Multiple stimuli-responsive upconversion luminescence nanocrystal magnetic microspheres and preparation method thereof

Abstract

This invention discloses a multi-stimulus-responsive upconversion luminescent nanocrystalline magnetic microsphere and its preparation method, belonging to the field of biomedical polymer materials and nanobiotechnology. The microsphere's main body is a porous copolymer matrix, encapsulating a core of magnetite (Fe3O4) magnetic nanocrystals. Rare-earth-doped fluoride upconversion luminescent nanocrystals are coupled to the surface and pores. The copolymer side chains of the microsphere incorporate azide groups, acidic stimulus-responsive groups, and photostimulation-responsive groups, constructing an independent and non-interfering triple orthogonal logic gating system on the surface. The preparation method includes preparing an oil phase containing copolymers, magnetic nanoparticles, and a volatile porogen; pre-emulsifying and homogenizing the oil phase in an acidic aqueous phase using an SPG membrane; inducing phase separation by controlled solvent evaporation to form porous microspheres; and finally coupling the upconversion nanocrystals via a click chemistry reaction. The magnetic microspheres of this invention eliminate background fluorescence interference from complex samples from a physical perspective. Their porous structure endows the microspheres with excellent quasi-levitation properties and magnetic responsiveness, effectively solving problems such as single surface function and signal crosstalk of microspheres. They are suitable for in vitro clinical diagnosis and complex analysis of multiple targets.

Description

Technical Field

[0001] This invention belongs to the field of biomedical polymer materials and nanobiotechnology, specifically relating to a smart magnetic microsphere based on a rigid copolymer skeleton, possessing multiple orthogonal logic gating functions and upconversion luminescence encoding characteristics, and its preparation method. Background Technology

[0002] Polymer microspheres, as important functional micro / nanomaterials, typically range in size from nanometers to micrometers. With their enormous specific surface area, excellent biocompatibility, abundant and tunable surface functional groups, and good physicochemical stability, polymer microspheres play an irreplaceable role in many cutting-edge fields such as biomedicine, clinical diagnostics, environmental monitoring, and industrial catalysis. Particularly in biotechnological applications such as in vitro diagnostics (IVD), immunoassay, nucleic acid extraction, cell sorting, and targeted drug delivery, polymer microspheres are often used as high-performance solid-phase carriers. By modifying their surface with specific biological ligands, they can efficiently achieve the specific capture, enrichment, and separation of target molecules, making them a core consumable in modern precision bioassay technologies.

[0003] Currently available commercial microspheres or conventionally reported polymer microspheres typically exhibit a "single-channel" surface chemical modification characteristic, meaning they are modified with only a single type of functional group (such as carboxyl, amino, epoxy, or streptavidin). While this single surface functional design satisfies basic "bind-elution" operations, it falls short when facing the increasingly complex detection demands of modern biomedicine. Especially in high-end applications involving multi-step cascade reactions, multi-target synergistic capture, or multiplexed detection, microspheres are often required to possess "time-sharing," "step-sharing," or "condition-triggered" intelligent response characteristics. Existing technologies lack orthogonal logic gating mechanisms capable of independently identifying and processing different biochemical signals, leading to signal crosstalk or non-specific binding between different functional groups, making it impossible to construct precise molecular logic circuits to execute complex biological instructions.

[0004] Furthermore, to endow microspheres with coding and recognition functions, existing technologies mostly employ organic fluorescent dyes or quantum dots for labeling. However, these materials suffer from poor lightfastness and are prone to photobleaching, and are primarily excited by ultraviolet or visible light. Complex biological samples (such as serum and whole blood) often exhibit strong autofluorescence in these wavelengths, which generates significant background noise and drastically reduces the signal-to-noise ratio of detection. Although upconversion nanoparticles possess the unique advantages of near-infrared excitation and the absence of background interference, how to efficiently and stably integrate them onto the surface of polymer microspheres while overcoming the fluorescence quenching problem during the dispersion and modification process remains a technical bottleneck restricting their application. Therefore, there is an urgent need to develop a smart magnetic microsphere that combines multiple orthogonal chemical logic response functions, background-free optical coding capabilities, and excellent quasi-levitation properties. Summary of the Invention

[0005] To overcome the shortcomings and deficiencies of the existing technologies, this invention provides a smart magnetic microsphere with multiple stimulus responses and upconversion nanocrystal luminescence, as well as its preparation method. By constructing a copolymer matrix with triple orthogonal logic gating, combined with core-shell structured upconversion nanocrystals and a porous process, the intelligent function, background-free encoding, and quasi-suspended physical properties of the microspheres are achieved.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: a multi-stimulus-responsive upconversion luminescent nanocrystalline magnetic microsphere, characterized in that the microsphere is a porous polymer microsphere with an internal magnetic nanocrystalline core and an upconversion luminescent nanocrystalline surface layer coupled to its surface and pores; the microsphere contains the following structural and functional components:

[0007] (a) A logic-gated functional copolymer matrix having a general structural formula as shown in formula (I):

[0008]

[0009] Formula (I): Styrene-N-phenylmaleimide-functionalized methacrylamide copolymer

[0010] Where R1 represents: Where 150≤X≤200, 150≤Y≤200, and 120≤Z≤160.

[0011] (b) An acidic stimulus-responsive group R2, having the general structural formula shown in formula (II):

[0012]

[0013] Formula (II): TIPS protected acetylene group.

[0014] (c) The photostimulation-responsive group R3 has the general structural formula shown in formula (III):

[0015]

[0016] Formula (III): thiol side chain protected by o-nitrobenzyl.

[0017] Where 3≤n≤6, and n is a positive integer.

[0018] (d) Flexible chain spacer arm R4, having a general structural formula as shown in equation (IV):

[0019]

[0020] Formula (IV): Carboxymethyl-terminated polyethylene glycol side chain.

[0021] Where 24≤n≤45, and n is a positive integer.

[0022] (e) The magnetic nanocrystalline core is iron(III) oxide.

[0023] (f) The surface layer of the upconversion luminescent nanocrystal is a rare earth ion-doped fluoride upconversion nanocrystal.

[0024] As a further technical solution of the present invention, the microspheres have the following composition by mass percentage: logic-gated functional copolymer matrix: 10% - 25%; acidic stimulus-responsive group R2: 0.5% - 2.5%; photostimulation-responsive group R3: 0.5% - 2.5%; flexible chain spacer arms R4: 40% - 70%; magnetic nanocrystalline core: 10% - 15%; upconversion luminescent nanocrystalline surface layer: 3% - 8%.

[0025] As a further technical solution of the present invention, the matrix of the upconversion luminescent nanocrystal is hexagonal sodium yttrium fluoride, and the doping ions are ytterbium sensitizer and thulium activator; and the nanocrystal has a core-shell structure, with the core layer being β-NaYF4:Yb,Tm and the shell layer being pure β-NaYF4.

[0026] As a further technical solution of the present invention, the microspheres have a wet apparent density of 1.02 g / cm³ to 1.10 g / cm³ in an aqueous dispersion system; and are superparamagnetic with a saturation magnetization of 2.5 emu / g to 8.5 emu / g and a remanence of less than 0.1 emu / g, enabling them to be separated under an external magnetic field; in an aqueous dispersion system at pH 7.4, the zeta potential of the microspheres is -20 mV to -45 mV.

[0027] This invention also provides a method for preparing multi-stimulus-responsive upconversion luminescent nanocrystalline magnetic microspheres, comprising the following steps:

[0028] (1) Preparation of oil phase: Styrene-N-phenylmaleimide-functionalized methacrylamide copolymer, hydrophobically modified magnetic iron oxide nanoparticles and volatile pore-forming agent are dissolved and dispersed in an organic solvent that is immiscible with water to form an oil phase dispersion. The dispersion is ultrasonically dispersed for 30 minutes until a uniform black oil phase dispersion without precipitation is formed.

[0029] (2) Pre-emulsification and membrane homogenization: The oil phase dispersion is pre-emulsified in an acidic aqueous phase, and then homogenized by circulating through an SPG membrane under nitrogen pressure to obtain a monodisperse water-in-oil emulsion.

[0030] (3) Curing and pore formation: The obtained emulsion is subjected to solvent evaporation under controlled conditions. The difference in evaporation rate between the organic solvent and the pore-forming agent is used to induce phase separation, forming polymer microspheres with porous structures containing magnetic particles.

[0031] (4) Surface modification: The cured microspheres are dispersed in the reaction medium, and upconversion luminescent nanoparticles with reactive groups complementary to the R1 group are added. They are then coupled to the normally open connection sites on the surface of the microspheres through click chemical reaction.

[0032] As a further technical solution of the present invention, the styrene-N-phenylmaleimide-functionalized methacrylamide copolymer in step (1) has the following synthesis steps:

[0033] (1) Precursor polymerization: Styrene, N-phenylmaleimide and pentafluorophenyl methacrylate are dissolved in toluene, and azobisisobutyronitrile is added. Free radical polymerization is carried out under anaerobic conditions. The polymerization temperature is 60-80℃ and the reaction time is 12-24 hours to prepare a polymer precursor containing active ester side groups.

[0034] (2) Side chain functionalization: The polymer precursor obtained in step (1) is dissolved in a good solvent, and a mixture of amine ligands containing functional groups R1, R2, R3, and R4 is added. The mixture is subjected to an aminolysis reaction in the presence of the alkaline catalyst triethylamine to convert the active ester side group into a functionalized amide side group. Then, a small molecule primary amine is added as a capping agent to eliminate the residual unreacted active ester groups after the functionalized ligand reaction is completed.

[0035] (3) Purification and drying: After the reaction is completed, the copolymer is obtained by precipitation, washing and vacuum drying.

[0036] In step (1), the molar ratio of styrene to N-phenylmaleimide is controlled between 1:1 and 1.2:1 to promote the formation of alternating copolymer structures by utilizing the charge transfer complex mechanism.

[0037] The amine ligand mixture described in step (2) comprises: 3-azidopropylamine, 3-(triisopropylsilyl)prop-2-yn-1-amine, ω-aminoalkyl-S-(o-nitrobenzyl) sulfide, and amino-polyethylene glycol-carboxylic acid, with molar ratios of 5%-10%, 5%-10%, 5%-10%, and 70%-85%, respectively.

[0038] In step (3), the detergent is methanol, and the drying temperature is 60-80℃.

[0039] As a further technical solution of the present invention, the organic solvent in step (1) is dichloromethane, the volatile porogen is n-heptane or n-hexane, and the volume ratio of organic solvent to volatile porogen is 6:4-5:5.

[0040] As a further technical solution of the present invention, the hydrophobically modified magnetic iron oxide nanoparticles in step (1) are oleic acid-modified iron oxide nanoparticles with a particle size of 8-15 nm. The mass ratio of styrene-maleimide functional copolymer to magnetic iron oxide nanoparticles should be 6:1 to 8:1.

[0041] As a further technical solution of the present invention, the acidic aqueous phase in step (2) contains 1.0%-2.0% polyvinyl alcohol with a pH value of 4.0-5.0; the SPG membrane has a pore size of 2-10 μm; and the nitrogen pressure is 15 kPa.

[0042] As a further technical solution of the present invention, the control conditions in step (3) are as follows: first, the organic solvent is evaporated at 20-25°C for 12-16 hours to form a shell, and then the pores are evaporated at 45-60°C for 3-5 hours to form a cavity.

[0043] As a further technical solution of the present invention, the UCNPs in step (4) are modified with dibenzocyclooctylene or terminal alkyne groups, and specifically bind to the R1 site on the surface of the microspheres through steric hindrance screening.

[0044] Compared with the prior art, the beneficial effects of the present invention are:

[0045] 1. This invention constructs a precise triple orthogonal logic gating system on the surface of microspheres, effectively enhancing the signal processing capabilities of microspheres in complex biological environments. By introducing azide groups (R1), TIPS-protected alkynyl groups (R2), and o-nitrobenzyl-protected thiol groups (R3) into the copolymer side chains, three independent chemical channels—"normally open," "acid-responsive," and "photoresponsive"—are constructed, respectively, without interference. This design effectively solves the problem of existing microsphere surfaces having single functions and being difficult to multiplex for detection, avoiding signal crosstalk between different functional groups. This allows for the construction of precise molecular logic circuits, providing an ideal carrier for achieving high-precision cascaded capture / release.

[0046] The orthogonality of the microsphere's logical channels makes it superior in multi-step reactions, facilitating more precise and controlled release in applications such as drug delivery and gene analysis. Click chemistry sites on the microsphere surface can improve modification efficiency and enhance linkage stability. The diversity of functional groups on the microsphere surface helps improve specific binding to different targets, making it suitable for complex clinical diagnostics. Through a dual acid-and-light stimulation response mechanism, in-situ regulation of the microsphere surface properties can be achieved, enhancing the microsphere's adaptability in intelligent detection devices.

[0047] 2. This invention introduces core-shell structured upconversion nanocrystals as optical encoding units, eliminating background interference from a physical perspective. Addressing the challenge of strong autofluorescence in biological samples (such as serum and whole blood) under UV / Vis light, this invention employs near-infrared (980nm) excited rare-earth upconversion luminescent materials. Since the biological matrix exhibits almost no absorption and does not emit light in the near-infrared region, the encoded signal of these microspheres has extremely low background noise, significantly improving the signal-to-noise ratio for trace detection. Furthermore, the "active core / inert shell" structure designed in this invention effectively blocks surface energy quenching, greatly enhancing the luminescence quantum yield.

[0048] 3. The microsphere preparation method of this invention effectively solves the kinetic contradiction between magnetic responsiveness and suspension stability through a phase separation process induced by a volatile porogen. During the preparation process, by controlling the ratio of dichloromethane to n-heptane and the temperature program for stepwise volatilization, the wet apparent density of the microspheres is precisely locked within the "quasi-suspension" range of 1.02-1.10 g / cm³. This density is slightly greater than that of water, ensuring that the microspheres do not float; yet it is light enough to allow them to maintain Brownian motion in solution for a long time without settling, similar to biological cells. This characteristic ensures efficient contact between the microspheres and the sample, as well as uniformity during automated pipetting, while retaining rapid separation capability under an external magnetic field, perfectly meeting the dual requirements of sensitivity and repeatability for high-end clinical diagnostics. Attached Figure Description

[0049] Figure 1Optical microscope image of the magnetic microspheres with porous structure obtained in Example 1.

[0050] Figure 2 Scanning electron microscope image of the magnetic microspheres with porous structure obtained in Example 1.

[0051] Figure 3 Hysteresis loop diagram of the upconversion luminescent nanocrystalline magnetic microspheres obtained in Example 1.

[0052] Figure 4 X-ray diffraction patterns of porous polymer microspheres (uncoupled upconversion nanocrystals) and copolymer precursors internally encapsulated with magnetic particles prepared in step (3) of Example 1.

[0053] Figure 5 The upconversion luminescence shift spectra of NaYF4:Yb,Tm nanocrystals with different sensitizer doping concentrations are shown.

[0054] Figure 6 Dynamic light scattering particle size distribution of the multi-stimulus-responsive upconversion luminescent nanocrystalline magnetic microspheres prepared in Example 1.

[0055] Figure 7 The images shown are combined images of the multi-logic-response magnetic microspheres in Example 9 under different logic triggering states using laser scanning confocal microscopy: (a) is the red fluorescence image of the normally open channel (R1), (b) is the green fluorescence image after the light-response channel (R3) is turned on, and (c) is the combined image after the multiple channels are superimposed.

[0056] Figure 8 These are CLSM images of the microspheres in different logic states in Embodiment 9 of the present invention, where (a) is a bright-field image, and (b)-(d) are fluorescence images in normally-on (R1), phototriggered (R3), and acid-triggered (R2) states, respectively. Detailed Implementation Plan

[0057] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are described clearly and completely. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments. The purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0058] Example 1

[0059] (1) Weigh 4.16 g (0.04 mol) styrene, 6.92 g (0.04 mol) N-phenylmaleimide and 10.08 g (0.04 mol) pentafluorophenyl methacrylate into a round-bottom flask containing 50 mL of anhydrous 1,4-dioxane. Sonicate for 10 min to completely dissolve the monomers, then add 68 mg of azobisisobutyronitrile as an initiator. Purge the flask with high-purity nitrogen for 30 min to replace the oxygen, then seal the flask and place it in a constant-temperature oil bath at 70 °C. React for 24 hours with magnetic stirring. After the reaction, drop the viscous polymer solution into 500 mL of excess methanol to precipitate the product. Filter and collect the white powder, wash under reflux for 24 h using a Soxhlet extractor (methanol solvent), and vacuum dry to obtain the active ester polymer precursor.

[0060] (2) Weigh 1.8 g of the polymer precursor prepared in step (1) and dissolve it in 100 mL of tetrahydrofuran. Prepare a mixed solution of amine ligands: 3-azidopropylamine (R1 precursor, 35 mg), 3-(triisopropylsilyl)prop-2-yn-1-amine (R2 precursor, 75 mg), ω-aminoalkyl-S-(o-nitrobenzyl) sulfide (R3 precursor, 80 mg), and amino-polyethylene glycol-carboxylic acid (R4 precursor, Mw=2000, 5.0 g) dissolved in 20 mL of tetrahydrofuran. Add the amine mixture dropwise to the polymer solution and add 1.0 mL of triethylamine as a catalyst. Stir the reaction at 40 °C for 24 hours. After the reaction is complete, add 0.5 mL of n-butylamine to cap the reaction for 2 hours. Finally, precipitate the reaction solution in diethyl ether and dry it under vacuum to obtain the functionalized copolymer.

[0061] (3) Weigh 600 mg of the logic-gated functional copolymer obtained in step (2) and dissolve it in a mixed solvent of 6 mL dichloromethane and 4 mL n-heptane (volume ratio 6:4) to form a polymer matrix solution. Then add 90 mg of oleic acid-modified iron oxide nanoparticles (average particle size 10 nm) and ultrasonically disperse for 30 min to obtain a uniform black oil phase dispersion. Separately measure 200 mL of deionized water, add 3.0 g of polyvinyl alcohol, heat to dissolve, and adjust the pH to 4.5 to obtain a continuous aqueous phase.

[0062] (4) The above oil phase dispersion was poured into a storage tank and, under nitrogen pressure (15 kPa), was forced through a hydrophilic SPG membrane with a pore size of 5.0 μm into a slowly flowing continuous aqueous phase to form a homogeneous water-in-oil emulsion. The emulsion was placed in a fume hood and mechanically stirred at 300 rpm at 25°C for 14 hours; then the temperature was raised to 55°C and stirring was continued for 4 hours, utilizing the phase separation and volatilization of n-heptane to form a porous structure inside the microspheres.

[0063] (5) The solidified microsphere slurry was centrifuged at 3000 rpm for 5 min to separate the microspheres, and washed three times alternately with ethanol and deionized water. 100 mg of the washed porous microspheres were dispersed in 10 mL of PBS buffer, and 5 mg of core-shell upconversion nanoparticles (β−NaYF4:Yb,Tm@NaYF4) with a surface modified with dibenzocyclooctylene were added. The mixture was incubated at room temperature with shaking for 4 hours to allow the R1 azide group on the surface of the microspheres to react with the DBCO group on the surface of the UCNPs without copper click. After the reaction, the nanoparticles were removed by magnetic separation using a magnet, and then freeze-dried under vacuum to obtain quasi-suspended magnetic microspheres.

[0064] Testing revealed that the microspheres prepared in this embodiment had an average particle size of 2.8 μm (narrow particle size distribution, exhibiting excellent monodispersity), a wet apparent density of 1.05 g / cm³, and showed no sedimentation after standing in water for 8 hours. The microspheres exhibited typical superparamagnetism, with remanence and coercivity approaching zero. Their saturation magnetization was approximately 2.8 emu / g, and the magnetic separation time under an applied magnetic field was 31 seconds; the Zeta potential was -26 mV.

[0065] Example 2

[0066] (1) The preparation method of the active ester polymer precursor in this embodiment is exactly the same as that in Example 1.

[0067] (2) The side chain functionalization operation method in this embodiment is basically the same as that in Example 1. The difference is that the ratio of the amine ligand mixture solution is adjusted so that the molar ratio of 3-azidopropylamine (R1 precursor), 3-(triisopropylsilyl)prop-2-yn-1-amine (R2 precursor), ω-aminoalkyl-S-(o-nitrobenzyl) sulfide (R3 precursor) and amino-polyethylene glycol-carboxylic acid (R4 precursor) is precisely controlled to be 5%:5%:5%:85%.

[0068] (3) The preparation method of the oil phase and the water phase in this embodiment is basically the same as that in Example 1. The difference is that: a mixed solvent of 5 mL dichloromethane and 5 mL n-hexane (volume ratio 5:5) is used to dissolve the copolymer; 100 mg of oleic acid modified iron oxide nanoparticles (average particle size 8 nm) are added; polyvinyl alcohol is added to the continuous acidic aqueous phase to make its mass fraction 2.0%, and the pH value is adjusted to 4.0.

[0069] (4) The operation method of pre-emulsification and solidification pore formation in this embodiment is basically the same as that in Example 1. The difference is that a hydrophilic SPG membrane with a pore size of 2.0 μm is used; the control conditions are adjusted to first evaporate the organic solvent at 20°C for 16 hours to form a shell, and then evaporate the pore-forming agent (n-hexane) at 45°C for 5 hours to form a cavity.

[0070] (5) The operation methods of the washing, surface modification and separation steps in this embodiment are exactly the same as those in Embodiment 1.

[0071] The microspheres prepared in this embodiment have a wet apparent density of 1.10 g / cm³ in an aqueous dispersion system, as tested. Due to the use of the highest proportion of polyethylene glycol flexible chain spacer arms R4, the zeta potential of the microspheres in an aqueous dispersion system at pH 7.4 precisely reaches -45 mV. The microspheres exhibit superparamagnetism with a saturation magnetization of 4.2 emu / g and a remanence of less than 0.1 emu / g, enabling rapid separation under an applied magnetic field.

[0072] Example 3

[0073] (1) The preparation method of the active ester polymer precursor in this embodiment is basically the same as that in Example 1, except that the polymerization reaction temperature is increased to 80°C, the reaction time is shortened to 12 hours, and the product is vacuum dried at 60°C after washing.

[0074] (2) The side chain functionalization operation method in this embodiment is basically the same as that in Example 1. The difference is that the ratio of the amine ligand mixture solution is adjusted so that the molar ratio of 3-azidopropylamine (R1 precursor), 3-(triisopropylsilyl)prop-2-yn-1-amine (R2 precursor), ω-aminoalkyl-S-(o-nitrobenzyl) sulfide (R3 precursor) and amino-polyethylene glycol-carboxylic acid (R4 precursor) is precisely controlled to be 10%:10%:10%:70%.

[0075] (3) The preparation method of the oil phase and the water phase in this embodiment is basically the same as that in Example 1. The difference is that 75 mg of oleic acid-modified iron oxide nanoparticles (average particle size 15 nm, so that the mass ratio of copolymer to magnetic particles reaches 8:1) are added; polyvinyl alcohol is added to the continuous acidic aqueous phase to make its mass fraction 1.0%, and the pH value is adjusted to 5.0.

[0076] (4) The operation method of pre-emulsification and solidification pore formation in this embodiment is basically the same as that in Example 1. The difference is that: under nitrogen pressure, a hydrophilic SPG membrane with a pore size of 10.0 μm is used; the control conditions are adjusted to first evaporate the organic solvent at 25°C for 12 hours to form a shell, and then evaporate the pore-forming agent at 60°C for 3 hours to form a cavity.

[0077] (5) The washing, surface modification and separation steps are exactly the same as in Example 1.

[0078] Testing showed that the copolymer prepared in this embodiment had a number-average molecular weight of 50,000 (lower limit); the average particle size of the microspheres was approximately 4.8 μm, and the wet apparent density was precisely 1.02 g / cm³. In an aqueous dispersion system at pH 7.4, the Zeta potential was -25 mV. The microspheres exhibited superparamagnetism with a saturation magnetization of 8.2 emu / g, and were capable of rapid separation under an applied magnetic field.

[0079] Example 4

[0080] (1) The polymerization reaction conditions were adjusted as follows: the sealed flask was placed in a constant temperature oil bath at 60°C, the reaction time was extended to 24 hours, and after washing, it was vacuum dried at 80°C. The remaining precursor preparation steps were the same as in Example 1.

[0081] (2) In the preparation of the amine ligand mixed solution, the molar ratio of R1, R2, R3 and R4 is precisely controlled to be 5%:5%:10%:80% respectively. The remaining side chain functionalization steps are the same as in Example 1.

[0082] (3) Weigh 600 mg of the obtained copolymer and dissolve it in a mixed solvent of 5 mL dichloromethane and 5 mL n-heptane (volume ratio 5:5). Add 100 mg of oleic acid-modified iron oxide nanoparticles (average particle size 8 nm, mass ratio 6:1). Add 4.0 g of polyvinyl alcohol (mass fraction 2.0%) to the continuous aqueous phase and adjust the pH to 4.0.

[0083] (4) The pore size of the SPG membrane was adjusted to 2.0 μm. The emulsion evaporation control conditions were as follows: first, mechanical stirring was carried out at 20℃ for 16 hours to form a shell layer; then the temperature was raised to 45℃ and stirring was continued for 5 hours to form a cavity.

[0084] (5) The washing and surface finishing steps are exactly the same as in Example 1.

[0085] Tests showed that the copolymer prepared in this embodiment had a number-average molecular weight of 300,000, the average particle size of the microspheres was 1.2 μm, the wet apparent density was 1.10 g / cm³, and the Zeta potential was -42 mV. Due to the use of a very high proportion and small-particle-size magnetic core, its saturation magnetization was 4.0 emu / g and the remanence was less than 0.1 emu / g.

[0086] Example 5

[0087] (1) Adjusting the feed ratio: Weigh 5.0 g (0.048 mol) styrene, 6.92 g (0.04 mol) N-phenylmaleimide (feed molar ratio controlled at 1.2:1) and 10.08 g pentafluorophenyl methacrylate. Place in an oil bath at 70℃ and react for 18 hours. Vacuum dry at 70℃ to obtain the polymer precursor.

[0088] (2) In the preparation of the amine ligand mixed solution, the molar ratio of R1, R2, R3 and R4 is controlled to be 8%:7%:5%:80% respectively. The remaining steps are the same as in Example 1.

[0089] (3) Weigh 600 mg of the obtained copolymer and dissolve it in a mixed solvent of 5.5 mL dichloromethane and 4.5 mL n-heptane. Add 85 mg of oleic acid-modified iron oxide nanoparticles (average particle size 10 nm). The mass fraction of polyvinyl alcohol in the continuous aqueous phase is 1.5%, and the pH is adjusted to 4.5.

[0090] (4) The SPG membrane pore size is selected as 5.0 μm. Volatilization conditions: stir at 22℃ for 14 hours, then raise the temperature to 50℃ and continue stirring for 4 hours.

[0091] (5) The surface modification steps were the same as in Example 1, with 6.5 mg of upconversion nanoparticles added for click reaction.

[0092] The copolymer prepared in this embodiment has a number-average molecular weight of approximately 180,000, an average microsphere size of 2.9 μm, a wet apparent density of 1.06 g / cm³, a zeta potential of -36 mV, and a saturation magnetization of 5.6 emu / g.

[0093] Example 6

[0094] (1) The precursor polymerization steps are exactly the same as in Example 1.

[0095] (2) In the preparation of the amine ligand mixed solution, the molar ratio of R1, R2, R3 and R4 is controlled to be 5%:10%:5%:80% respectively. The remaining steps are the same as in Example 1.

[0096] (3) Replace the volatile porogen: Weigh 600 mg of the copolymer and dissolve it in a mixed solvent of 6 mL dichloromethane and 4 mL n-hexane (instead of n-heptane). Add 90 mg of oleic acid-modified iron oxide nanoparticles (average particle size 12 nm). The continuous aqueous phase polyvinyl alcohol mass fraction is 1.2%, and the pH is adjusted to 4.2.

[0097] (4) The pore size of the SPG membrane was selected as 8.0 μm. Volatilization conditions: Stir at 24℃ for 15 hours, then raise the temperature to 55℃ and continue stirring for 3.5 hours.

[0098] (5) The washing and surface finishing steps are the same as in Example 1.

[0099] Tests showed that, in this embodiment, using a hexane-based pore-forming system, the average particle size of the microspheres was 3.8 μm, the wet apparent density was 1.04 g / cm³, and the porous morphology was uniformly distributed.

[0100] Example 7

[0101] (1) The preparation method of the active ester polymer precursor in this embodiment is exactly the same as that in Example 1.

[0102] (2) In the preparation of the amine ligand mixed solution, the molar ratio of R1, R2, R3 and R4 is controlled to be 10%: 5%: 10%: 75% respectively. The remaining steps are the same as in Example 1.

[0103] (3) Adjusting the oil and aqueous phases: Weigh 600 mg of the copolymer and dissolve it in a mixed solvent of 5.5 mL dichloromethane and 4.5 mL n-heptane. Add 95 mg of oleic acid-modified iron oxide nanoparticles (average particle size 12 nm). The continuous aqueous phase polyvinyl alcohol mass fraction is 1.8%, and the pH is adjusted to 4.3.

[0104] (4) The pore size of the SPG membrane is selected as 7.0 μm. Volatilization conditions: stir at 25℃ for 14 hours, then raise the temperature to 50℃ and continue stirring for 4 hours.

[0105] (5) The washing and surface finishing steps are the same as in Example 1.

[0106] The microspheres prepared in this embodiment have an average particle size of approximately 3.2 μm and a wet apparent density of 1.07 g / cm³. In an aqueous dispersion system at pH 7.4, the Zeta potential is -32 mV. The microspheres exhibit superparamagnetism with a saturation magnetization of 6.5 emu / g, demonstrating both excellent suspension and magnetic responsiveness.

[0107] Example 8

[0108] This embodiment aims to test the high loading capacity of microspheres on the surface of upconversion luminescent nanocrystals.

[0109] (1) The precursor polymerization steps are exactly the same as in Example 1.

[0110] (2) The side chain functionalization steps are exactly the same as in Example 1.

[0111] (3) Adjusting the ratio of matrix to magnetic core: Weigh 600 mg of copolymer and add 100 mg of oleic acid-modified iron oxide nanoparticles. The continuous aqueous phase and pre-emulsification process are the same as in Example 1.

[0112] (4) The curing and pore-forming steps are the same as in Example 1.

[0113] (5) Take 100 mg of the cured and washed porous microspheres and add 8.7 mg of core-shell upconversion nanoparticles with a surface modified with dibenzocyclooctylene (so that the mass fraction in the final microspheres is close to 8%). Extend the shaking incubation at room temperature for 8 hours to ensure sufficient covalent coupling.

[0114] The tests in this embodiment verified the maximum load limit of the upconversion luminescent layer. The luminescence intensity of the microspheres under 980 nm excitation was extremely high, and thanks to the huge specific surface area of ​​the porous framework, the UCNPs were uniformly distributed on the surface and in the pores of the microspheres, without the occurrence of severe particle aggregation that would lead to fluorescence quenching.

[0115] Example 9

[0116] This embodiment is used to verify the triple orthogonal logic gating performance of the microspheres prepared in Example 1.

[0117] (1) Normally Open Channel (R1) Test: Take 1 mg of microsphere dispersion and add 10 μL of red fluorescent probe with DBCO group. After shaking for 30 min, magnetic separation and washing are performed. Strong red fluorescence was observed on the surface of the microspheres under a fluorescence microscope, proving that the R1 click site is in a normally open active state.

[0118] (2) Photoresponse Channel (R3) Test: The microspheres were irradiated with a 365 nm portable UV lamp (3 W) for 10 min to remove the o-nitrobenzyl protection from the R3 group. Then, 10 μL of a green fluorescent probe with a maleimide group was added. After shaking for 30 min, the microspheres were washed. Detection revealed that the microspheres exhibited superimposed green fluorescence, confirming that the photolock was successfully opened (e.g., ...). Figure 7 (As shown).

[0119] (3) Acid-responsive channel (R2) test: Take the microspheres from step (2) and treat them with acetate buffer at pH 4.0 for 15 min to remove the TIPS silica protection from the R2 groups. Then add 10 μL of a blue fluorescent probe with an azide group and a trace amount of copper catalyst. After washing, the surface of the microspheres exhibits superimposed red, green, and blue fluorescence (e.g., Figure 8 (As shown). Experiments have confirmed that the three logic channels work independently without interfering with each other.

[0120] Example 10

[0121] This embodiment aims to test the photolysis efficiency of the photostimulation responsive group (R3) at the longest carbon chain limit n=6.

[0122] (1) The precursor polymerization steps are exactly the same as in Example 1.

[0123] (2) Replacement of the photoresponsive precursor: In the amine ligand mixture solution, 6-aminohexyl-S-(o-nitrobenzyl) sulfide was used to replace the short-chain precursor in the original example. The molar ratio of each ligand was the same as in Example 1.

[0124] (3) The preparation of the oil phase and water phase is the same as in Example 1.

[0125] (4) The curing and pore-forming steps are the same as in Example 1.

[0126] (5) The washing and surface finishing steps are the same as in Example 1.

[0127] Testing in this embodiment verified the upper limit of the alkyl chain length of the photoresponsive group in the claims. Logic gate triggering experiments showed that the use of a longer carbon chain as a spacer arm significantly reduced the steric hindrance near the ortho-nitrobenzyl group. Under the same intensity of ultraviolet light irradiation, the on-time of its photoresponsive channel (R3) was increased by approximately 15% compared to Example 1, further enhancing the response sensitivity of the microsphere in rapid diagnostics.

[0128] Example 11

[0129] This embodiment aims to test the suspension and antifouling performance of the flexible chain spacer arm (R4) at a very low degree of polymerization ($n=24$).

[0130] (1) The precursor polymerization steps are exactly the same as in Example 1.

[0131] (2) Replacement of flexible chain precursor: In the amine ligand mixture solution, the molecular weight of amino-polyethylene glycol-carboxylic acid was replaced from Mw=2000 to Mw=1000 (i.e., n=24 in general formula IV). The molar ratio of each ligand was the same as in Example 1.

[0132] (3) The preparation of the oil phase and water phase is the same as in Example 1.

[0133] (4) The curing and pore-forming steps are the same as in Example 1.

[0134] (5) The washing and surface finishing steps are the same as in Example 1.

[0135] Testing in this embodiment verified the lower limit of the flexible chain length in the claims. The average particle size of the microspheres was 2.7 μm, and the Zeta potential remained at -20 mV under the modification of extremely short PEG chains. The short chains still provided sufficient steric hindrance, and the microspheres showed no significant sedimentation after standing in pure water for 6 hours. Furthermore, they exhibited excellent resistance to nonspecific adsorption when biological serum samples containing contaminating proteins were subsequently added.

[0136] Comparative Example 1

[0137] This comparative example aims to illustrate the effect of volatile porogens on microsphere density. The only difference from Example 1 is that, in step (3) of oil phase preparation, n-heptane is not added; instead, 10 mL of pure dichloromethane is used to dissolve the polymer and magnetic particles. The remaining film emulsification, curing, and modification steps are exactly the same as in Example 1.

[0138] The prepared comparative microspheres were tested and found to have an average particle size of approximately 2.1 μm, but a high wet apparent density of 1.35 g / cm³. When these microspheres were dispersed in water, significant gravity settling occurred within 3 minutes after stirring was stopped, causing them to accumulate at the bottom of the container. This indicates that the lack of a pore-forming agent resulted in a solid structure for the microspheres, leading to increased volume shrinkage and excessively high density, thus failing to achieve the "quasi-suspended" characteristic required by this invention.

[0139] Comparative Example 2

[0140] This comparative example aims to illustrate the necessity of orthogonal protecting groups for constructing logic-gated systems. The only difference from Example 1 is that, during the side-chain functionalization process in step (2), the composition of the amine ligand mixture was changed, replacing the ligand with a stimulus-responsive function.

[0141] 1. Use common propyneamine instead of the R2 precursor (3-(triisopropylsilyl)prop-2-yn-1-amine);

[0142] 2. Use ordinary cysteine ​​instead of the R3 precursor (ω-aminoalkyl-S-(o-nitrobenzyl) sulfide); the remaining steps (copolymerization, pore formation, assembly) are consistent with those in Example 1.

[0143] The microspheres prepared in the comparative example were subjected to logic function testing according to the method in Example 2, and the results are as follows:

[0144] 1. Photoresponse Channel Test: Maleimide fluorescent probes were added directly without UV light stimulation. Because the thiol groups on the microsphere surface lack the "photolock" protection of o-nitrobenzyl groups, the probe immediately reacts with the exposed thiol groups, causing the microspheres to display green fluorescence even without a trigger signal. This represents a serious logical error (false positive), proving that the microspheres have lost their ability to control the "conditional triggering" of light signals.

[0145] 2. Acid response channel test: Similarly, when azide probe and copper catalyst are added under neutral pH conditions, the click reaction occurs rapidly due to the lack of "acid lock" protection of TIPS on the alkyne group, and the microspheres show blue fluorescence even without receiving an acidic signal.

[0146] 3. Stability test: In addition, since the thiol groups were not protected, during the storage of the microspheres, a significant non-specific aggregation phenomenon was observed between the microspheres due to the formation of disulfide bonds by the oxidation of thiol groups, resulting in a significant decrease in dispersibility.

[0147] The preferred embodiments of the present invention disclosed above are merely illustrative of the invention. These preferred embodiments do not exhaustively describe all details, nor do they limit the invention to the specific implementations described. Obviously, although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that many modifications and variations can be made without departing from the principles and spirit of the invention. This specification selects and specifically describes these embodiments to better explain the principles and practical applications of the invention, thereby enabling those skilled in the art to better understand and utilize the invention. The invention is limited only by the claims and their full scope and equivalents.

Claims

1. A multi-stimulus-responsive upconversion luminescent nanocrystalline magnetic microsphere, characterized in that, The microspheres are porous polymer microspheres with an internal magnetic nanocrystal core and an upconversion luminescent nanocrystal surface layer coupled to the surface and pores; the microspheres contain the following structural and functional components: (a) A logic-gated functional copolymer matrix having the following general structural formula: Where R1 represents: Where 150≤X≤200, 150≤Y≤200, and 120≤Z≤160. R1 can be replaced by R2, R3, and R4. (b) The acidic stimulus-responsive group R2 has the following general structural formula: (c) The photostimulation-responsive group R3 has the following general structural formula: Where 3≤n≤6, and n is a positive integer. (d) Flexible chain spacer arm R4, having the following general structural formula: Where 24≤n≤45, and n is a positive integer. (e) The magnetic nanocrystalline core is iron(III) oxide. (f) The surface layer of the upconversion luminescent nanocrystal is a rare earth ion-doped fluoride upconversion nanocrystal.

2. The multi-stimulus-responsive upconversion luminescent nanocrystalline magnetic microsphere as described in claim 1, characterized in that, The microspheres have the following composition by mass percentage: Logic-gated functional copolymer matrix: 10% - 25% Acidic stimulus-responsive group R2: 0.5% - 2.5% Photostimulation responsive group R3: 0.5% - 2.5% Flexible chain spacer arm R4: 40% - 70% Magnetic nanocrystalline core: 10% - 15% Upconversion luminescent nanocrystalline surface layer: 3% - 8%.

3. The multi-stimulus-responsive upconversion luminescent nanocrystalline magnetic microspheres according to claim 1, characterized in that, The number average molecular weight of the copolymer is from 50,000 to 300,000.

4. The multi-stimulus-responsive upconversion luminescent nanocrystalline magnetic microspheres according to claim 1, characterized in that, The magnetic nanocrystal core is oleic acid-modified iron oxide nanocrystals with a particle size of 8-15 nm.

5. The multi-stimulus-responsive upconversion luminescent nanocrystalline magnetic microspheres according to claim 1, characterized in that, The matrix of the upconversion luminescent nanocrystal is hexagonal sodium yttrium fluoride, and the doping ions are ytterbium sensitizer and thulium activator; and the nanocrystal has a core-shell structure, with a core layer of β-NaYF4:Yb,Tm and a shell layer of pure β-NaYF4.

6. The multi-stimulus-responsive upconversion luminescent nanocrystalline magnetic microspheres according to claim 1, characterized in that, The glass transition temperature is above 200℃.

7. The multi-stimulus-responsive upconversion luminescent nanocrystalline magnetic microspheres according to claim 1, characterized in that, The wet apparent density of the microspheres in the aqueous dispersion system ranged from 1.02 g / cm³ to 1.10 g / cm³.

8. The multi-stimulus-responsive upconversion luminescent nanocrystalline magnetic microspheres according to claim 1, characterized in that, The microspheres are superparamagnetic, with a saturation magnetization of 2.5 emu / g to 8.5 emu / g and a remanence of less than 0.1 emu / g, enabling them to separate under an applied magnetic field.

9. The multi-stimulus-responsive upconversion luminescent nanocrystalline magnetic microspheres according to claim 1, characterized in that, In an aqueous dispersion system at pH 7.4, the zeta potential of the microspheres ranges from -20 mV to -45 mV.

10. A method for preparing multi-stimulus-responsive upconversion luminescent nanocrystalline magnetic microspheres according to claim 1, characterized in that... Includes the following steps: (1) Preparation of oil phase: Styrene-N-phenylmaleimide-functionalized methacrylamide copolymer, hydrophobically modified magnetic iron oxide nanoparticles and volatile pore-forming agent are dissolved and dispersed in an organic solvent that is immiscible with water to form an oil phase dispersion; (2) Pre-emulsification and membrane homogenization: The oil phase dispersion is pre-emulsified in an acidic aqueous phase, and then homogenized by circulating through an SPG membrane under nitrogen pressure to obtain a monodisperse water-in-oil emulsion. (3) Curing and pore formation: The obtained emulsion is subjected to solvent evaporation under controlled conditions. The difference in evaporation rate between the organic solvent and the pore-forming agent is used to induce phase separation, forming polymer microspheres with porous structures containing magnetic particles. (4) Surface modification: The cured microspheres are dispersed in the reaction medium, and upconversion luminescent nanoparticles with reactive groups complementary to the R1 group are added. The nanoparticles are covalently coupled to the R1 group on the surface of the microspheres through click chemical reaction.

11. The preparation method according to claim 10, characterized in that, The organic solvent mentioned in step (1) is dichloromethane, and the volatile porogen is n-heptane or n-hexane. The volume ratio of the organic solvent to the volatile porogen is 6:4-5:

5.

12. The preparation method according to claim 10, characterized in that, The acidic aqueous phase in step (2) contains 1.0%-2.0% polyvinyl alcohol and has a pH value of 4.0-5.0; the SPG membrane has a pore size of 2-10 μm; and the nitrogen pressure is 15 kPa.

13. The preparation method according to claim 10, characterized in that, The control conditions described in step (3) are as follows: first, the organic solvent is volatilized at 20-25°C to form a shell, and then the pore-forming agent is volatilized at 45-60°C to form a cavity.

14. The preparation method according to claim 10, characterized in that: In step (4), the UCNPs are modified with dibenzocyclooctylene or terminal alkyne groups, which are specifically bound to the R1 site on the surface of the microspheres by steric hindrance screening.

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