Method for cross-linking auxiliary cluster engineering construction of room temperature phosphorescent polymer microspheres and application thereof

Abstract

The application discloses a method for constructing room-temperature phosphorescent polymer microspheres by cross-linking auxiliary clusters and application thereof, and belongs to the technical field of luminescent materials. The monomer UPyMA and the cross-linking agent MBA are used to prepare polymer microsphere precursors through precipitation polymerization and free radical polymerization, and then the room-temperature phosphorescent polymer microspheres with adjustable RTP emission are obtained through hydrothermal treatment. The phosphorescent performance is accurately controlled by systematically adjusting the cross-linking degree, the microsphere size, the heteroatom density and the polymer chain flexibility. The optimized cross-linking microsphere RTP lifetime reaches 324.79 ms, and the total quantum yield reaches 31.4%. The microspheres can be used as energy donors to realize multicolor afterglow emission (the energy transfer efficiency is up to 84.49%) through the PRET strategy, and are applied to time-resolved anti-counterfeiting. Further, through the antibody-induced PET quenching and the antigen-triggered phosphorescence recovery mechanism, a "turn-on" type RTP immunoassay method for cardiac troponin I (cTnI) is constructed, the detection limit is 0.22 ng / mL, the method has good selectivity and reproducibility, and has application prospects in the field of instant diagnosis.

Description

Technical Field

[0001] This invention belongs to the field of luminescent materials technology, and particularly relates to a method for constructing room temperature phosphorescent polymer microspheres by cross-linked assisted cluster engineering and its application. Background Technology

[0002] Photoluminescent materials have become indispensable tools in modern bioanalysis and information security, with wide applications in bioimaging, immunoassay, optical encryption, and anti-counterfeiting. Traditional luminescent materials typically rely on large π-conjugated structures to achieve high emission efficiency and tunable emission colors, but these methods suffer from complex synthesis processes, high costs, pollutant generation, and unavoidable biological and immunotoxic effects, which limit their practical applications.

[0003] Clusteroluminescence (CL) originates from the spatial aggregation of non-conjugated groups (such as carbonyl, amide, ester, and hydroxyl groups). Through through-space conjugation (TSC), fluorescence / phosphorescence emission can be generated under photoexcitation. The cluster-triggered emission (CTE) mechanism shows that the aggregation of unconventional chromophores can effectively narrow the band gap, enhance electron delocalization, and create new emission pathways. Benefiting from their metal-free properties, structural diversity, ease of preparation, and excellent biocompatibility, clusteroluminescent materials have become an emerging research frontier in the field of luminescent materials.

[0004] Early cluster-luminescence systems mostly exhibited short-lived fluorescence emission, making them susceptible to background fluorescence interference in practical applications. Room-temperature phosphorescence (RTP), with lifetimes ranging from microseconds to seconds, can effectively eliminate short-lived background interference through time-resolved techniques, offering unique advantages in biosensing and information security. The generation of efficient RTP relies on efficient intersystem crossing (ISC) processes and stable triplet excitons, typically employing strategies such as heavy atom effects and rigid confinement to enhance spin-orbit coupling (SOC) and suppress nonradiative transitions.

[0005] Cluster-based room-temperature phosphorescent (CL-RTP) materials combine the advantages of long-lifetime phosphorescence with the simplicity of non-conjugated system structures, offering new opportunities for the development of environmentally friendly and biocompatible phosphorescent materials. However, the performance of CL-RTP materials is still far inferior to that of traditional RTP systems. In particular, the relationship between cluster evolution, polymer microstructure, and triplet stabilization remains unclear, hindering the rational design of high-performance CL-RTP materials.

[0006] Polymer microspheres offer an attractive platform for addressing this challenge. Due to their well-defined morphology, tunable composition, and excellent processing properties, polymer microspheres have been widely used in the fabrication of sensing, catalytic, and optical materials. More importantly, the confined polymer network within the microsphere can effectively restrict molecular motion, suppress oxygen diffusion, and stabilize excited states, thus creating favorable conditions for the generation of RTPs. Furthermore, the microsphere structure provides abundant opportunities for surface functionalization and biomolecular coupling, making it particularly attractive for multifunctional luminescent systems. However, existing RTP microspheres are mainly constructed through guest-host doping strategies, and developing CL-RTP microspheres with controllable cluster formation and expanding their applications remains a significant challenge. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention proposes a method for constructing room-temperature phosphorescent polymer microspheres using cross-linked assisted cluster engineering and its applications. This invention provides a highly efficient method for preparing CL-RTP polymer microspheres through a cross-linked assisted cluster engineering strategy, achieving synergistic regulation of luminescent clusters and cross-linking networks within the polymer microspheres. This results in multifunctional polymer microspheres with long-lifetime room-temperature phosphorescence and high quantum yield, and expands their applications in time-resolved anti-counterfeiting and biosensoring.

[0008] To achieve the above objectives, the present invention provides the following technical solution: A method for constructing room-temperature phosphorescent polymer microspheres using cross-linking-assisted cluster engineering includes the following steps: (1) The monomer 2-(3-(6-methyl-4-oxo-1,4-dihydropyrimidin-2-yl)ureido) ethyl methacrylate (UPyMA) and the crosslinking agent N,N'-methylenebis(acrylamide) (MBA) were dissolved in the reaction solvent, and an initiator was added. The polymer microsphere precursor (PUM) was prepared by precipitation polymerization and free radical polymerization. (2) The polymer microsphere precursor obtained in step (1) is dispersed in a solvent and subjected to hydrothermal treatment to obtain room temperature phosphorescent polymer microspheres with tunable RTP emission.

[0009] Further, in step (1), the molar ratio of the monomer 2-(3-(6-methyl-4-oxo-1,4-dihydropyrimidin-2-yl)ureido)methyl methacrylate and the crosslinking agent N,N'-methylenebis(acrylamide) is 1:(5~10). This invention further clarifies the influencing factors of cluster luminescence by systematically adjusting the amount of crosslinking agent MBA, microsphere size, electron-rich density, and polymer chain flexibility.

[0010] Further, in step (1), the molar ratio of the monomer 2-(3-(6-methyl-4-oxo-1,4-dihydropyrimidin-2-yl)ureido) ethyl methacrylate and the initiator is 1:0.1.

[0011] Furthermore, the initiator is azobisisobutyronitrile (AIBN).

[0012] Further, in step (1), the reaction solvent is a mixed solution of ethanol and water, the reaction temperature is 60~75℃, and the reaction time is 12~24 hours.

[0013] Furthermore, in step (2), the temperature of the hydrothermal treatment is 100~160℃ and the time is 3 hours.

[0014] Furthermore, in step (2), the solvent is water.

[0015] The present invention also provides a room-temperature phosphorescent polymer microsphere prepared according to the above method.

[0016] The present invention also provides an application of the above-mentioned room temperature phosphorescent polymer microspheres in time-resolved anti-counterfeiting and preparation of a biosensor for detecting cardiac troponin I (cTnI).

[0017] The room-temperature phosphorescent polymer microspheres provided by this invention can be used for phosphorescent resonance energy transfer (PRET), which can then be used for time-resolved anti-counterfeiting, and can also enable biological detection of cTnI.

[0018] The present invention also provides a method for detecting cardiac troponin I (cTnI) using the above-mentioned room-temperature phosphorescent polymer microspheres, comprising the following steps: (1) Anti-cTnI antibody was coupled to the surface of room temperature phosphorescent polymer microspheres, and phosphorescence quenching was induced by antibody-induced photo-induced electron transfer (PET) mechanism; (2) Add cTnI antigen from the sample to be tested. Through antigen-antibody specific recognition and competitive binding, a cTnI-anti-cTnI immune complex is formed, which causes the cTnI-anti-cTnI immune complex to detach from the surface of the microspheres, inhibiting the PET process and realizing phosphorescence recovery. (3) The concentration of cTnI was quantitatively detected based on the degree of phosphorescence recovery. The detection limit was 0.22 ng / mL and the detection range was 0~80 ng / mL.

[0019] Compared with the prior art, the present invention has the following advantages and technical effects: (1) This invention establishes a cross-linked assisted cluster engineering strategy, constructs organic RTP polymer microspheres through free radical polymerization and hydrothermal treatment, and systematically studies the regulation of phosphorescence emission by cross-linking degree, microsphere size, heteroatom density and polymer chain flexibility, providing clear design principles for the construction of cluster-triggered RTP microspheres.

[0020] (2) The highly cross-linked RTP polymer microspheres prepared in this invention have long lifetime RTP emission (324.79 ms) and high quantum yield (31.4%), which are superior to existing cluster-luminescent RTP materials (for example, the phosphorescence lifetime of polystyrene-maleic anhydride derivatives prepared by Professor Y. Yang, B. Liu of the National University of Singapore (Long-lived room temperature phosphorescence from amorphous non-traditional intrinsic clusteroluminescence polymers, J. Mater. Chem. C, 2022,10, 16420.) is only 39.88 ms, and the total quantum yield is 11.4%).

[0021] (3) The present invention achieves efficient energy transfer (efficiency up to 84.49%) through PRET strategy and obtains adjustable multicolor afterglow emission, which can be applied to time-resolved anti-counterfeiting.

[0022] (4) The "turn-on" type RTP immunoassay method developed in this invention, based on antibody-induced PET quenching / antigen-triggered phosphorescence recovery, achieves high sensitivity and high selectivity for the detection of cardiac troponin I (detection limit of 0.22 ng / mL), meets the requirements of clinical testing, and has application prospects in the field of point diagnosis.

[0023] (5) The polymer microsphere preparation method of the present invention is simple, low cost, free of metal elements, biocompatible, and can be mass-produced. Attached Figure Description

[0024] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 The diagram shows the preparation process of room temperature phosphorescent polymer microspheres and the loading process of rhodamine B, as well as the process of using room temperature phosphorescent polymer microspheres for multicolor microsphere preparation and "off-on" cTnI antigen detection. a shows the preparation process of room temperature phosphorescent polymer microspheres and the loading process of rhodamine B, and b shows the process of using room temperature phosphorescent polymer microspheres for multicolor microsphere preparation and "off-on" cTnI antigen detection. Figure 2 The diagram shows the preparation process of the room temperature phosphorescent polymer microspheres of the present invention and the scanning electron microscope images of the microspheres under different hydrothermal treatment conditions. a is a schematic diagram of the preparation process of the room temperature phosphorescent polymer microspheres, b is a SEM image of the PUM polymer microsphere precursor, c is a SEM image of PUM-1 microspheres, d is a SEM image of PUM-2 microspheres, and e is a SEM image of PUM-3 microspheres. Figure 3 The following are spectral performance diagrams of the room-temperature phosphorescent polymer microspheres of the present invention: a) phosphorescence emission spectrum of the precursor polymer microsphere PUM at different excitation wavelengths; b) phosphorescence emission spectrum of PUM-1 microspheres obtained after hydrothermal treatment of the precursor microsphere PUM at 100 °C for 3 h at different excitation wavelengths; c) phosphorescence emission spectrum of PUM-2 microspheres obtained after hydrothermal treatment of the precursor microsphere PUM at 140 °C for 3 h at different excitation wavelengths; d) phosphorescence emission spectrum of PUM-2 microspheres obtained after hydrothermal treatment of the precursor microsphere PUM at 160 °C for 3 h at different excitation wavelengths; e) phosphorescence lifetime of the precursor microsphere PUM under 400 nm excitation; f) phosphorescence lifetime of PUM-1 microspheres under 400 nm excitation; g) phosphorescence lifetime of PUM-2 microspheres under 400 nm excitation; and h) phosphorescence lifetime of PUM-3 microspheres under 400 nm excitation. Figure 4 In Example 4, a is the fluorescence spectrum of the highly cross-linked room temperature phosphorescent polymer microspheres under different excitation wavelengths; b is the phosphorus spectrum of the highly cross-linked room temperature phosphorescent polymer microspheres under different excitation wavelengths; c is the phosphorescence lifetime of the highly cross-linked room temperature phosphorescent polymer microspheres under 400 nm excitation; d is a schematic diagram of the preparation process of PUA, the linear polymer microsphere precursor in Comparative Example 1; e is the fluorescence spectrum of PUA-2, the linear polymer microsphere in Comparative Example 1, under different excitation wavelengths; f is the phosphorus spectrum of PUA-2, the linear polymer microsphere in Comparative Example 1, under different excitation wavelengths; and g is the phosphorescence lifetime of PUA-2, the linear polymer microsphere in Comparative Example 1, under 400 nm excitation. Figure 5 In Figure a, the phosphorescence emission and absorption overlap of the highly cross-linked room temperature phosphorescent polymer microspheres in Example 4 are shown in Figure b. The fluorescence changes of the phosphorescent microspheres after adsorbing different mass concentrations of Rhodamine B are shown in Figure c. The phosphorescence changes of the phosphorescent microspheres after adsorbing different mass concentrations of Rhodamine B are shown in Figure d. The lifetime changes of the donor phosphorescent microspheres after adsorbing different mass concentrations of Rhodamine B are shown in Figure e. The lifetime changes of the acceptor Rhodamine B after adsorbing different mass concentrations of Rhodamine B are shown in Figure f. The energy transfer efficiency is shown in Figure f. Figure 6 The graph shows the trend of Zeta potential changes of highly cross-linked room temperature phosphorescent polymer microspheres loaded with anti-cTnI in Example 4. The concentrations of anti-cTnI loaded from a to f are 0.05 μg / mL, 0.1 μg / mL, 0.5 μg / mL, 1 μg / mL, 1.5 μg / mL, and 3 μg / mL, respectively. Figure 7In Example 4, a is the fluorescence spectrum of cTnI at concentrations of 0-80 ng / mL after loading anti-cTnI onto highly cross-linked room-temperature phosphorescent polymer microspheres; b is the phosphorescence spectrum of cTnI at concentrations of 0-80 ng / mL after loading anti-cTnI onto highly cross-linked room-temperature phosphorescent polymer microspheres; c is the phosphorescence lifetime spectrum of cTnI at concentrations of 0-80 ng / mL after loading anti-cTnI onto highly cross-linked room-temperature phosphorescent polymer microspheres; d is the repeatability test graph; e is the phosphorescence intensity curve fitted by four parameters; f is the specificity detection graph; g is the fabricated paper-based phosphorescence sensor graph; and h is the grayscale value curve fitted by four parameters. Detailed Implementation

[0025] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0026] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0027] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0028] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0029] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0030] Embodiments of the present invention provide a method for constructing room-temperature phosphorescent polymer microspheres using cross-linked assisted cluster engineering, comprising the following steps: (1) Dissolve monomer UPyMA and crosslinking agent MBA in reaction solvent, add initiator, and prepare polymer microsphere precursor (PUM) through precipitation polymerization and free radical polymerization reaction. (2) The polymer microsphere precursor obtained in step (1) is dispersed in a solvent and subjected to hydrothermal treatment to obtain room temperature phosphorescent polymer microspheres with tunable RTP emission.

[0031] In a preferred embodiment of the present invention, in step (1), the molar ratio of monomer UPyMA to crosslinking agent MBA is 1:(5~10), preferably 1~10. By increasing the amount of crosslinking agent MBA, the crosslinking density of the polymer microspheres can be increased, further enhancing the phosphorescence lifetime and quantum yield of the room temperature phosphorescent polymer microspheres.

[0032] In a preferred embodiment of the present invention, in step (1), the molar ratio of monomer UPyMA and initiator is 1:0.1.

[0033] In a preferred embodiment of the present invention, the initiator is AIBN.

[0034] In a preferred embodiment of the present invention, in step (1), the reaction solvent is a mixture of ethanol and water in a volume ratio of 1:1, the reaction temperature is 60~75°C, and the reaction time is 12~24 hours; preferably, the reaction temperature is 70°C and the reaction time is 16 hours.

[0035] In a preferred embodiment of the present invention, in step (2), the temperature of the hydrothermal treatment is 100~160°C and the time is 3 hours; preferably, the temperature of the hydrothermal treatment is 140~160°C, and more preferably, the temperature of the hydrothermal treatment is 140°C.

[0036] In a preferred embodiment of the present invention, in step (2), the solvent is water.

[0037] The embodiments of the present invention also provide a room temperature phosphorescent polymer microsphere prepared according to the above method, which has a carbon-oxygen double bond (C=O) enriched luminescent clusters and a cross-linked enhanced conformational rigidity structure, with an RTP lifetime of 324.79 ms and a total yield of 31.4%.

[0038] The embodiments of the present invention also provide an application of the above-mentioned room temperature phosphorescent polymer microspheres in time-resolved anti-counterfeiting and preparation of a biosensor for detecting cardiac troponin I. The above-mentioned room temperature phosphorescent polymer microspheres serve as an energy donor, and organic fluorescent dyes are adsorbed onto the surface of the microspheres through electrostatic adsorption to achieve multicolor afterglow emission with an energy transfer efficiency of up to 84.49%, which is applied to time-resolved anti-counterfeiting.

[0039] In a preferred embodiment of the present invention, the organic fluorescent dye is Rhodamine B, and the optimal doping weight ratio is 1.50%.

[0040] Figure 1 The diagrams show the preparation process of room-temperature phosphorescent polymer microspheres and the loading process of Rhodamine B, as well as the process of using room-temperature phosphorescent polymer microspheres for multicolor microsphere preparation and "off-on" cTnI antigen detection. a shows the preparation process of room-temperature phosphorescent polymer microspheres and the loading process of Rhodamine B, and b shows the process of using room-temperature phosphorescent polymer microspheres for multicolor microsphere preparation and "off-on" cTnI antigen detection.

[0041] Embodiments of the present invention also provide a method for detecting cardiac troponin I (cTnI) using the above-mentioned room-temperature phosphorescent polymer microspheres, comprising the following steps: (1) Anti-cTnI antibody was coupled to the surface of room temperature phosphorescent polymer microspheres, and phosphorescence quenching was induced by antibody-induced photo-induced electron transfer (PET) mechanism; (2) Add cTnI antigen from the sample to be tested. Through antigen-antibody specific recognition and competitive binding, a cTnI-anti-cTnI immune complex is formed, which causes the cTnI-anti-cTnI immune complex to detach from the surface of the microspheres, inhibiting the PET process and achieving phosphorescence recovery. (3) The concentration of cTnI was quantitatively detected based on the degree of phosphorescence recovery. The detection limit was 0.22 ng / mL, and the linear range was 0~80 ng / mL.

[0042] The method of this invention can be applied to paper-based sensors to achieve convenient and visual detection of cTnI.

[0043] This invention prepares polymer microsphere precursors from monomers UPyMA and crosslinking agent MBA through precipitation polymerization and free radical polymerization, followed by hydrothermal treatment at 100-160℃ to obtain room-temperature phosphorescent polymer microspheres with tunable RTP emission. Precise control of phosphorescence performance is achieved by systematically adjusting the degree of crosslinking, microsphere size, heteroatom density, and polymer chain flexibility. The optimized microspheres achieve an RTP lifetime of 324.79 ms and a total yield of 31.4%. These microspheres can be used as energy donors to achieve multicolor afterglow emission (energy transfer efficiency up to 84.49%) through a PRET strategy, applicable to time-resolved anti-counterfeiting. Furthermore, a "turn-on" RTP immunoassay method for cardiac troponin I (cTnI) is constructed through antibody-induced PET quenching and antigen-triggered phosphorescence recovery mechanisms, with a detection limit of 0.22 ng / mL, exhibiting good selectivity and reproducibility, and showing promising application prospects in the field of point-of-care diagnostics.

[0044] Unless otherwise specified, the room temperature in this invention is 25±2℃.

[0045] All raw materials used in the embodiments of this invention were purchased commercially. For example, the anti-cTnI antibody was purchased from Qitai Biotechnology Co., Ltd., model number MXJ30; the cTnI antigen was purchased from Qitai Biotechnology Co., Ltd., model number XJ03.

[0046] It should be noted that any aspects not described in detail in this invention are conventional practices in the field and are not the focus of this invention.

[0047] The technical solution of the present invention will be further illustrated by the following embodiments.

[0048] Example 1 A method for constructing room-temperature phosphorescent polymer microspheres using cross-linking-assisted cluster engineering, comprising the following steps: (1) Preparation of PUM polymer microsphere precursor The monomer UPyMA (0.5 mmol) and crosslinking agent MBA (2.5 mmol) were dissolved in 50 mL of a mixed solvent of ethanol and water (volume ratio 1:1). After sonication, the initiator AIBN (0.05 mmol) was added. The resulting mixed solution was transferred to a three-necked flask and stirred at 70 °C for 16 hours under nitrogen protection. After the reaction was completed, the product was collected by centrifugation, washed three times with ethanol, and dried under vacuum to obtain the PUM polymer microsphere precursor.

[0049] (2) Preparation of microspheres with RTP properties by hydrothermal treatment The PUM polymer microsphere precursor (50 mg) obtained in step (1) was dispersed in 10 mL of deionized water, transferred to a high-pressure reactor, and subjected to hydrothermal treatment at 100 °C for 3 hours. After natural cooling to room temperature, the product was collected by centrifugation, washed three times with ethanol, and dried under vacuum to obtain room temperature phosphorescent polymer microspheres (which are room temperature phosphorescent polymer microspheres with tunable RTP emission), denoted as PUM-1 microspheres.

[0050] Example 2 A method for constructing room-temperature phosphorescent polymer microspheres by cross-linking assisted cluster engineering is the same as in Example 1, except that the hydrothermal treatment temperature is 140°C, and the resulting room-temperature phosphorescent polymer microspheres are denoted as PUM-2 microspheres.

[0051] Example 3 A method for constructing room-temperature phosphorescent polymer microspheres by cross-linking assisted cluster engineering is the same as in Example 1, except that the hydrothermal treatment temperature is 160°C, and the resulting room-temperature phosphorescent polymer microspheres are denoted as PUM-3 microspheres.

[0052] Figure 2This diagram illustrates the preparation process of the room-temperature phosphorescent polymer microspheres of this invention and shows scanning electron microscope (SEM) images of the microspheres under different hydrothermal treatment conditions. a) is a schematic diagram of the preparation process of the room-temperature phosphorescent polymer microspheres; b) is an SEM image of the PUM polymer microsphere precursor; c) is an SEM image of PUM-1 microspheres; d) is an SEM image of PUM-2 microspheres; and e) is an SEM image of PUM-3 microspheres. The particle size of PUM-1 microspheres is 334.25 nm, that of PUM-2 microspheres is 281.57 nm, and that of PUM-3 microspheres is 246.47 nm. As the hydrothermal treatment temperature increases, the microsphere particle size gradually decreases, indicating that the hydrothermal environment drives the polymer chains to form a more thermodynamically stable close-packed conformation and promotes further cross-linking.

[0053] The three room-temperature phosphorescent polymer microspheres prepared in Examples 1 to 3 all lack large conjugated structures, which reduces chemical toxicity and increases their biocompatibility.

[0054] Accompanying these structural changes, the phosphorescence properties of the microspheres evolved significantly. The untreated PUM microspheres exhibited a phosphorescence lifetime of only 5.51 ms and a total quantum yield of 6.2%. After hydrothermal treatment at 100 °C, the phosphorescence lifetime of PUM-1 microspheres increased to 183.45 ms, and the quantum yield increased to 10.8%. When the hydrothermal temperature was increased to 140 °C, the phosphorescence lifetime and total quantum yield of PUM-2 microspheres reached 270.67 ms and 14.1%, respectively. Further increasing the temperature to 160 °C, the phosphorescence lifetime and total quantum yield of PUM-3 microspheres reached 137.87 ms and 9.6%, respectively. PUM-2 microspheres exhibited the best phosphorescence performance. Figure 3 ).

[0055] Example 4 A method for constructing room temperature phosphorescent polymer microspheres by crosslinking-assisted cluster engineering is the same as in Example 2, except that the amount of crosslinking agent MBA is increased to 5.0 mmol. The room temperature phosphorescent polymer microspheres finally obtained in this example are highly crosslinked room temperature phosphorescent polymer microspheres.

[0056] The room-temperature phosphorescent polymer microspheres obtained in this embodiment have a phosphorescence lifetime of 324.79 ms and a total yield of 31.4%, which is significantly better than that of PUM-2 microspheres.

[0057] Comparative Example 1 Monomers UPyMA (0.5 mmol) and acrylamide (AM, 2.5 mmol) were dissolved in 50 mL of a mixture of ethanol and water (volume ratio 1:1). After sonication, initiator AIBN (0.05 mmol) was added. The resulting mixture was transferred to a three-necked flask and reacted at 70 °C for 16 hours under nitrogen protection. After the reaction was completed, the product was collected by centrifugation, washed three times with ethanol, and vacuum dried to obtain the PUA polymer microsphere precursor (preparation process as follows). Figure 4 (as shown in d) The PUA polymer microsphere precursor (50 mg) was dispersed in 10 mL of deionized water, transferred to a high-pressure reactor, and subjected to hydrothermal treatment at 140 °C for 3 hours. After natural cooling to room temperature, the product was collected by centrifugation, washed three times with ethanol, and dried under vacuum to obtain linear room temperature phosphorescent polymer microspheres, denoted as PUA-2.

[0058] The linear room-temperature phosphorescent polymer microspheres prepared in this comparative example had a phosphorescence lifetime of only 50.38 ms and a total yield of only 1.6%, which is far lower than that of the PUM-2 microspheres prepared in Example 2 and the highly cross-linked room-temperature phosphorescent polymer microspheres prepared in Example 4 (all room-temperature phosphorescent polymer microspheres prepared in Examples 1-4 were cross-linked microspheres). These results confirm that the cross-linked network within the polymer microspheres protects triplet excitons and suppresses oxygen quenching, thereby promoting RTP emission.

[0059] Comparative Example 2 Same as in Example 2, keeping the same feed ratio, but replacing the AIBN initiator with azobisisobutyramidine hydrochloride (AIBA) initiator to prepare microspheres with a particle size of 125 nm, with phosphorescence lifetime and total yield of 44.33 ms and 14.9%, respectively.

[0060] Comparative Example 3 Same as in Example 2, keeping the same feed ratio, but replacing the monomer UPyMA with the monomer isocyanate methacrylate (IEM) which has a lower electron-rich density, the resulting microspheres had a phosphorescence lifetime of 37.47 ms and a total yield of 3.9%.

[0061] Comparative Example 4 Same as in Example 2, keeping the same feed ratio, but replacing the flexible crosslinking agent MBA with the rigid monomer N,N'-p-styrene diacetamide (PBA), the resulting microspheres had a phosphorescence lifetime of 180.94 ms and a total yield of 1.2%.

[0062] The results of Comparative Examples 2 to 4 confirmed that factors such as microsphere size, electron-rich density, and precursor flexibility significantly affect the cluster luminescence performance of microspheres.

[0063] Test Example 1: PRET Multicolor Permeability Control Five mg of highly cross-linked room-temperature phosphorescent polymer microspheres from Example 4 were dispersed in 10 mL of deionized water. Rhodamine B (RhB) was added at different weight ratios (0.05%–2.5%), and the mixture was stirred at room temperature for 2 hours to allow the fluorescent dye to be adsorbed onto the surface of the microspheres via electrostatic adsorption. Unadsorbed dye was removed by centrifugation and washing to obtain composite microspheres.

[0064] Figure 4 In Example 4, a represents the fluorescence spectrum of the highly cross-linked room-temperature phosphorescent polymer microspheres under different excitation wavelengths; b represents the phosphorus spectrum of the highly cross-linked room-temperature phosphorescent polymer microspheres under different excitation wavelengths; c represents the phosphorescence lifetime of the highly cross-linked room-temperature phosphorescent polymer microspheres under 400 nm excitation; d represents a schematic diagram of the preparation process of the linear polymer microsphere precursor PUA in Comparative Example 1; e represents the fluorescence spectrum of the linear polymer microsphere PUA-2 in Comparative Example 1 under different excitation wavelengths; f represents the phosphorus spectrum of the linear polymer microsphere PUA-2 in Comparative Example 1 under different excitation wavelengths; and g represents the phosphorescence lifetime of the linear polymer microsphere PUA-2 in Comparative Example 1 under 400 nm excitation.

[0065] The absorption of RhB showed good spectral overlap with the RTP emission of the highly cross-linked room-temperature phosphorescent polymer microspheres in Example 4, and electrostatic adsorption brought the donor and acceptor close together, satisfying the necessary conditions for PRET. As the RhB doping weight ratio increased from 0.05% to 2.5%, the fluorescence intensity of the energy acceptor increased while the fluorescence intensity of the energy donor decreased. Figure 5 (b) The optimal RhB doping weight ratio is 1.50%. Figure 5 (c) The RTP lifetime of the highly crosslinked room temperature phosphorescent polymer microspheres decreased from 324.79 ms (highly crosslinked room temperature phosphorescent polymer microspheres in Example 4) to 50.37 ms ( Figure 5 (d), while the receptor exhibited a longer lifetime of 122.76 ms (d). Figure 5 (e). Energy transfer efficiency is calculated by measuring the donor lifetime or quantum yield in the presence and absence of the acceptor, reaching a maximum of 84.49% ( Figure 5 (f)

[0066] Test Example 2: cTnI Detection Application 5 mg of the highly cross-linked room-temperature phosphorescent polymer microspheres (1 mg / mL) from Example 4 were dispersed in 5 mL of PBS buffer (pH 7.4), and different concentrations of anti-cTnI antibody (0.05–3 μg / mL) were added. The mixture was incubated at room temperature for 1 hour. Unbound antibody was removed by centrifugation and washing to obtain the anti-cTnI complex loaded with room-temperature phosphorescent polymer microspheres from Example 4. Zeta potential ( Figure 6Tests showed that the antibody was successfully immobilized on the surface of the microspheres.

[0067] Different concentrations of cTnI antigen (0–80 ng / mL) were added to the anti-cTnI-loaded complex of the room-temperature phosphorescent polymer microspheres prepared in Example 4, and the mixture was incubated at room temperature for 30 minutes. As the cTnI concentration increased, the phosphorescence intensity gradually recovered, and the lifetime also gradually returned to its original value. Figure 7 (a~c). A fitting curve was constructed using phosphorescence intensity (I-I0) / I0 versus cTnI concentration (0~80 ng / mL), where I is the emission intensity (au) at a certain cTnI concentration, I0 is the initial intensity (au), and the correlation coefficient R is... 2 The value was 0.986, and the limit of detection was 0.22 ng / mL. Figure 7 (e). Ten repeated measurements of 1.0 ng / mL cTnI yielded a relative standard deviation (RSD) of 0.033, indicating that the method of this invention has good reproducibility. Figure 7 (d).

[0068] The anti-cTnI complex loaded with phosphorescent microspheres from Example 4 was supplemented with 80 ng / mL of common serum interfering agents (C-reactive protein, serum albumin, myoglobin, creatine kinase) for specificity testing. Figure 7 (f) indicates that common serum interfering agents (C-reactive protein, serum albumin, myoglobin, creatine kinase) do not show significant phosphorescence enhancement, and the method has good selectivity for cTnI. Further, the method was applied to a paper-based sensor (Phos-LFTA). A nitrocellulose membrane was attached to a PVC board and cut into strips of 0.5*3 cm. The phosphorescent microspheres loaded with the anti-cTnI complex from Example 4 were uniformly coated onto the membrane, with the coating area being a 0.5*0.5 cm square region near the bottom. The membrane was then dried in a vacuum oven at 37 ℃. Subsequently, different concentrations (0~80 ng / mL) of cTnI were dropped onto the sample area on the membrane. After standing for 15 min, convenient and visual detection of cTnI was achieved. Figure 7 (g and h).

[0069] The above are merely preferred embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for constructing room-temperature phosphorescent polymer microspheres using cross-linked auxiliary cluster engineering, characterized in that, Includes the following steps: (1) The monomer 2-(3-(6-methyl-4-oxo-1,4-dihydropyrimidin-2-yl)ureido) ethyl methacrylate and the crosslinking agent N,N'-methylenebis(acrylamide) were dissolved in the reaction solvent, and an initiator was added. The polymer microsphere precursor was prepared by precipitation polymerization and free radical polymerization. (2) The polymer microsphere precursor obtained in step (1) is dispersed in a solvent and subjected to hydrothermal treatment to obtain the room temperature phosphorescent polymer microsphere.

2. The method for constructing room-temperature phosphorescent polymer microspheres by cross-linking-assisted cluster engineering according to claim 1, characterized in that, In step (1), the molar ratio of the monomer 2-(3-(6-methyl-4-oxo-1,4-dihydropyrimidin-2-yl)ureido) ethyl methacrylate and the crosslinking agent N,N'-methylenebis(acrylamide) is 1:(5~10).

3. The method for constructing room-temperature phosphorescent polymer microspheres using cross-linked assisted cluster engineering according to claim 1, characterized in that, In step (1), the molar ratio of the monomer 2-(3-(6-methyl-4-oxo-1,4-dihydropyrimidin-2-yl)ureido) ethyl methacrylate and the initiator is 1:0.

1.

4. The method for constructing room-temperature phosphorescent polymer microspheres by cross-linking-assisted cluster engineering according to claim 3, characterized in that, The initiator is azobisisobutyronitrile.

5. The method for constructing room-temperature phosphorescent polymer microspheres by cross-linking-assisted cluster engineering according to claim 1, characterized in that, In step (1), the reaction solvent is a mixed solution of ethanol and water, the reaction temperature is 60~75℃, and the reaction time is 12~24 hours.

6. The method for constructing room-temperature phosphorescent polymer microspheres by cross-linking-assisted cluster engineering according to claim 1, characterized in that, In step (2), the temperature of the hydrothermal treatment is 100~160℃ and the time is 3 hours.

7. The method for constructing room-temperature phosphorescent polymer microspheres by cross-linking-assisted cluster engineering according to claim 1, characterized in that, In step (2), the solvent is water.

8. A room-temperature phosphorescent polymer microsphere, characterized in that, It is prepared according to any one of claims 1 to 7.

9. The application of the room-temperature phosphorescent polymer microspheres as described in claim 8 in time-resolved anti-counterfeiting.

10. The application of the room-temperature phosphorescent polymer microspheres as described in claim 8 in the preparation of a biosensor for detecting cardiac troponin I.

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