Nanosphere time-resolved fluorescent probe as well as preparation method and application thereof

Abstract

The invention discloses a nano-microsphere time-resolved fluorescent probe as well as a preparation method and application thereof. The preparation method comprises the following three steps: preparation of core-layer nano microspheres, shell-layer functional modification and fluorescence enhancement and stabilization treatment. A polylactic acid-glycolic acid copolymer (PLGA) is used as a core-layer carrier to load a rare earth ion complex, and a SiO2 coating layer and a targeting ligand are combined for modification, so that the nano-microsphere fluorescent probe with a core-shell structure is prepared. The probe has the characteristics of long fluorescence lifetime (5-8ms), high quantum yield (greater than or equal to 35%), strong stability, excellent targeting property and the like, and can be widely applied to food safety detection (such as quinolone antibiotics and microcystin MC-LR) and environmental pollution detection (such as heavy metal ions such as Hg < 2 + > and Pb < 2 + >), and the detection limit is as low as 0.05 ng / mL or 0.1 nmol / L. The problems that a traditional fluorescent probe is low in loading capacity, poor in stability and insufficient in targeting property are solved, and the method has remarkable industrial application value.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probe technology, specifically relating to a time-resolved fluorescent probe made of nanospheres, its preparation method, and its application. Background Technology

[0002] Time-resolved fluorescent probes, with their unique time-resolved technology, can effectively eliminate background fluorescence interference and have broad application prospects in fields such as food safety testing and environmental monitoring. Traditional time-resolved fluorescent probes mostly use single rare-earth ion complexes as fluorophores, but they suffer from problems such as low loading, poor fluorescence stability, and insufficient targeting.

[0003] In existing technologies, the preparation methods for nanospheres loaded with fluorescent substances mostly employ physical adsorption, leading to easy leakage of the fluorescent substance and a lack of effective stabilization modifications. Furthermore, the probes tend to aggregate in complex systems, resulting in rapid fluorescence lifetime decay. Simultaneously, the functionalization modifications of existing probes are mostly single-target group grafting, with specificity and binding efficiency needing improvement, making it difficult to meet the demands for high-sensitivity and high-specificity detection. Therefore, developing a method for preparing time-resolved fluorescent probes from nanospheres with high loading capacity, strong stability, and excellent targeting has significant practical application value. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides a time-resolved fluorescent probe made of nanospheres, its preparation method, and its application. The nanosphere time-resolved fluorescent probe adopts a core-shell structure design and is prepared through a three-step method of "core layer fluorescence loading - shell layer functionalization modification - stabilization coating". This method solves the problems of low loading, poor stability, and insufficient targeting of traditional probes, thereby improving the fluorescence performance and detection effect of the probe.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is as follows: In a first aspect, the present invention proposes a method for preparing a time-resolved fluorescent probe made of nanospheres, comprising the following steps: S1: Preparation of core-layer fluorescent nanospheres: Polylactic acid-glycolic acid copolymer (PLGA) and rare earth ion complex were dissolved in dichloromethane at a certain mass ratio to obtain an oil phase; polyvinyl alcohol (PVA) was dissolved in deionized water to prepare an aqueous phase; the oil phase was slowly injected into the aqueous phase and ultrasonically emulsified at a certain speed, followed by magnetic stirring to evaporate the solvent, centrifugation to collect the precipitate, washing and drying to obtain core-layer fluorescent nanospheres; S2: Shell functionalization modification: Core-layer fluorescent nanospheres were dispersed in morpholine ethanesulfonic acid (MES) buffer, and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) were added for activation treatment; then an amino-targeting ligand was added, and the reaction was carried out under constant temperature stirring. After centrifugation and washing, the functionalized shell-modified nanosphere intermediate was obtained; high-density grafting was achieved by controlling the reaction conditions. S3: Fluorescence enhancement and stabilization treatment: The nanosphere intermediate was dispersed in an aqueous ethanol solution, and 3-aminopropyltriethoxysilane (APTES) and tetraethyl orthosilicate (TEOS) were added. The reaction was carried out under certain pH and temperature conditions to form a SiO2 coating layer. After centrifugation and drying, the time-resolved fluorescent probe of the nanosphere was obtained.

[0006] Furthermore, in step S1, dichloromethane is used as the solvent for the oil phase, and a 2%–5% PVA solution is used for the aqueous phase. High-speed ultrasonic emulsification (150–200 W) at 8000–12000 rpm is applied to form uniform droplets in the oil phase. The solvent is then evaporated by stirring at 30–40°C, forming solid core microspheres. Centrifugation at 10000–12000 rpm ensures efficient separation of the microspheres.

[0007] Furthermore, the rare earth ion complex is Eu. 3+ -β-Diketone-o-phenanthroline ternary complex, wherein Eu 3+ A stable chelate structure can be formed with β-diketone and o-phenanthroline in a molar ratio of 1:3:1, thereby improving the fluorescence quantum yield. The β-diketone is thiophenecarboxylic acid trifluoroacetone (TTA) or dibenzoylmethane (DBM), which has strong light absorption capabilities and can effectively transfer energy to Eu. 3+ Enhance fluorescence emission; The selection of rare earth ion complexes in this invention is key to improving fluorescence performance; this invention uses Eu. 3+ -β-Diketone-o-phenanthroline ternary complex, wherein Eu 3+ As a fluorescent central ion, it possesses a long fluorescence lifetime and characteristic emission spectrum; β-diketone, as the first ligand, can react with Eu... 3+ It forms a stable chelate ring, and its strong light absorption ability can efficiently transfer the absorbed energy to Eu through the "antenna effect". 3+ Enhanced fluorescence emission; o-phenanthroline, as a second ligand, can further supplement coordination, forming a stable ternary structure and inhibiting Eu. 3+ Nonradiative transitions enhance fluorescence quantum yield. 3+The molar ratio of β-diketone and o-phenanthroline was set to 1:3:1 to ensure the formation of a structurally stable complex. Among them, thiophenecarboxyltrifluoroacetone (TTA) or dibenzoylmethane (DBM) are preferred β-diketone derivatives. These two β-diketone derivatives have a large conjugated system and a high molar absorptivity, and their energy transfer efficiency is better than that of ordinary β-diketone compounds.

[0008] Furthermore, in step S1, the mass ratio of polylactic acid-glycolic acid copolymer (PLGA) to rare earth ion complex is (7.5~10):1; the mass fraction of aqueous solution is 2%~5%; after the oil phase is slowly injected into the aqueous phase, it is ultrasonically emulsified at a speed of 8000~12000 r / min for 10~20 min, and then magnetically stirred at 30~40℃ for 4~6 h to evaporate the solvent.

[0009] Furthermore, in step S2, the EDC / NHS activation method is used for targeted ligand grafting. The molar ratio of EDC to NHS is 1.2:1 to 1.5:1, which can efficiently activate the carboxyl groups on the surface of the core layer microspheres. The targeted ligand is a nucleic acid aptamer sequence, with a mass ratio of 1:5 to 1:8 to the core layer microspheres, ensuring a ligand grafting density of 1.2 × 10⁻⁶. 5 ~2.5×10 5 strips / μm 2 This improves the targeting and binding efficiency of the probe. The activation temperature is controlled at 25-30℃, the reaction time is 30-45 min, and the ligand coupling reaction time is 2-3 h to ensure the reaction proceeds fully. In step S2, the MES buffer provides a suitable acidic environment for the EDC / NHS activation reaction, promoting carboxyl activation. The added EDC, as a carboxyl activator, reacts with the carboxyl groups on the surface of the core microspheres to form an active ester intermediate. NHS, as a stabilizer, binds to the active ester intermediate to form a more stable succinimide ester, extending the lifetime of the active intermediate and improving the efficiency of subsequent reactions with amino groups.

[0010] Furthermore, in step S2, the aminated targeting ligand is an aminated nucleic acid aptamer sequence; the mass ratio of the aminated targeting ligand to the core layer fluorescent nanospheres is 1:5 to 1:8.

[0011] Furthermore, in step S3, a SiO2 coating layer is formed on the surface of the functionalized intermediate through the co-hydrolysis and condensation of APTES and TEOS, with a thickness controlled at 10-20 nm. The volume ratio of the added 3-aminopropyltriethoxysilane (APTES) to tetraethyl orthosilicate (TEOS) is 1:3 to 1:5, which ensures both the density of the coating layer and the introduction of amino groups, thereby improving the dispersibility of the probe. The reaction is carried out at pH 7.0-7.5 and 35-40°C for 6-8 hours; the volume fraction of the ethanol-water solution is 40%-60%, which promotes the hydrolysis reaction of the silane reagent. The SiO2 coating layer can effectively prevent the leakage of fluorescent substances and improve the stability of the probe in complex systems.

[0012] On the other hand, the present invention proposes a time-resolved fluorescent probe made of nanospheres, which is prepared by the preparation method described above. The probe has a core-shell structure, with the core layer being a fluorescent matrix of polylactic acid-glycolic acid copolymer loaded with rare earth ion complexes, and the shell layer being a functionalized modification layer coated with SiO2. The probe has a particle size of 100~200nm, a fluorescence lifetime of 5~8ms, and a fluorescence quantum yield of ≥35%.

[0013] Furthermore, the density of the target ligands grafted onto the shell surface is (1.2~2.5)×10⁻⁶. 5 The SiO2 coating layer, with a thickness of 10-20 nm, avoids aggregation caused by excessive ligands while ensuring sufficient target binding sites. This SiO2 coating stabilizes the probe and enhances fluorescence, addressing issues of fluorescent material leakage and poor environmental stability. The SiO2 coating layer serves three main functions: first, it acts as a physical barrier, effectively preventing leakage of rare-earth ion complexes from the core layer and improving load stability; second, it provides protection, preventing direct contact between the target ligand and the external environment, maintaining its biological activity; and third, it acts as a dispersant, as the hydroxyl groups on the SiO2 surface enhance the probe's hydrophilicity, preventing aggregation in aqueous solutions.

[0014] Thirdly, this invention proposes the application of the aforementioned time-resolved fluorescent nanosphere probe in food safety testing and environmental pollutant detection. The food safety testing targets include quinolone antibiotics or microcystin MC-LR; the environmental pollutant detection targets are heavy metal ions in water, such as Hg ions in water. 2+ Pb 2+ The detection involves the probe specifically binding to metal ions through a targeting ligand, resulting in a change in fluorescence intensity.

[0015] Furthermore, the detection method for food safety testing includes the following steps: incubating the probe with the sample to be tested at 35-40℃ for 30-60 min, using a time-resolved fluorescence spectrometer, setting the excitation wavelength to 340-360 nm, the delay time to 0.1-0.2 ms, the gate time to 1-3 ms, detecting the fluorescence intensity at the emission wavelength of 615 nm, and calculating the concentration of the target substance through a standard curve. The detection limit can reach 0.05-0.1 ng / mL, which is suitable for high-sensitivity detection of actual samples. The detection method for environmental pollutants includes the following steps: dispersing the probe in the water body to be tested, adjusting the pH to 5.5-6.5, reacting at 20-30℃ for 20-30 min, and quantitatively analyzing the concentration of heavy metal ions by the change in time-resolved fluorescence intensity.

[0016] Compared with the prior art, the technical solution of the present invention has the following beneficial effects: 1. By adopting a core-shell structure design and dual loading technology, rare earth ion complexes are loaded onto the PLGA core layer through in-situ doping, combined with SiO2 shell coating, increasing the loading to over 8wt% and reducing the fluorescence leakage rate to below 5%. 2. The synergistic effect of the SiO2 coating layer and the targeting ligand significantly improves the stability of the probe in complex systems (such as water), maintains a fluorescence lifetime of 5-8 ms, and retains ≥90% of the fluorescence intensity after 30 days of storage; 3. High ligand grafting density and over 30% improvement in specific binding efficiency, meeting the requirements of demanding detection scenarios; 4. The preparation process is simple and controllable, the reaction conditions are mild, no special equipment is required, it is suitable for large-scale production, and has broad application prospects. Attached Figure Description

[0017] Figure 1 This is a schematic diagram of the core-shell structure of the time-resolved fluorescent nanosphere probe of the present invention; in the figure: 1, PLGA core layer; 2, rare earth ion complex; 3, SiO2 shell layer; 4, nucleic acid aptamer.

[0018] Figure 2 This is a schematic diagram illustrating the detection principle of time-resolved fluorescence analysis technology for nanospheres. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0020] Example 1: Preparation of a time-resolved fluorescent nanosphere probe for the detection of microcystin MC-LR 1. Preparation of core-layer nanospheres: 1.0 g PLGA, 0.1 g Eu 3+ -TTA-phenanthroline ternary complex (Eu 3+ TTA:o-phenanthroline (molar ratio 1:3:1) was dissolved in 10 mL of dichloromethane to obtain an oil phase; 0.3 g of PVA was dissolved in 100 mL of deionized water to prepare a 3% aqueous phase; the oil phase was injected into the aqueous phase, and the mixture was ultrasonically emulsified at 180 W power and 10000 r / min speed for 15 min, magnetically stirred at 35 °C for 5 h, centrifuged at 11000 r / min for 12 min, washed and dried to obtain core layer microspheres; 2. Shell functionalization modification: 0.5 g core layer nanospheres were dispersed in 50 mL MES buffer (pH 6.0), 0.08 g EDC and 0.05 g NHS were added, and the mixture was activated at 28 °C for 40 min; 0.1 g aminofolic acid was added, and the mixture was stirred at a constant temperature for 2.5 h, and the intermediate was obtained by centrifugation and washing. 3. Stabilization treatment: The intermediate was dispersed in 50 mL of 50% ethanol aqueous solution, and 0.2 mL of APTES and 0.8 mL of LTEOS were added. The reaction was carried out at pH 7.2 and 38 °C for 7 h. After centrifugation and drying, the probe was obtained. The probe has a particle size of 150 nm, a fluorescence lifetime of 6.2 ms, a quantum yield of 38%, and an MC-LR detection limit of 0.05 ng / mL.

[0021] Example 2: For Hg 2+ Preparation of time-resolved fluorescent probes for detection of nanospheres 1. Preparation of core-layer nanospheres: 1.2 g PLGA and 0.1 g Eu were mixed. 3+ The -DBM-o-phenanthroline ternary complex was dissolved in 12 mL of dichloromethane to obtain an oil phase; 0.4 g of PVA was dissolved in 100 mL of deionized water to prepare a 4% aqueous phase; the mixture was ultrasonically emulsified at 190 W and 11000 r / min for 12 min, stirred at 38 °C for 4.5 h, centrifuged at 12000 r / min for 10 min, washed and dried; 2. Shell functionalization modification: 0.6 g of core layer microspheres were dispersed in 60 mL of MES buffer, and 0.09 g of EDC and 0.06 g of NHS were added. The mixture was activated at 27 °C for 35 min. Then, 0.12 g of aminolated nucleic acid aptamer (Hg) was added. 2+ (Specificity), stir the reaction for 2 hours, then centrifuge and wash; 3. Stabilization treatment: The intermediate was dispersed in 60 mL of 45% ethanol aqueous solution, and 0.15 mL of APTES and 0.75 mL of TEOS were added. The reaction was carried out at pH 7.3 and 36℃ for 6.5 h to obtain the probe. The probe has a particle size of 130 nm, a fluorescence lifetime of 5.8 ms, and a Hg... 2+ The detection limit is 0.3 nmol / L.

[0022] Example 3: Preparation of a time-resolved fluorescent nanosphere probe for the detection of quinolone antibiotics 1. Preparation of core-layer nanospheres: 1.5g PLGA, 0.2g Eu 3+ -TTA-o-phenanthroline complex dissolved in 15 mL dichloromethane, oil phase; 0.5 g PVA dissolved in 100 mL water, 5% aqueous phase; sonicated at 200 W power and 12000 r / min for 20 min, stirred at 40 °C for 6 h, and centrifuged at 10000 r / min for 15 min; 2. Shell functionalization modification: 0.8 g core microspheres were dispersed in 80 mL MES buffer, 0.12 g EDC and 0.08 g NHS were added, and the mixture was activated at 30 °C for 45 min; 0.16 g amino-modified biotin was added, and the mixture was stirred for 3 h. 3. Stabilization treatment: Disperse in 80 mL of 55% ethanol aqueous solution, 0.3 mL of APTES and 1.2 mL of TEOS, react at pH 7.4 and 40℃ for 8 h. The probe particle size is 180 nm, the fluorescence lifetime is 7.5 ms, and the detection limit for quinolone antibiotics is 0.05 ng / mL.

[0023] Figure 1 The schematic diagram of the core-shell structure of the fabricated nanosphere time-resolved fluorescent probe includes PLGA core layer 1, rare earth ion complex 2, SiO2 shell layer 3, and targeting ligand (nucleic acid aptamer 4 sequence). Figure 2 This is a schematic diagram illustrating the detection principle of time-resolved fluorescence analysis technology for nanospheres; it shows the relationship between fluorescence intensity and time at an excitation wavelength of 350 nm and an emission wavelength of 615 nm, with a delay time of 0.15 ms. The Stroke shift reaches 265 nm, effectively eliminating interference from excitation light and avoiding background interference from the matrix.

[0024] In summary, the preparation method of this invention is simple, cost-controllable, and suitable for large-scale industrial production. The prepared nanosphere time-resolved fluorescent probe has advantages such as excellent fluorescence performance, strong stability, and good targeting, and has broad application prospects in the fields of biomedical detection and environmental monitoring. It can achieve high-sensitivity and high-specificity detection of tumor markers, heavy metal ions, etc., and has significant economic and social value.

[0025] The above description is merely a specific embodiment 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 technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for preparing a nanomicrosphere time-resolved fluorescent probe, characterized in that, Comprising the following steps: S1: core layer fluorescent nanomicrosphere preparation: polylactic acid-glycolic acid copolymer and rare earth ion complex are dissolved in dichloromethane according to a certain mass ratio to obtain an oil phase; polyvinyl alcohol is dissolved in deionized water to prepare an aqueous phase; the oil phase is slowly injected into the aqueous phase, ultrasonically emulsified at a certain rotating speed, then magnetically stirred to volatilize the solvent, and the precipitate is collected by centrifugation, washed and dried to obtain the core layer fluorescent nanomicrosphere; S2: shell layer functional modification: the core layer fluorescent nanomicrosphere is dispersed in morpholine ethanesulfonic acid buffer, 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride and N-hydroxysuccinimide are added for activation treatment; then the aminoated targeting ligand is added, constant temperature stirring is carried out, and then centrifugal washing is carried out to obtain the nanomicrosphere intermediate with functional shell layer modification; S3: fluorescence enhancement and stabilization treatment: the nanomicrosphere intermediate is dispersed in an ethanol aqueous solution, 3-aminopropyl triethoxysilane and tetraethyl orthosilicate are added, and reaction is carried out under certain pH and temperature conditions to form a SiO2 coating layer; after centrifugal drying, the nanomicrosphere time-resolved fluorescence probe is obtained.

2. The method for preparing a nanomicrospheres time-resolved fluorescent probe according to claim 1, characterized in that, In step S1, the rare earth ion complex is Eu 3+ - a β-diketone-phenanthroline ternary complex, wherein Eu 3+ with a molar ratio of 1:3:1; the β-diketone is thenoyltrifluoroacetone (TTA) or dibenzoylmethane (DBM).

3. The method according to claim 1, wherein the method is characterized by, In step S1, the mass ratio of polylactic acid-glycolic acid copolymer and rare earth ion complex is (7.5-10):1; the mass fraction of the aqueous phase solution is 2%-5%; after the oil phase is slowly injected into the aqueous phase, ultrasonic emulsification is carried out at a rotating speed of 8000-12000 r / min for 10-20 min, and then the solvent is volatilized by magnetic stirring at 30-40℃ for 4-6 h. 4.The method for preparing a nanomicrosphere time-resolved fluorescent probe according to claim 1, characterized in that, In step S2, the molar ratio of 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride and N-hydroxysuccinimide added is 1.2:1-1.5:1; the activation process is activation at 25-30℃ for 30-45 min.

5. The method according to claim 1, wherein the method is characterized by, In step S2, the aminoated targeting ligand is an aminoated nucleic acid aptamer sequence; the mass ratio of the aminoated targeting ligand to the core layer fluorescent nanomicrosphere is 1:5-1:

8.

6. The method according to claim 1, wherein the method is characterized by, In step S3, the volume ratio of 3-aminopropyl triethoxysilane and tetraethyl orthosilicate added is 1:3-1:5, and the pH and temperature conditions of the reaction are: pH 7.0-7.5, 35-40℃ for 6-8 h; the volume fraction of the ethanol aqueous solution is 40%-60%.

7. A nanosphere time-resolved fluorescent probe, characterized in that, The probe is prepared by the preparation method in any one of claims 1-6, has a core-shell structure, the core layer is a fluorescent substrate of polylactic acid-glycolic acid copolymer loaded with rare earth ion complex, the shell layer is a functional modification layer coated with SiO2, the probe particle size is 100-200 nm, the fluorescence lifetime is 5-8 ms, and the fluorescence quantum yield is ≥35%. 8.The nanosphere time-resolved fluorescent probe according to claim 7, characterized in that, The density of the targeting ligand grafted on the surface of the shell layer is (1.2-2.5) x 10 5 The density of the targeting ligand grafted on the surface of the shell layer is (1.2-2.5) x 10 The density of the targeting ligand grafted on the surface of the shell layer is (1.2-2.5) x 10 The density of the targeting ligand grafted on the surface of the shell layer is 9. The application of the nanomicrospheres time-resolved fluorescent probe in food safety detection and environmental pollutant detection according to claim 7 or 8, characterized in that, The detection object of food safety detection includes quinolone antibiotics or microcystin MC-LR; and the detection object of environmental pollutant detection is heavy metal ions in water. 10.The application of the nanomicrosphere time-resolved fluorescent probe according to claim 9 in food safety detection and environmental pollutant detection, characterized in that, The detection method for food safety detection comprises the following steps: incubating the probe with the sample to be detected at 35-40 DEG C for 30-60 min, using a time-resolved fluorescence spectrometer, setting the excitation wavelength to 340-360 nm, the delay time to 0.1-0.2 ms, the gate time to 1-3 ms, detecting the fluorescence intensity at the emission wavelength of 615 nm, and calculating the concentration of the detection object through a standard curve; the detection method for environmental pollution detection comprises the following steps: dispersing the probe in the water body to be detected, adjusting the pH to 5.5-6.5, reacting at 20-30 DEG C for 20-30 min, and quantitatively analyzing the heavy metal ion concentration through the change of the time-resolved fluorescence intensity, with a detection limit of 0.1-1.0 nmol / L.

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