剂或吸光剂，尤其是本发明所列的那些发光剂或吸光剂物质。根据本发明的光化学缓存剂能够协助参与光化学反应，在发光剂和吸光剂之间构建能量交换和存储的桥梁。在光化学反应中经过加成、重排或断键的反应步骤，激活在能级间跃迁的能量提取过程。
根据本发明的光化学缓存剂优选为非聚合物的小分子化合物，分子量优选小于2000g mol -1，更优选小于1000g mol -1。所述不是聚合物的化合物是指化合物不是通过常规的聚合反应得到，优选该化合物中不包含或包含不超过2个的重复单元。
特别的，本发明人发现，有一些特定的缓存剂化合物特别适合于制备稳定且长余辉发光性能良好的纳米微球。适用于本发明的长余辉发光苯乙烯聚合物微球中的缓存剂选自如下的结构式(I)：
Figure PCTCN2019123220-appb-000018
其中，
G和T为选自O,S,Se和N的杂原子；
R 1′和R 2′以及R 4′到R 8′各自独立地选自H、羟基、羧基、氨基、巯基、酯基、硝基、磺酸基、卤素、酰胺基，或具有1-50、优选1-24、如2-14个碳原子的烷基、烯基、炔基、烷氧基、烷硫基、烷氨基、芳基、芳烷基、具有N、O或S的杂芳基或杂芳烷基，或者它们的组合，其中所述芳基、芳烷基、杂芳基或杂芳烷基任选具有一个或多个取代基L；和
L选自羟基、羧基、氨基、巯基、酯基、硝基、磺酸基、卤素、酰胺基，或具有1-50、优选1-24、如2-14个或6-12个碳原子的烷基、烯基、炔基、烷氧基和烷氨基，或者它们的组合；和
R 3′为吸电子基团或包含吸电子基团的芳基。
在本申请上下文中，“芳基”表示与脂族化合物相区别的芳香族化合物形成的基团或环，它们通过一个或多个单键而直接与另一结构基团相连接或者与另一环结构稠合，因此区分于通过亚烷基或酯基等间隔基与另一结构基团相连的基团例如“芳烷基”或“芳氧基”或“芳酯基”。类似的，也适用于“杂芳基”，它们可以看做是用杂原子N、S、Se或O代替芳基上的环碳原子或用所述杂原子代替脂族环如环烯烃上的碳原子而形成的基团。此外，如无相反指示，则所述“芳基”或“杂芳基”还包括用芳基、杂芳基取代或稠合的芳基或杂芳基，如联苯基、苯基噻吩基或苯并噻唑基。另外，所述“芳基”或“杂芳基”还可以包括具有例如醚基或羰基的官能基团的芳族或杂芳族化合物形成的基团，如蒽酮、二苯醚或噻唑酮等。有利的，根据本发明的“芳基”或“杂芳基”具有4-30个、更优选5-24、例如6-14或6-10个碳原子。术语“稠合”则表示两个芳环具有公共的边。
在本申请上下文中，术语“烷基”、“烷氧基”或“烷硫基”指的是直链、支化或环状的饱和的脂族烃基，其通过单键、氧基或硫基与其他基团相连接，其优选具有1-50、更优选1-24、如1-18个碳原子。术语“烯基”或“炔基”指的是直链、支化或环状的具有一个或多个C-C双键或三键的不饱和的脂族烃基，优选具有2-50、更优选2-24、如4-18个碳原子。
在本申请上下文中，术语“烷氨基”指的是一个或多个烷基取代的氨基，包括单烷氨基或二烷基氨基，如甲基氨基、二甲基氨基、二乙基氨基、二丁基氨基等。
在本申请上下文中，术语“卤素”包括氟、氯、溴和碘。
在本申请上下文中，术语“吸电子基团”理解为当该基团取代了芳族或杂芳族环上的氢后会使得环上电子云密度降低的基团。这样的基团在化学领域中是广泛公知的。优选的，在本发明中，所述吸电子基团选自硝基、卤素、卤代烷基、磺酸基、氰基、酰基、羧基和/或它们的组合。
此外，在本申请上下文中，所列举的各个取代基定义中的选择基团可以相互组合而形成符合价键原则的新取代基，这意味着例如由烷基、酯基与乙烯基相互组合形成的例如C1-C6烷基酯基乙烯基(C 1-6烷基-O-C(＝O)-C＝C-)也在相关取代基的定义中。
在一个优选的实施方式中，环部分
Figure PCTCN2019123220-appb-000019
可以选自
Figure PCTCN2019123220-appb-000020
Figure PCTCN2019123220-appb-000021
更优选地，G和T选自S和O，最优选地G和T之一为S而另一个为O。
在一个优选的实施方式中，R 1′和R 2′以及R 4′到R 8′各自独立地选自具有1-18、优选1-12、更优选1-16个碳原子的烷基、烷氧基、烷氨基或芳基或者它们的组合，其中所述芳基可以被一个或多个基团L取代或未取代并且优选是被一个或多个L取代或未取代的苯基。
优选地，L选自羟基，磺酸基、卤素、硝基、具有1-12个、更优选1-6个碳原子的直链或支化的烷基、烷氧基、烷氨基、氨基，或者它们的组合。
更优选地，基团R 1′和R 2′以及R 4′到R 8′选自甲氧基、乙氧基、二甲氨基、二乙氨基、二丁氨基、甲基、乙基、丙基、丁基、叔丁基，或者它们的组合。
更优选地，基团R 3′选自吸电子基团或包含吸电子基团的芳基，所述吸电子基团优选自硝基、氰基、卤素、卤代烷基和/或它们的组合。相应的，包含吸电子基团的芳基优选包括环上具有一个或多个选自硝基、氰基、卤素和/或卤代烷基的取代基的芳基，优选苯基，如氟代苯基或全氟苯基。
在一个尤其优选的实施方式中，光化学缓存剂选自例如如下这些化合物：
Figure PCTCN2019123220-appb-000022
Figure PCTCN2019123220-appb-000023
Figure PCTCN2019123220-appb-000024
载体介质
本发明的长余辉苯乙烯聚合物微球中，除了上述组分A)吸光剂、组分B)发光剂和组分C)光化学缓存剂之外，还必须含有苯乙烯聚合物微球作为组分D)载体介质。任选地，除了这些还可以包括用于纳米微球制备的其他加工助剂，或者进一步改善长余辉发光效果的组分。
根据本发明，苯乙烯聚合物微球能够特别良好地吸附如上所述的特定的组分A)到C)，从而形成稳定的负载组分A)到C)的纳米微球。
在本申请中，所述“苯乙烯聚合物微球”由苯乙烯聚合物形成。所述的“苯乙烯聚合物”指的是苯乙烯的均聚物或其与其他可共聚单体形成的高分子共聚物。这样的可共聚单体的例子包括烯烃、炔烃、烯属不饱和羧酸或其酸酐或酰胺或酯等、以及它们的具有一个或多个取代基的衍生物形式，如丁二烯、马来酸酐、(甲基)丙烯酸或(甲基)丙烯酰胺等。在此，所述的“烯烃”或“炔烃”指的是直链、支化或环状的具有一个或多个C-C双键或三键的不饱和脂族烃，优选具有2-50、更优选2-24、如4-18个碳原子。所述的“不饱和羧酸”尤其指的是脂族的烯属不饱和羧酸，即具有式Y-COOH，其中Y是具有一个或多个C-C双键取代C-C单键的C2-C18、如C3-C8的直链、支化或环状的脂族烷基，更优选(甲基)丙烯酸。所述“取代基”包括卤素、氨基、酰胺基、醛基和/或羧基等。
优选的，苯乙烯聚合物微球表面可以含有选自氨基、酰胺基、羧基、和/或醛基的偶联基团，从而本发明的纳米微球表面可以利用这些基团而更好地偶联上抗体或适配体，该抗体或适配体能够与特定抗原发生免疫反应。因此，优选的共聚单体具有一个或多个选自氨基、酰胺基、羧基和/或醛基的基团，如(甲基)丙烯酸、(甲基)丙烯酰胺或氨基取代的烯烃等。
根据本发明的一个有利的实施方式，形成所述微球的苯乙烯聚合物中包含按所有单体总重量计的1％-15％，更优选2％-10％的上述共聚单体。优选的，根据本发明的苯乙烯聚合物或其微球含有基于聚合物总重量计0.05％-5％，更优选为0.1％-2％的如上所 述的偶联基团。这样的优选的苯乙烯聚合物易于获得并且特别适合于免疫检测应用。
在一个更优选的实施方式中，所述苯乙烯聚合物是由苯乙烯、(甲基)丙烯酸或其酯或(甲基)丙烯酰胺以及任选的其他共聚单体形成的共聚物。
此外，根据本发明的苯乙烯聚合物微球的粒径的变异系数小于10％、优选小于5％和更优选小于3％。变异系数(CV)表示数据标准偏差与平均值的比值，它是对数据点在一系列数据中围绕平均值的离散度的统计度量。在本发明范畴内，苯乙烯聚合物微球粒径的变异系数越小越有利于在实际应用中获得稳定可重复的检测效果。本领域技术人员熟悉变异系数的测量方法及所需使用的测量仪器。
本发明范畴内，作为苯乙烯聚合物载体介质，可以直接采用本身以微球形式合成得到的苯乙烯聚合物。苯乙烯聚合物可以采用本领域技术人员已知的合适的自由基聚合反应来制备。取决于不同的聚合和处理工艺，微球状的载体介质可以包括如下的微球结构：核壳结构、水包油结构、油包水结构、介孔结构、中空结构、可溶胀结构等。优选地，苯乙烯聚合物微球的结构选自中空结构、介孔结构和核壳结构。不同结构可根据负载量进行选择，例如选择中空结构的苯乙烯聚合物微球可以吸附更多的A)、B)和C三组分。一般而言，随着苯乙烯聚合物微球的粒径增大，单个微球中含有的组分A)、B)和C)的数量或质量增多，由此单个微球的长余辉发光增强对免疫层析过程中测试信号的高效检出是有利的；但是粒径太大则不利于微球在试纸条上的侧向层析。因此为了获得理想的免疫层析检测效果，本发明的长余辉发光苯乙烯聚合物微球有利地具有5nm–1000nm，更优选50nm–800nm，最优选100nm–500nm范围内的粒径。
在一个有利的实施方式中，所述组分A)到C)的总含量以组分A)到D)的总质量计为0.1％到30％、优选0.2％到25％、更优选0.5％到20％和最优选1％到15％。当组分A)到C)的总含量过低时，即相当于D)组分的含量过高，导致长余辉发光的亮度降低，使得无法基于长余辉发光信号进行有效的免疫检测。当组分A)到C)的总含量过高时，即相当于D)组分的含量过低，导致所形成的纳米微球的分散性和稳定性差，例如容易发生粒子团聚和沉降现象，甚至材料无法形成单分散的纳米结构，不能满足免疫检测的应用需求。
另外，在根据本发明的长余辉材料组合物中，调整吸光剂与发光剂的摩尔比例在合适的范围内能够进一步改善长余辉的效果。在一个有利的实施方式中，吸光剂与发光剂的摩尔比在1:2到1:10000，优选1:10到1:8000或1:50到1:6000，更优选1:100 到1:4000或1:200到1:2000。在一个有利的实施方式中，光化学缓存剂以材料三个组分A)到C)总质量计，其含量可以为0.1％到80％，优选0.3％到60％，更优选0.5％到40％，最优选1％到20％。
当吸光剂比例过高时，会产生长余辉发光被吸光剂吸收而减弱的不利影响。当吸光剂比例过低时，吸收的激发光能量比较有限，也会导致长余辉发光较弱。另外，当光化学缓存剂过少时，能量缓存能力较弱，导致长余辉发光的性能受到不利影响，例如影响到长余辉发光的稳定性和发光亮度等。当体系中添加的缓存剂过多时，会阻碍各组分之间的碰撞传能，缓存的能量无法有效地传输出去而被耗散掉，使得长余辉发光性能降低。
本发明的长余辉材料可以从溶液直接加工，制备成长余辉发光苯乙烯聚合物微球，从而方便地应用于免疫层析试纸条检测领域。
本发明的长余辉发光材料，体系的激发与发射波长容易调控，可以覆盖紫、蓝、绿、黄、红和近红外的光谱区域。通过选取吸光剂或发光剂的种类和必要时适当的结构修饰，使得激发和发射的可操作范围都非常宽，所以实际的激发与发射性质的组合非常丰富。优选地，激发光的波长的可调节范围为300nm至1000nm。另外，长余辉发光既可以为基于上转换机制的发光，也可以为基于下转换机制的发光，还可以为零斯托克斯位移的发光。当使用波段为λ1的光进行激发，长余辉发光的发射光波段λ2灵活分布，长余辉发光可以涵盖紫外可见到近红外的所有波段。当λ1<λ2时，较短波长的光激发而实现较长波长的光发射，即是激发光波长比发射光波长红移，属于常规的下转换发光模式；当λ1>λ2时，较长波长的光激发而实现较短波长的光发射，即是激发光波长比发射光波长蓝移，属于上转换发光模式；当λ1＝λ2时，即是激发光波长与发射光波长处于相同波段，属于零斯托克斯位移的发光模式。
可以使用多种光源对本发明的长余辉发光材料激发充能。常见的光源照明设备、点光源、环形光源、室内及室外自然光照，都可以对基于光化学机制的长余辉发光剂体系进行激发充能。在一种优选的方案中，这些光源包括固体激光、气体激光、半导体激光、光电二极管、D65标准光源、苯乙烯聚合物发光二极管、紫外灯、手电筒、闪光灯、氙灯、钠灯、汞灯、钨丝灯、白炽灯、日光灯和自然太阳光，以及这些光源的组合。在一种更优选的方案中，激光器、发光二极管被用作激发光源，这些光源输出光的单色性较好、发光亮度较高，能够选择性地快速激发充能，在实际应用中光源发射出的光可以为聚焦、发散、环形、准直的光束。激发光源的光输出强度可以具有 很宽的功率密度范围(1μW cm -2–1000W cm -2)，激发时间也具有很宽的动态范围(1μs–1h)。另外，光源输出的激发光可以是连续光、脉冲光或组合模式的输出模式，其中脉冲光是可调制的且具有很宽的调制频率范围(0.001Hz–100KHz)。在一种有利的方案中，根据本发明的超亮长余辉发光材料所需的激发时间短，激发光的照射时间为0.1s–100s，优选0.5s–60s，更优选1s–30s，最优选2s–10s。
第二方面，本发明涉及一种包含上述长余辉发光苯乙烯聚合物微球的探针。该探针包括如上所述的长余辉发光苯乙烯聚合物微球以及负载或偶联于其上的抗体或适配体。
在一个有利的实施方案中，所述抗体或适配体在探针中的含量以整个探针的质量计优选为1％–20％，更优选2％–15％，最优选5％–12％。
合适的抗体或适配体理论上并没有特别的限制。优选它们能够与包括真菌毒素、致病菌、病毒、炎性因子或肿瘤标志物等待检测目标抗原发生特异性的免疫结合，优选自C反应蛋白(CRP)抗体、血清淀粉样蛋白(SAA)抗体、降钙素原(PCT)抗体、甲胎蛋白(AFP)抗体、癌胚抗原(CEA)抗体、前列腺特异抗原(PSA)抗体、心肌肌钙蛋白(CTn-I)抗体、人绒毛膜促性腺激素(HCG)、抗链球菌溶血素O(ASO)、类风湿因子(RF)和/或寡聚核苷酸片段。
第三方面，本发明涉及一种制备如上所述的长余辉发光苯乙烯聚合物微球的方法，其包括如下步骤：
(1)提供组分A)到C)；和
(2)在分散液或溶液中将组分A)到C)分散和吸附到载体介质组分D)上。
在此，可以有利地将组分A)到C)首先彼此混合，然后将其分散或溶解到合适的溶剂中，或者将组分A)到C)依次分散或溶解到合适的溶剂中，以形成溶液。合适的溶剂没有特别的限制，只要能使其形成稳定的溶液或分散液，所述溶剂可以是例如液体石蜡、苯乙醇-乙二醇和水的混合物、均三甲苯和乙醇的混合物、四氢呋喃、二氯甲烷等。
在制得包含组分A)到C)的溶液或分散液之后，可以向其中加入载体介质微球或其溶液或分散液。或者，也可以将包含组分A)到C)的溶液或分散液加入到包含载体介质微球的溶液或分散液中。可以采用水或其他适当的溶剂来分散载体介质，例如去离子水、磷酸盐缓冲液(PBS)、硼酸盐缓冲液(BBS)等。
在制备溶液或分散液的过程中，需要时可以使用超声波、高压均质机等辅助设备， 或进行适当的加热并且伴随搅拌。
在步骤(2)得到稳定的包含组分A)到D)的长余辉发光苯乙烯聚合物微球的分散液之后，可以根据需要无需处理而将其直接进行后续的利用，如用于制备适于免疫检测的试纸。或者，可以进一步在得到的纳米微球上吸附或修饰抗体或适配体，从而得到根据本发明的探针。
探针的制备方法本身是已知的或者可以由本领域技术人员根据已知技术稍加改进而获得。该制备方法主要包括将如上所述的长余辉发光苯乙烯聚合物微球与抗体或适配体通过功能性反应基团如羧基、氨基、醛基进行生物偶联。例如，可以羧基-氨基反应形成偶联或醛基-氨基反应形成偶联。通常，根据纳米微球表面功能基团的情况选择对应的偶联方法。
因此，本发明的第四个方面涉及如上所述的探针的制备方法。
第五方面，本发明提供一种包含如上所述的长余辉发光苯乙烯聚合物微球或如上所述探针的用于免疫层析检测的试纸。所述试纸包括样品垫、结合垫、测试线和质控线，其中所述结合垫上设置有如上所述的长余辉发光苯乙烯聚合物微球或如上所述的探针。
用于免疫层析检测技术的试纸本身的结构是已知的。样品垫、结合垫、测试线和质控线三者可以贴附于底板上，如PVC底板。对于这样的试纸的结构，可以参考例如已公开的专利文献CN105929155A，在此将其全部内容引入本申请中。
在如图3所示的一个示例性结构中，所述试纸包括PVC底板1，底板1上依次设有样品垫2、结合垫3、硝酸纤维素膜4和吸水垫5，其中硝酸纤维素膜4上沿着从样品垫2到吸水垫5的方向，还依次设有测试线6和质控线7。
免疫层析技术主要有双抗夹心和竞争法。双抗夹心法主要用于检测蛋白等大分子类物质，如肿瘤标志物、病毒和炎性因子等。这些检测方法本身是已知的。在一种示例性实施方式中，该方法使用一对针对抗原不同表位的配对抗体，将捕获抗体固定在NC膜的T线上，检测抗体偶联修饰的纳米探针则固定于结合垫上，羊抗鼠(或驴抗鼠、羊抗兔、兔抗鼠等)二抗作为质控线固定在NC膜的C线上。检测过程中，将样品滴加在样品垫上，通过毛细作用从左向右泳动，依次通过结合垫，T线和C线发生特异性免疫反应。竞争法主要用于小分子物质检测。在该方法中例如可以将全抗原(小分子与大分子的偶联产物)固定在NC膜上形成T线，抗体偶联修饰的纳米探针则固定于结合垫上，羊抗鼠(或驴抗鼠、羊抗兔、兔抗鼠等)二抗作为C线。检测过程中， 样品滴加在样品垫上，通过毛细作用依次通过结合垫，T线和C线，固定在T线的抗原会和样本中游离的抗原与抗体发生竞争性结合。免疫层析检测方法的一般程序是本领域技术人员已知的，并且在本申请实施例中例示性给出了该方法的实施方式。
最后，本发明还涉及一种免疫层析检测的方法，其包括如下步骤：
(1)提供如上所述的长余辉发光苯乙烯聚合物微球或探针或试纸；
(2)用激发光照射所述苯乙烯聚合物微球或探针或试纸；和
(3)停止照射，读取发光信号。
与采用无机长余辉材料的类似技术如CN105929155A相比，本发明的免疫层析检测方法更具优点。首先，激发波长的可选择范围更宽，包括紫外光、可见光和近红外光的波长区间。其次，本发明的长余辉发光苯乙烯聚合物微球吸收截面更大几个数量级，这使得光照射充能的时间可以更短，例如最优选2s–10s。另外，本发明的长余辉发光苯乙烯聚合物微球长余辉发光亮度更高，超过肉眼可见的亮度水平，可供选择的检测设备更普遍。优选地，所述检测中用于读取发光信号的仪器为智能手机、发光成像系统、专业长余辉发光检测设备等。更优选地，检测设备为常见的商业化手机，其中配备有信号读取的软件，可以对手机拍照的图片进行信号强度的数据分析。
附图说明
图1为包含根据本发明的长余辉发光苯乙烯聚合物微球的探针的结构示意图。其中可见，组分A)到C)都吸附在载体介质纳米微球上，同时载体介质上还偶联有抗体或适配体。
图2为根据本发明的长余辉发光纳米材料的发光机制示意图。
图3为本发明的免疫层析试纸条示意图，包括PVC底板1，底板1上依次设有样品垫2、结合垫3、硝酸纤维素膜4和吸水垫5；硝酸纤维素膜4上沿着从样品垫2到吸水垫5的方向，还依次设有测试线6和质控线7，图中箭头的指示方向为侧向层析方向。
图4为实施例1合成的300nm的具有羧基的苯乙烯聚合物纳米微球的透射电镜图
图5为实施例2的长余辉苯乙烯聚合物纳米微球的透射电镜图像。
图6基于本发明实施例29测试的长余辉苯乙烯聚合物纳米微球的存放稳定性。
图7基于本发明实施例33的长余辉发光纳米材料的C反应蛋白(CRP)检测的 标准曲线。
图8长余辉免疫层析试纸条检测C反应蛋白(CRP)的效果图，图片均使用手机拍摄。免疫层析试纸条中所用的长余辉信号指示探针不同，左图(a)为基于本发明实施例33的长余辉发光纳米材料的CRP检测效果图，右图(b)为基于对比实施例12制备的无机长余辉SrAl 2O 4:Eu 2+,Dy 3+纳米材料的CRP检测效果图。
图9基于本发明实施例34的长余辉发光纳米材料的血清淀粉样蛋白(SAA)检测的标准曲线。
图10基于本发明实施例35的长余辉发光纳米材料的降钙素原(PCT)检测的标准曲线。
图11基于本发明实施例36的长余辉发光纳米材料的甲胎蛋白(AFP)检测的标准曲线。
图12基于本发明实施例37的长余辉发光纳米材料的癌胚抗原(CEA)检测的标准曲线。
图13基于本发明实施例38的长余辉发光纳米材料的前列腺特异抗原(PSA)检测的标准曲线。
图14基于本发明实施例39的长余辉发光纳米材料的心肌肌钙蛋白(cTn-I)检测的标准曲线。
图15基于本发明实施例40的长余辉发光纳米材料的人绒毛膜促性腺激素(HCG)检测的标准曲线。
图16基于本发明实施例41的长余辉发光纳米材料的抗链球菌溶血素O(ASO)检测的标准曲线。
图17基于本发明实施例42的长余辉发光纳米材料的类风湿因子(RF)检测的标准曲线。
实施例
1、性能测试方法
在本发明的长余辉发光测试中，当激光作为激发光源时，使用美国Opotek,Inc.公司波长可调谐的激光器(Opolette 355)。特定情况下，也使用发光二极管(LED)作为激发光源，激发光的功率密度保持一致。特定波长的激发光照射样品进行充能，照射充能的时间为3s。充能结束后关闭激光，开始测试发光性能。使用英国爱丁堡仪 器公司的荧光光谱仪(Edinburgh FS-5)进行长余辉发光强度的测试。使用北京奥博迪光电技术有限公司的长余辉测试系统(OPT-2003)进行长余辉发光亮度的测试。本发明使用商业化智能手机或常见的数码相机拍照，记录明场及长余辉发光图片。
本文使用的短语“肉眼可见”是长余辉发光材料领域的专业名词，意味着材料的发光亮度大于或等于0.32mcd·m -2，可见光在处于该亮度的辐射水平及以上时通常能够被肉眼看见。本文使用的短语“发光时间”是长余辉发光材料领域的专业名词，表示材料的发光亮度衰减到肉眼可见水平时所经历的时间。本文使用的短语“蓝色长余辉发光”是对材料的长余辉发光颜色的表述，意味着在蓝色的波长区间内有明显的长余辉发光产生；同理，该描述对应地也适用于本文使用的对其他颜色的描述。在实际的情况中，由于观察方法上的不同或受到个体差异的影响，发光颜色或发光时间等观测结果可能会存在误差。
2、所用原料列表
Figure PCTCN2019123220-appb-000025
Figure PCTCN2019123220-appb-000026
Figure PCTCN2019123220-appb-000027
Figure PCTCN2019123220-appb-000028
Figure PCTCN2019123220-appb-000029
3、长余辉发光纳米材料的制备
实施例1
合成苯乙烯聚合物纳米微球，作为长余辉发光材料的载体介质。该苯乙烯聚合物纳米微球是通过乳液聚合的方法合成的：首先取39.3g苯乙烯、2.1g甲基丙烯酸和0.5g十二烷基苯磺酸钠分散到100mL超纯水中，将该溶液加入到500mL的三口烧瓶中，通入氮气，温度保持在25℃，持续搅拌30分钟。然后把温度缓慢升到70℃，立即加入0.3g过硫酸钾(溶解在25g超纯水中)，继续反应4小时。反应结束后，用超纯水和乙醇清洗，常温放置备用。根据所述方法所制备的苯乙烯聚合物纳米微球表面含有羧基，通过电导滴定法测得羧基的含量以重量计占苯乙烯聚合物微球总重量的0.2％。所制备苯乙烯聚合物纳米微球的扫描电子显微镜图像如图4所示，粒径为300nm且非常均匀，据统计纳米粒子之间的变异系数<5％。对于该专业领域内的技术人员来说，上述苯乙烯聚合物纳米微球的合成及调控方法是容易理解的，通过调节合成原料的加入量可以得到其它不同粒径的苯乙烯聚合物纳米微球。
实施例2
制备长余辉发光的苯乙烯聚合物微球。将吸光剂PdOEP、发光剂Eu-1和光化学缓存剂CA-1加入到5mL的苯甲醇-乙二醇-水(v:v:v，1:8:1)溶液中，其中PdOEP 浓度为5μmol L -1，CA-1浓度为3mmol L -1，Eu-1浓度为5mmol L -1。各组分超声分散后，加入50mg的实施例1中300nm表面带有羧基的苯乙烯聚合物(PS)纳米微球，在110℃加热30min。然后，冷却至室温，使用乙醇和水离心清洗3次，最后将纳米粒子分散到水中保存，其扫描电子显微镜下的形貌如图5所示。由电镜图可见，吸光剂、发光剂和光化学缓存剂吸附到苯乙烯聚合物纳米载体的过程中，苯乙烯聚合物纳米微球的外表仍旧维持原样，形貌没有遭到破坏。对所制备的长余辉发光纳米粒子进行余辉性能测试，将长余辉发光纳米粒子配制成1mg mL -1浓度的水溶液。首先，使用365nm波长的激发光照射3s进行充能，充能完成后关闭光源，测试结果如表1所示。
对比实施例1(C1)
由商业化的无机长余辉材料制备无机长余辉苯乙烯聚合物微球。该制备过程中，采用专业领域内技术人员已知的分散聚合方法。在已商业化的无机长余辉材料中，SrAl 2O 4:Eu 2+,Dy 3+是目前亮度最高的绿色长余辉发光材料，应用非常广泛。商品化的SrAl 2O 4:Eu 2+,Dy 3+材料是经高温烧结后再研磨所得的长余辉粉体。首先，通过离心分离获得粒径约250nm的SrAl 2O 4:Eu 2+,Dy 3+无机长余辉纳米微球，采用十六烷基三甲基溴化胺(CTAB)在超声辅助下对无机长余辉纳米微球进行亲油处理。然后，取15g苯乙烯和1g甲基丙烯酸分散到80mL乙醇-水混合溶液(v:v，1:3)中，将该溶液加入到250mL的三口烧瓶中，加入CTAB处理后的无机长余辉纳米微球1g，通入氮气，温度保持在25℃，持续搅拌30分钟。然后把温度缓慢升到70℃，立即加入0.2g偶氮二异丁腈(溶解在25mL乙醇中)，继续反应4小时。反应结束后，用超纯水和乙醇清洗，常温放置备用。通过梯度离心和过滤，最后获得苯乙烯聚合物包裹的无机长余辉纳米微球，粒径约300nm。按照实施例2的方法，将无机长余辉苯乙烯聚合物微球配制成1mg mL -1浓度的水溶液，对纳米微球进行余辉性能测试，结果经肉眼观察没有看到任何的余辉光，借助仪器测试的长余辉发光强度如表1所示。
实施例3-12
重复实施例2的操作，其中吸光剂、光化学缓存剂、发光剂这三种组分的摩尔比例保持为1:600:1000，不同之处显示在表1中。
对比实施例2-3(C2和C3)
重复实施例1的操作，其中吸光剂、光化学缓存剂、发光剂这三种组分的摩尔比例保持为1:600:1000，不同之处显示在表1中。
对比实施例4(C4)
以NCBS作为吸光剂，PFVA作为发光剂，DO作为光化学缓存剂，使用苯乙烯聚合物纳米微球作为纳米载体，制备长余辉纳米微球。将吸光剂NCBS、发光剂PFVA和光化学缓存剂DO加入到5mL的苯甲醇-乙二醇-水(v:v，1:8:1)溶液中，其中PdOEP浓度为5μmol L -1，CA-1浓度为3mmol L -1，Eu-1浓度为5mmol L -1。各组分超声分散后，加入50mg实施例1中的300nm表面带有羧基的苯乙烯聚合物纳米微球，在110℃加热30min。然后，冷却至室温，使用乙醇和水离心清洗3次，最后将纳米粒子分散到水中保存。按照实施例2的方法对所制备的长余辉发光苯乙烯聚合物微球进行余辉性能测试，将长余辉发光苯乙烯聚合物微球配制成1mg mL -1浓度的水溶液。首先，使用808nm波长的激发光照3s进行充能，充能完成后关闭光源，结果经肉眼观察没有看到任何的余辉光，借助仪器测试的长余辉发光强度如表1所示。
对比实施例5(C5)
重复对比实施例4的操作，不同之处显示在表1中。
表1
Figure PCTCN2019123220-appb-000030
Figure PCTCN2019123220-appb-000031
实施例13
制备长余辉发光的苯乙烯聚合物微球。将吸光剂PdPc、发光剂Eu-2和光化学缓存剂CA-1加入到5mL的苯甲醇-乙二醇-水(v:v，1:8:1)溶液中，其中PdPc浓度为200μmol L -1，CA-1浓度为2mmol L -1，Eu-2浓度为10mmol L -1。各组分超声分散后，加入50mg的300nm表面带有羧基的苯乙烯聚合物纳米微球，在110℃加热30min。然后，冷却至室温，使用乙醇和水离心清洗3次，最后将纳米粒子分散到水中保存。对所制备的长余辉发光纳米粒子进行余辉性能测试，将长余辉发光纳米粒子配制成1mg mL -1浓度的水溶液。首先，使用730nm波长的激发光照射3s进行充能，充能完成后关闭光源，测试结果如表1所示。
实施例14-19
重复实施例13的操作，不同之处显示在表2中，其中发光剂Eu-2的浓度为10mmol L -1。
对比实施例6-7(C6和C7)
重复实施例13的操作，不同之处显示在表2中，其中吸光剂PdPc的浓度为10mmol L -1。
表2
Figure PCTCN2019123220-appb-000032
实施例20
将吸光剂PdPc、发光剂Eu-2和光化学缓存剂CA-1混合在二氯甲烷中，使用超声波辅助各组分的溶解，最后形成均匀透明的溶液。在该溶液中，吸光剂PdPc、光化学缓存剂CA-1、发光剂Eu-2这三种组分的摩尔比例为1:600:2000。然后，除去二氯甲烷溶剂得到A)、B)、C)三组分的油状混合物。称取2mg所述的三组分混合物，加入到含有50mg苯乙烯聚合物纳米微球的5mL苯甲醇-乙二醇-水(v:v，1:8:1)溶液中，在110℃加热30min。然后，冷却至室温，使用乙醇和水离心清洗3次，最后将纳米粒子分散到水中保存。将长余辉发光苯乙烯聚合物微球配制成1mg mL -1浓度的水溶液，对所制备的长余辉发光苯乙烯聚合物微球进行余辉性能测试。首先，使用730nm波长的激发光照射3s进行充能，充能完成后关闭光源，测试结果如表3所示。其中，ABC三组分在微球总重量中占的质量分数是按照如下方法测得。将所制得的长余辉苯乙烯聚合物微球充分溶解在四氢呋喃中，然后分离出A)、B)和C)三组分，并计算出三组分在纳米微球中所占的质量分数。另外，观察所得的长余辉纳米微球在水溶液中静置一个月后是否有沉淀生成。
实施例21-23
重复实施例20的操作，不同之处在于纳米材料中载体介质及组分含量(如表3中 所示)，通过调节加入油状三组分混合物的质量获得ABC三组分在微球总重量中不同的质量分数。测试结果如表3所示。
对比实施例8-9(C8和C9)
重复实施例20的操作，不同之处在于纳米材料中载体介质及组分含量(如表3中所示)，通过调节加入油状三组分混合物的质量获得ABC三组分在微球总重量中不同的质量分数。测试结果如表3所示。
对比实施例10(C10)
将吸光剂PdPc、发光剂Eu-2和光化学缓存剂CA-1混合在二氯甲烷中，使用超声波辅助各组分的溶解，最后形成均匀透明的溶液。在该溶液中，吸光剂PdPc、光化学缓存剂CA-1、发光剂Eu-2这三种组分的摩尔比例为1:600:2000。然后，除去二氯甲烷溶剂得到A)、B)、C)三组分的油状混合物。称取10mg所述的三组分混合物，加入到10mL的均三甲苯-乙醇(v:v，1:1)溶液中。各组分超声分散后，加入50mg表面带有氨基的硅纳米微球(粒径为300nm)，在80℃加热2小时。然后，冷却至室温，使用乙醇和水离心清洗3次，最后将纳米微球分散到水中保存。对所制备的长余辉发光苯乙烯聚合物微球进行余辉性能测试，将长余辉发光苯乙烯聚合物微球配制成1mg mL -1浓度的水溶液。首先，使用730nm波长的激发光照射3s进行充能，充能完成后关闭光源，测试结果如表3所示。其中，ABC三组分在微球总重量中占的质量分数是按照如下方法测得。将所制得的长余辉硅纳米微球溶解在甲苯中，充分超声将吸附的ABC三组分溶解，然后分离出A)、B)和C)三组分，并计算出三组分在纳米微球中所占的质量分数。
对比实施例11(C11)
将吸光剂PdPc、发光剂Eu-2和光化学缓存剂CA-1混合在二氯甲烷中，使用超声波辅助各组分的溶解，最后形成均匀透明的溶液。在该溶液中，吸光剂PdPc、光化学缓存剂CA-1、发光剂Eu-2这三种组分的摩尔比例为1:600:2000。然后，除去二氯甲烷溶剂得到A)、B)、C)三组分的油状混合物。称取10mg所述的三组分混合物，加入到含有50mg嵌段共聚物F127的四氢呋喃中，充分溶解，然后除去四氢呋喃，使用超声波将所获得的组合物分散在2mL的水中，通过离心和过滤得到水中均匀分 散的长余辉纳米微球。按照实施例2的方法对所制备的长余辉发光苯乙烯聚合物微球进行余辉性能测试，将长余辉发光苯乙烯聚合物微球配制成1mg mL -1浓度的水溶液。首先，使用730nm波长的激发光照3s进行充能，充能完成后关闭光源，测试结果如表3所示。其中，ABC三组分在微球总重量中占的质量分数是按照如下方法测得。将所制得的长余辉苯乙烯聚合物微球充分溶解在四氢呋喃中，然后分离出A)、B)和C)三组分，并计算出三组分在纳米微球中所占的质量分数。
表3
Figure PCTCN2019123220-appb-000033
实施例24
取80nm的具有羧基的苯乙烯聚合物微球离心，除去合成过程中的表面活性剂，接着将1g的PS微球固体复溶到100mL超纯水中，超声形成分散相。接着向PS微球水溶液中加入2％的十二烷基苯磺酸钠和1％乙二胺聚氧乙烯聚氧丙烯嵌段聚醚各1mL，并进行搅拌。取如表4中所示的组分A)、B)和C)分散于10mL四氢呋喃溶液中形成分散相，其中A)、B)和C)三个组分的浓度分别为5μmol L -1、2mmol L -1和10mmol L -1。在溶液配制完成后，将苯乙烯聚合物相迅速加入水相中，然后逐渐升温到50℃，持续搅拌10h。然后将得到的粒径80nm的长余辉PS微球离心，除去 多余的染料，再用超纯水和乙醇清洗两次，并保存于超纯水中，避光放于常温下备用。
实施例25-28
重复实施例24的操作，不同之处在于作为纳米载体介质的带有羧基的苯乙烯聚合物微球的粒径(如表4中所示)。测试结果如表4所示。
表4
Figure PCTCN2019123220-appb-000034
实施例29
取根据实施例3制备的长余辉发光的苯乙烯聚合物纳米微球10mg离心，复溶到9.5mL的超纯水中，充分超声使其分散均匀后，并向其中加入0.5mL吐温20(10wt％水溶液)，将配置好的溶液分装成10只1mL的溶液，避光静置在暗室中，每一个月进行离心，除掉上清液后用水重新溶解至1mL，然后分别测试其长余辉发光强度，测试结果如图6所示。测试结果表明，长余辉发光性能保持稳定，说明吸附进去的长余辉发光组分A)、B)和C)不会从苯乙烯聚合物纳米载体介质中泄漏。
实施例30
根据实施例3的制备方法，我们按照相同的操作重复10遍，制备获得10个批次的长余辉发光的苯乙烯聚合物微球。按照实施例3的方法，将所制备的长余辉发光纳米粒子分别配制成1mg mL -1浓度的水溶液，对这10个批次的长余辉发光的苯乙烯聚合物微球进行余辉性能测试。首先使用540nm波长的激发光照射3s进行充能，充能完成后关闭光源，得到10个不同批次之间的余辉光强度，测试结果如表5所示。分 析所得的10次长余辉发光的强度，结果表明偏差小于5％，说明该长余辉发光体系具有很好的稳定性和可重复性。
表5
批次 苯乙烯聚合物球粒径(nm) 长余辉发光强度(a.u.)
1 300 175650
2 300 181520
3 300 176254
4 300 178625
5 300 179534
6 300 177250
7 300 180462
8 300 180497
9 300 178564
10 300 176542
实施例31
通过荧光的长余辉纳米微球偶联甲胎蛋白(AFP)抗体AFP-Ab 1制备探针：
1)取根据实施例3制备的长余辉发光苯乙烯聚合物微球100mg离心，复溶到18mL的pH为7.4的BBS缓冲液中，充分超声使其分散均匀；2)向其中分别加入10mg的1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)和2.5mg的N-羟基琥珀酰亚胺磺酸钠盐(NHSS)，室温下反应2小时；3)反应结束后，离心洗涤，复溶到10mL的pH为7.4的BBS缓冲液中，向其中加入10mg的AFP-Ab 1型单克隆抗体，室温下反应4小时；4)反应结束后，离心洗涤，复溶到10mL的pH为7.4的BBS缓冲液中，向其中加入100mg的BSA，室温下反应2小时；5)反应结束后，离心洗涤，复溶到10mL的pH为7.4的BBS缓冲液中，4℃保存备用。
实施例32
通过长余辉纳米微球偶联前列腺特异抗原(PSA)适配体制备探针：
1)取10mg实施例3制备的长余辉发光的苯乙烯聚合物纳米微球离心，复溶到 1.8mL的BBS缓冲液中(pH 7.4)，充分超声使其分散均匀；2)向其中分别加入1mg的EDC和0.25mg的NHSS，室温下震荡反应2小时；3)反应结束后，离心洗涤，复溶到2mL的BBS缓冲液中，并向其中加入20μL含有2μmol mL -1的PSA适配体aptamer，其序列为(NH 2-ATTAAAGCTCGCCATCAAATAGCTGC)，室温下反应4小时；4)反应结束后，离心洗涤，复溶到2mL BBS缓冲液中，向其中加入10mg的BSA，室温下反应2小时；5)反应结束后，离心清洗2遍，复溶到4mL BBS缓冲液中(pH 7.4)，4℃保存备用。
实施例33
制备包含本发明长余辉发光苯乙烯聚合物微球的侧向层析免疫试纸条及其在C反应蛋白(CRP)检测中的应用
(1)用于检测C反应蛋白(CRP)的免疫层析试纸条制备：
1)如下所述使实施例1中的长余辉纳米微球偶联CRP-Ab 1：取实施例3制备的长余辉发光的苯乙烯聚合物微球100mg离心，复溶到18mL的pH为7.4的BBS缓冲液中，充分超声使其分散均匀；向其中分别加入10mg的EDC和2.5mg的NHSS，室温下反应2小时；反应结束后，离心洗涤，复溶到10mL的pH为7.4的BBS缓冲液中，向其中加入10mg的CRP-Ab 1型单克隆抗体，室温下反应4小时；反应结束后，离心洗涤，复溶到10mL的pH为7.4的BBS缓冲液中，向其中加入100mg的BSA，室温下反应2小时；反应结束后，离心洗涤，复溶到10mL的pH为7.4的BBS缓冲液中，4℃保存备用。
2)CRP免疫层析试纸条NC膜的制备：使用PBS缓冲液(1％的BSA，1％蔗糖，50mM的NaCl和0.5％的TWEEN 20)分别将CRP-Ab 2型单克隆抗体和驴抗鼠IgG分别以1mg mL -1和1mg mL -1的浓度且以8mm的间隔，用划膜仪划于硝酸纤维素膜上，放于37℃过夜烘干。
3)CRP免疫层析试纸条样品垫和结合垫的制备：CRP免疫层析试纸条的样品垫为玻璃纤维。CRP免疫层析试纸条结合垫也为玻璃纤维材质，区别在于玻璃纤维上喷涂有长余辉发光苯乙烯聚合物纳米探针。
玻璃纤维上的探针喷涂流程：取如步骤1)中制备好的荧光探针，离心处理，用喷膜缓冲液复溶为20mg mL -1，通过喷膜仪将荧光探针溶液倒吸入仪器，以1.2μL cm -1的速度将荧光探针喷于玻璃纤维上，并于37℃烘烤过夜。
4)CRP免疫层析试纸条试纸条的组装：在白色PVC底板上依次相互交错3mm地贴上样品垫，标记了CRP-Ab 1型单克隆抗体的长余辉探针的玻璃纤维(结合垫)，划有CRP-Ab 2的T线和驴抗鼠IgG的C线的NC膜，最后贴上吸水纸。接着将组装好的层析板通过高速斩切机切割成3.8mm宽的试纸条，然后再用配套的上下两个塑料卡壳固定试纸条，即得到免疫层析试纸条。
(2)CRP标准曲线的建立：
1)将CRP抗原储备液稀释为不同浓度的全血CRP抗原溶液，浓度分别为0μg mL -1、0.1μg mL -1、0.5μg mL -1、5μg mL -1、20μg mL -1、40μg mL -1、160μg mL -1和320μg mL -1。
2)取1μL样本CRP抗原溶液加入到99μL的PBS缓冲液中(含有1％的BSA、0.1％的SDS和0.1％的B66)，并充分混合。
3)将混合均匀的100μL混合液加入到免疫层析试纸条加样孔处，液体会通过毛细作用依次通过样品区、检测区和吸水区。检测样本中含有抗原溶液时，抗原首先与样品区的长余辉发光探针结合形成免疫复合物，然后随着液体泳动到测试线(T线)与CRP-Ab 2形成夹心免疫复合物，多余的长余辉发光探针则泳动到控制线(C线)与驴抗鼠二抗结合。而当检测样本中没有抗原时，则会带动长余辉发光探针直接泳动到C线与驴抗鼠二抗结合。
4)反应进行5分钟后，将免疫层析试纸条使用长余辉发光检测仪检测。激发光照射时间为3s，停止激发后开始采集试纸条上的长余辉发光信号。测定T线和C线的长余辉发光强度，再计算出强度的比值，通过比值与抗原浓度的对应关系建立标准曲线(图7)。所述长余辉发光检测仪为日常使用的商业化智能手机，其中配备有信号读取的软件，可以对手机拍照的图片进行信号强度的数据分析。
(3)实际样本中CRP的免疫层析检测：
所制备的长余辉发光纳米指示探针，仅需要在读数前使用激发光照射激发，而在后续的读数过程中激发光处于关闭状态，该方法消除了背景荧光信号的干扰，可实现待测物的高灵敏定量检测。在对含有CRP样本检测中发现，基于本发明的长余辉发光苯乙烯聚合物微球的免疫层析试纸条(图8左)在检测灵敏度上比基于无机长余辉的检测体系(图8右)提高了100倍以上，但对检测样品处理要求上没有明显变化，而且检测用时部分在激发时间上只需要3s。根据本发明的长余辉发光免疫层析试纸条的检测结果，样本中含有21μg mL -1的CRP抗原。
对比实施例12(C12)
重复实施例33的操作，不同之处为：在第一步中，使用对比实施例1中的无机长余辉纳米微球偶联CRP-Ab 1。据此制备的无机长余辉纳米的免疫层析试纸条，长余辉发光信号弱，肉眼不可见且使用手机拍摄不到信号，如图8中右图所示。
实施例34-42
重复实施例33的操作，获得基于长余辉侧向层析免疫试纸条的抗原检测标准曲线，不同之处在于所检测的目标抗原分别更换为：SAA(实施例34，图9)、PCT(实施例35，图10)、AFP(实施例36，图11)、CEA(实施例37，图12)、PSA(实施例38，图13)、cTn-I(实施例39，图14)、HCG(实施例40，图15)、ASO(实施例41，图16)和RF(实施例42，图17)。
实施例43
将从中检所购买的CRP抗原标准品稀释到21μg mL -1，对该样本进行30次平行检测。取待检测样本平均分成30份，重复实施例33的操作分别进行检测，所得的30次检测结果展示在表6中。对所得的30次检测结果进行分析，发现检测结果的变异系数小于5％，说明本发明的长余辉发光探针及免疫层析试纸条检测方法具有很好的检测准确性和可重复性。
表6
Figure PCTCN2019123220-appb-000035

Claims (31)

  1. 一种长余辉发光苯乙烯聚合物微球，其包含
    A)至少一种吸光剂，
    B)至少一种发光剂，所述发光剂是单体的非聚合的化合物且其分子量小于10000g mol -1，
    C)至少一种式(I)的光化学缓存剂，
    Figure PCTCN2019123220-appb-100001
    其中，
    G和T为选自O,S,Se和N的杂原子；
    R 1′和R 2′以及R 4′到R 8′各自独立地选自H、羟基、羧基、氨基、巯基、酯基、硝基、磺酸基、卤素、酰胺基，或具有1-50、优选1-24、如2-14个碳原子的烷基、烯基、炔基、烷氧基、烷硫基、烷氨基、芳基、芳烷基、具有N、O或S的杂芳基或杂芳烷基，或者它们的组合，其中所述芳基、芳烷基、杂芳基或杂芳烷基任选具有一个或多个取代基L；和
    L选自羟基、羧基、氨基、巯基、酯基、硝基、磺酸基、卤素、酰胺基，或具有1-50、优选1-24、如2-14个或6-12个碳原子的烷基、烯基、炔基、烷氧基和烷氨基，或者它们的组合；和
    R 3′为吸电子基团或包含吸电子基团的芳基，优选地所述吸电子基团选自硝基、卤素、卤代烷基、磺酸基、氰基、酰基、羧基和/或它们的组合；和
    D)用于吸附组分A)到C)的载体介质，所述载体介质是苯乙烯聚合物微球；
    其中，所述吸光剂与发光剂是结构不同的化合物，并且所述组分A)到C)的总含量以组分A)到D)的总质量计为0.1％到30％、优选0.2％到25％、更优选0.5％到20％和最优选1％到15％。
  2. 根据权利要求1所述的长余辉发光苯乙烯聚合物微球，其特征在于，环部分
    Figure PCTCN2019123220-appb-100002
    可以选自
    Figure PCTCN2019123220-appb-100003
    更优选地G和T选自S和O，最优选地G和T之一为S而另一个为O。
  3. 根据权利要求1或2所述的长余辉发光苯乙烯聚合物微球，其特征在于，基团R 1′和R 2′以及R 4′到R 8′各自独立地选自具有1-18、优选1-12、更优选1-16个碳原子的烷基、烷氧基、烷氨基或芳基或者它们的组合，其中所述芳基可以被一个或多个基团L取代或未取代并且优选是被一个或多个L取代或未取代的苯基。
  4. 根据前述权利要求任一项所述的长余辉发光苯乙烯聚合物微球，其特征在于，L选自羟基，磺酸基，卤素，硝基，具有1-12个、更优选1-6个碳原子的直链或支化的烷基、烷氧基、烷氨基、氨基，或者它们的组合；
    更优选地选自卤素，具有1-12个、更优选1-6个碳原子的直链或支化的烷基、烷氧基、烷氨基，或者它们的组合。
  5. 根据前述权利要求任一项所述的长余辉发光苯乙烯聚合物微球，其特征在于，基团R 1′和R 2′以及R 4′到R 8′选自甲氧基、乙氧基、二甲氨基、二乙氨基、二丁氨基、甲基、乙基、丙基、丁基、叔丁基，或者它们的组合。
  6. 根据前述权利要求任一项所述的长余辉发光苯乙烯聚合物微球，其特征在于，基团R 3′选自吸电子基团或包含吸电子基团的苯基，所述吸电子基团优选自硝基、氰基、卤素、卤代烷基和/或它们的组合，和所述包含吸电子基团的芳基选自一个或多个硝基、氰基、卤素和/或卤代烷基取代的苯基。
  7. 根据前述权利要求任一项所述的长余辉发光苯乙烯聚合物微球，其特征在于，所述光化学缓存剂选自如下这些化合物的一种或多种：
    Figure PCTCN2019123220-appb-100004
    Figure PCTCN2019123220-appb-100005
  8. 根据前述权利要求任一项所述的长余辉发光苯乙烯聚合物微球，其特征在于，所述苯乙烯聚合物微球表面含有选自氨基、酰胺基、羧基和/或醛基的偶联基团。
  9. 根据前述权利要求任一项所述的长余辉发光苯乙烯聚合物微球，其特征在于，所述苯乙烯聚合物包括苯乙烯的均聚物或其与其他可共聚单体形成的共聚物，其中所述可共聚单体包括烯烃、炔烃、烯属不饱和羧酸或其酸酐或酰胺或酯、以及它们的具有一个或多个取代基的衍生物形式，并且所述取代基选自卤素、氨基、酰胺基、醛基和/或羧基。
  10. 根据前述权利要求任一项所述的长余辉发光苯乙烯聚合物微球，其特征在于，所述苯乙烯聚合物选自苯乙烯与脂族的烯属不饱和羧酸或其酸酐或酰胺或酯的共聚物，优选是苯乙烯与式Y-COOH的烯属不饱和羧酸或其酰胺的共聚物，其中Y是具有一个或多个C-C双键取代C-C单键的C2-C18、如C3-C8的直链、支化或环状的脂族烷基，更优选所述苯乙烯聚合物是由苯乙烯与(甲基)丙烯酸和/或(甲基)丙烯酰胺形成的共聚物。
  11. 根据前述权利要求任一项所述的长余辉发光苯乙烯聚合物微球，其特征在于，所述的苯乙烯聚合物中包含按所有单体总重量计的1％-15％，更优选2％-10％的共聚单体。
  12. 根据权利要求8所述的长余辉发光苯乙烯聚合物微球，其特征在于，所述的苯乙烯聚合物含有基于聚合物总重量计0.05％-5％，更优选为0.1％-2％的偶联基团。
  13. 根据前述权利要求任一项所述的长余辉发光苯乙烯聚合物微球，其特征在于，所述苯乙烯聚合物载体介质的粒径的变异系数小于10％、优选小于5％和更优选小于3％。
  14. 根据前述权利要求任一项所述的长余辉发光苯乙烯聚合物微球，其特征在于，所述长余辉发光苯乙烯聚合物微球具有5nm–1000nm，更优选50nm–800nm，最优选100nm–500nm范围内的粒径。
  15. 根据前述权利要求任一项所述的长余辉发光苯乙烯聚合物微球，其特征在于，吸光剂与发光剂的摩尔比在1:2到1:10000，优选1:10到1:8000或1:50到1:6000，更优选1:100到1:4000或1:200到1:2000。
  16. 根据前述权利要求任一项所述的长余辉发光苯乙烯聚合物微球，其特征在于，光化学缓存剂以组分A)到C)总质量计，其含量为0.1％到80％，优选0.3％到60％，更优选0.5％到40％，最优选1％到20％。
  17. 根据前述权利要求任一项所述的长余辉发光苯乙烯聚合物微球，其特征在于，所述长余辉发光苯乙烯聚合物纳米微球由组分A)到D)组成。
  18. 根据前述权利要求任一项所述的长余辉发光有机纳米微球，其特征在于，所述发光剂可选自铱配合物、稀土配合物、并苯类化合物、氟硼二吡咯类化合物(BODIPY)、以及这些化合物的衍生物和共聚物。
  19. 探针，其包括根据权利要求1至18任一项所述的长余辉发光苯乙烯聚合物微球以及负载或偶联于其上的抗体或适配体。
  20. 根据权利要求19所述的探针，其特征在于，所述抗体或适配体在探针中的 含量以整个探针的质量计优选为1％–20％，更优选2％–15％，最优选5％–12％。
  21. 根据权利要求19或20所述的探针，其特征在于，所述抗体或适配体优选自C反应蛋白(CRP)抗体、血清淀粉样蛋白(SAA)抗体、降钙素原(PCT)抗体、甲胎蛋白(AFP)抗体、癌胚抗原(CEA)抗体、前列腺特异抗原(PSA)抗体、心肌肌钙蛋白(CTn-I)抗体、人绒毛膜促性腺激素(HCG)抗体、抗链球菌溶血素O(ASO)抗体、类风湿因子(RF)抗体和/或寡聚核苷酸片段。
  22. 制备根据权利要求1至18任一项所述的长余辉发光苯乙烯聚合物微球的方法，其包括如下步骤：
    (1)提供组分A)到C)；和
    (2)在分散液或溶液中将组分A)到C)分散和吸附到载体介质组分D)上。
  23. 根据权利要求22所述的方法，其特征在于，使用选自液体石蜡、苯乙醇-乙二醇和水的混合物、均三甲苯和乙醇的混合物、四氢呋喃和二氯甲烷的一种或多种溶剂分散或溶解组分A)到C)。
  24. 制备根据权利要求19所述的探针的方法，其中将抗体或适配体吸附或修饰于根据权利要求1至18任一项所述的长余辉发光苯乙烯聚合物微球上。
  25. 根据权利要求24的方法，其中包括将所述长余辉发光苯乙烯聚合物微球与抗体或适配体通过功能性反应基团如羧基、氨基、酰胺基、和/或醛基进行生物偶联。
  26. 用于免疫层析检测的试纸，其包含根据权利要求1至18任一项所述的长余辉发光苯乙烯聚合物微球或根据权利要求19至21任一项所述的探针。
  27. 根据权利要求26所述的试纸，试纸包括结合垫、测试线和质控线，其中所述结合垫上设置有所述的长余辉发光苯乙烯聚合物微球或所述的探针。
  28. 免疫层析检测的方法，其包括如下步骤：
    (1)提供根据权利要求1至18任一项所述的长余辉发光苯乙烯聚合物微球、根据权利要求19至21任一项所述的探针或者根据权利要求26至27任一项所述的试纸；
    (2)用激发光照射所述苯乙烯聚合物微球或探针或试纸；和
    (3)停止照射，读取发光信号。
  29. 根据权利要求28所述的检测方法，其特征在于，激发波长的可调区间300nm–1000nm。
  30. 根据权利要求28所述的检测方法，其特征在于，光照射时间为2s–10s。
  31. 根据权利要求28所述的检测方法，其特征在于，用于读取发光信号的仪器选自手机、发光成像系统和/或专业长余辉发光检测设备，更优选手机。
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