Nanometer fluorescent probe, preparation method and application thereof, and method for detecting microcystic toxins in liquid

Abstract

The invention relates to the field of bioanalytical chemistry and environmental monitoring, and discloses a nano fluorescent probe, a preparation method and application thereof, and a method for detecting microcystic toxins in liquid. The nano composite probe comprises an MOF material and nano particles loaded on the MOF material, the nano particles comprise carboxyl-modified up-conversion nano particles and nucleic acid aptamers covalently connected with the up-conversion nano particles through amido bonds, and the up-conversion nano particles contain a thulium element and an erbium element. The nano fluorescent probe has the advantages of high specificity, simplicity and rapidness in operation, high sensitivity, high interference resistance, simple preparation process and low cost, provides an efficient solution for rapid detection of liquid microcystin pollution, and has a remarkable market prospect.

Description

Technical Field

[0001] The present invention relates to the fields of bioanalytical chemistry and environmental monitoring, and in particular to a nano fluorescent probe, a preparation method and application thereof, and a method for detecting microcystin in liquid. Background Art

[0002] Microcystins (MCs) are a class of highly toxic, acutely harmful cyclic peptide hepatotoxins produced by freshwater blue-green algae, posing serious risks to aquatic life, drinking water safety, and human health. Among these, microcystin-(leucine-arginine) is the most common and acutely toxic microcystin. It is highly hepatotoxic and also has some toxicity to the heart, kidneys, spleen, and gastrointestinal tract. Currently, traditional methods for detecting MC-LR in liquids include high-performance liquid chromatography (HPLC) and HPLC-MS / MS, but these methods are expensive, cumbersome, and require high technical skills from the operator. Plant cell bioassays and protein phosphatase inhibition methods are relatively insensitive. Enzyme-linked immunosorbent assays (ELISAs) are more sensitive but have a narrow linear range.

[0003] Therefore, there is an urgent need to provide a nanofluorescent probe that can maintain high sensitivity and stability while also having high selectivity for microcystin, a wide linear range, strong anti-interference ability, and a simple and fast operation method. Summary of the Invention

[0004] The purpose of the present invention is to overcome the problem that the existing technology cannot maintain high sensitivity and stability while also having high selectivity for microcystins, a wide linear range and a simple and fast operation method. The present invention provides a nano fluorescent probe and its preparation method and application, and a method for detecting microcystins in liquids. The nano fluorescent probe has high specificity for microcystins, a wide linear range and simple and fast operation, while also maintaining high sensitivity and anti-interference ability (quantitative analysis of microcystins through changes in the dual-wavelength fluorescence intensity ratio, significantly improving detection stability and accuracy), and has significant market prospects.

[0005] In order to achieve the above-mentioned objectives, the first aspect of the present invention provides a nanocomposite probe, which includes an MOF material and nanoparticles loaded on the MOF material, wherein the nanoparticles include carboxyl-modified upconversion nanoparticles and nucleic acid aptamers covalently linked to the upconversion nanoparticles through amide bonds, and the upconversion nanoparticles contain thulium and erbium elements.

[0006] Preferably, the nucleic acid aptamer has the nucleotide sequence shown in SEQ ID NO.1.

[0007] Preferably, the preparation method of the MOF material comprises: mixing tetrakis(4-carboxyphenyl)porphine, zirconyl chloride octahydrate, benzoic acid and N,N-dimethylformamide solution for reaction, and then washing with N,N-dimethylformamide solution.

[0008] Preferably, the mass ratio of the tetrakis(4-carboxyphenyl)porphine, zirconyl chloride octahydrate, benzoic acid and N,N-dimethylformamide solution is 1:2-4:26-30:700-1400.

[0009] Preferably, the mixing reaction conditions include: temperature of 80-100° C. and time of 4-6 h.

[0010] Preferably, the method for preparing the nanoparticles comprises the following steps:

[0011] S1. Performing a stepwise coprecipitation reaction of 1-octadecene, oleic acid, gadolinium acetate hydrate, erbium acetate hydrate, ytterbium acetate hydrate, yttrium acetate hydrate, thulium acetate hydrate, NH4F, and NaOH. Subsequently, raising the system temperature to 290-310°C under an argon flowing atmosphere and maintaining the temperature for 0.6-1.5 hours to obtain upconversion nanoparticles.

[0012] S2, performing carboxyl modification on the upconversion nanoparticles described in step S1 to obtain carboxylated upconversion nanoparticles;

[0013] S3. Perform an amide reaction between the carboxyl-modified upconversion nanoparticles described in step S2 and the nucleic acid aptamer to obtain nanoparticles.

[0014] Preferably, in step S1, the volume ratio of the hydrated erbium acetate to the hydrated thulium acetate is 1:0.05-0.15.

[0015] Preferably, in step S2, the carboxylation modification step includes: before the upconversion nanoparticles are carboxyl-modified, mixing the upconversion nanoparticles with cyclohexane and octylphenol polyoxyethylene ether, and then performing a mixing reaction I with ammonia water and ethyl orthosilicate to obtain silica-coated upconversion nanoparticles; then performing a mixing reaction II with the silica-coated upconversion nanoparticles with ethanol and 3-aminopropyltriethoxysilane to obtain amino-modified silica-coated upconversion nanoparticles; finally, performing a mixing reaction III with the amino-modified silica-coated upconversion nanoparticles with N,N-dimethylformamide solution and a carboxylating agent to obtain carboxylated conversion nanoparticles.

[0016] Preferably, the carboxylating agent is selected from at least one of succinic anhydride, succinic anhydride and glutaric anhydride.

[0017] Succinic anhydride is more preferred.

[0018] Preferably, in step S3, the amide reaction process comprises: activating the carboxyl-modified upconversion nanoparticles described in step S2 and then mixing the activated carboxyl-modified upconversion nanoparticles with a HEPES buffer solution and a nucleic acid aptamer for reaction.

[0019] Preferably, the mixing reaction conditions include: time of 10-14 hours and temperature of 25-35°C.

[0020] The second aspect of the present invention provides a method for preparing a nanocomposite probe, the method comprising the following steps: preparing nanoparticles; loading the nanoparticles on a MOF material;

[0021] The nanoparticles include carboxyl-modified upconversion nanoparticles and nucleic acid aptamers covalently linked to the upconversion nanoparticles via amide bonds, and the upconversion nanoparticles contain thulium and erbium elements.

[0022] Preferably, the MOF material is PCN-224 nanoparticles, and the preparation method of the PCN-224 nanoparticles comprises: mixing tetrakis(4-carboxyphenyl)porphine, zirconyl chloride octahydrate, benzoic acid and N,N-dimethylformamide solution for reaction, and then washing with N,N-dimethylformamide solution.

[0023] Preferably, the mixing reaction conditions include: temperature of 80-100° C. and time of 4-6 h.

[0024] Preferably, the method for preparing the nanoparticles comprises the following steps:

[0025] S1. Performing a stepwise coprecipitation reaction of 1-octadecene, oleic acid, gadolinium acetate hydrate, erbium acetate hydrate, ytterbium acetate hydrate, yttrium acetate hydrate, thulium acetate hydrate, NH4F, and NaOH. Subsequently, raising the system temperature to 290-310°C under an argon flowing atmosphere and maintaining the temperature for 0.6-1.5 hours to obtain upconversion nanoparticles.

[0026] S2, performing carboxyl modification on the upconversion nanoparticles described in step S1 to obtain carboxylated upconversion nanoparticles;

[0027] S3. Perform an amide reaction between the carboxyl-modified upconversion nanoparticles described in step S2 and the nucleic acid aptamer to obtain nanoparticles.

[0028] Preferably, in step S1, the volume ratio of the hydrated erbium acetate to the hydrated thulium acetate is 1:0.05-0.15.

[0029] Preferably, in step S2, the carboxylation modification step includes: before the upconversion nanoparticles are carboxyl-modified, first mixing the upconversion nanoparticles with cyclohexane and octylphenol polyoxyethylene ether, and then mixing with ammonia water and ethyl orthosilicate for reaction I to obtain silica-coated upconversion nanoparticles, then mixing the silica-coated upconversion nanoparticles with ethanol and 3-aminopropyltriethoxysilane for reaction II to obtain amino-modified silica-coated upconversion nanoparticles, and then mixing the amino-modified silica-coated upconversion nanoparticles with N,N-dimethylformamide solution and a carboxylating agent for reaction II to obtain the carboxyl-modified upconversion nanoparticles.

[0030] Preferably, the carboxylating agent comprises succinic anhydride, succinic anhydride and glutaric anhydride.

[0031] Succinic anhydride is more preferred.

[0032] Preferably, in step S3, the amide reaction process comprises: activating the carboxyl-modified upconversion nanoparticles described in step S2 and then mixing the activated carboxyl-modified upconversion nanoparticles with a HEPES buffer solution and a nucleic acid aptamer for reaction.

[0033] Preferably, the mixing reaction time is 10-14 hours and the temperature is 25-35°C.

[0034] Preferably, the nucleic acid aptamer is a nucleic acid aptamer having the nucleotide sequence described in SEQ ID NO.1.

[0035] Preferably, the loading method comprises: mixing and incubating the nanoparticles, MOF material and buffer solution.

[0036] Preferably, the buffer solution is selected from any one of a sodium monohydrogen phosphate-potassium dihydrogen phosphate buffer solution, a potassium dihydrogen phosphate buffer solution and a disodium hydrogen phosphate buffer solution.

[0037] Preferably, the buffer solvent is a sodium monohydrogen phosphate-potassium dihydrogen phosphate buffer solution.

[0038] Preferably, the incubation conditions include: a temperature of 20-30° C. and a time of 10-20 min. Preferably, the mass ratio of the nanoparticles to the MOF material is 1:0.45-0.7.

[0039] The third aspect of the present invention provides use of the aforementioned nanocomposite probe and / or the nanocomposite probe prepared by the aforementioned method in detecting microcystins in liquid.

[0040] A fourth aspect of the present invention provides a method for detecting microcystins in liquid, comprising: contacting a sample containing microcystins with the nanofluorescent probe as described above and / or the nanofluorescent probe prepared by the method as described above.

[0041] Preferably, the concentration of microcystin in the sample is 0.5-80 nM.

[0042] Preferably, the contact conditions include: temperature of 20-30° C. and time of 15-35 min.

[0043] Through the above technical solution, the nanocomposite probe provided by the present invention includes a MOF material and nanoparticles supported on the MOF material. The nanoparticles include carboxyl-modified upconversion nanoparticles and a nucleic acid aptamer covalently linked to the upconversion nanoparticles via an amide bond. The upconversion nanoparticles contain thulium and erbium elements. While maintaining high sensitivity and stability, they also have high selectivity for microcystins, a wide linear range, and simple and rapid operation. This has significant market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 A is a transmission electron microscope image of the nucleus in test case 1; Figure 1 B is a transmission electron microscope image of the intermediate layer of Test Example 1; Figure 1 C is a transmission electron microscope image of the core-shell of Test Example 1; Figure 1 D in the middle is the transmission image of MOFs of Example 1; Figure 1 E in the middle is the scanning electron microscope image of MOFs of Example 1;

[0045] Figure 2 The XRD spectrum of NaGdF4:Yb,Er@NaYF4:Yb,Tm@NaYF4 in Test Example 2; the EDS spectrum of NaGdF4:Yb,Er@NaYF4:Yb,Tm@NaYF4;

[0046] Figure 3are the energy dispersive X-ray spectra (EDS), infrared spectra and Zeta potential diagrams in Test Example 2; wherein A is the EDS spectrum of NaGdF4:Yb,Er in Test Example 2; B is the EDS spectrum of NaGdF4:Yb,Er@NaYF4:Yb,Tm in Test Example 2; C is the EDS spectrum of NaGdF4:Yb,Er@NaYF4:Yb,Tm@NaYF4 in Test Example 2; D is the EDS spectrum of OA-UCNPs( a), infrared spectra of UCNPs@SiO2(b), UCNPs@SiO2-NH2(c) and UCNPs@SiO2-COOH(d); E is the Zeta potential diagram of UCNPs@SiO2, UCNPs@SiO2-NH2 and UCNPs@SiO2-COOH; F is the Zeta potential diagram of UCNPs-aptamer-MOFs and UCNPs-aptamer-MOFs-MC-LR;

[0047] Figure 4 is a spectral overlap diagram between the absorption spectrum and the upconversion emission spectrum of the MOF material in Test Example 3;

[0048] Figure 5 is a graph showing the fluorescence intensity spectrum and the linear relationship between the microcystin concentration and the fluorescence quenching intensity in Test Example 4; wherein A is a graph showing the luminescence recovery of the UCNPs-aptamer-MOFs nanocomposite probe at different MC-LR concentrations in Test Example 4; and B is a fitted standard linear graph showing the ratio of the luminescence intensity of the UCNPs-aptamer-MOFs nanocomposite probe at 550 nm to that at 812 nm at different MC-LR concentrations in Test Example 4;

[0049] Figure 6 This is a graph showing the selective specificity of the UCNPs-aptamer-MOFs nanocomposite probe for MC-LR in Test Example 5;

[0050] Figure 7 This is a schematic diagram of the principle of detecting microcystin by the nanocomposite probe in Example 2-1. DETAILED DESCRIPTION

[0051] The endpoints of the ranges and any values disclosed herein are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0052] In a first aspect, the present invention provides a nanocomposite probe comprising a metal organic framework (MOF) material and nanoparticles supported on the MOF material. The nanoparticles comprise carboxyl-modified upconversion nanoparticles and nucleic acid aptamers covalently linked to the upconversion nanoparticles via amide bonds. The upconversion nanoparticles contain thulium and erbium. Under near-infrared light excitation, the thulium and erbium elements in the nanocomposite probe emit fluorescence at 550 nm and 812 nm, respectively. When microcystin is present in the system, the 550 nm fluorescence intensity changes. The microcystin concentration is quantified by detecting the change in the ratio of the 550 nm to 812 nm fluorescence intensities. This ratiometric detection method effectively eliminates interference from nonspecific factors such as light source fluctuations, instrument drift, and sample concentration changes during the detection process, significantly improving the accuracy and stability of the detection.

[0053] The MOF material in the nanocomposite probe provided by the present invention participates in fluorescence quenching and works synergistically with carboxyl-modified upconversion nanoparticles and nucleic acid aptamers, so that the nanocomposite probe of the present invention not only maintains high sensitivity and stability, but also has high selectivity for microcystin, a wide linear range, strong anti-interference ability and a simple and fast operation method.

[0054] According to the present invention, preferably, the nucleic acid aptamer has the nucleotide sequence shown in SEQ ID NO. 1. The nanocomposite probe provided by the present invention, having the nucleotide sequence shown in SEQ ID NO. 1, uses a nucleic acid aptamer as a component for recognizing microcystins. This provides the nanocomposite probe with advantages such as a wider range of target molecules, more convenient in vitro screening, better stability, and insensitivity to temperature, thereby increasing the flexibility and specificity of the nanocomposite probe for microcystins.

[0055] According to the present invention, preferably, the method for preparing the MOF material comprises: mixing tetrakis(4-carboxyphenyl)porphine, zirconyl chloride octahydrate, benzoic acid, and an N,N-dimethylformamide solution for a reaction, followed by washing with an N,N-dimethylformamide solution. Preferably, the mass ratio of tetrakis(4-carboxyphenyl)porphine, zirconyl chloride octahydrate, benzoic acid, and N,N-dimethylformamide solution (DMF) is 1:2-4:26-30:700-1400, more preferably 1:2.5-3.5:27-29:800-1200. Preferably, the mixing reaction conditions include: a temperature of 80-100°C and a reaction time of 4-6 hours. Exemplary methods for preparing MOF materials include dissolving 100 mg of tetrakis(4-carboxyphenyl)porphine, 200-400 mg of zirconyl chloride octahydrate, 2.6 g-3 g of benzoic acid, and 80-150 mL of DMF in a round-bottom flask, and stirring the mixture at 80-100° C. for 4-6 hours. After the reaction is completed, the mixture is washed three times with DMF to obtain PCN-224 nanoparticles, which serve as the MOF material. The inventors have discovered that by employing the preferred implementation method, the MOF material can possess unique optical structures and properties. Its absorption spectrum can effectively overlap with the emission spectrum of upconversion nanoparticles (UCNPs), thereby achieving efficient fluorescence quenching. In the nanocomposite probe provided by the present invention, the fluorescence signal based on the upconversion nanoparticles (UCNPs) can be sensitively changed according to the presence or absence and concentration of microcystin, greatly improving the sensitivity of detection.

[0056] According to the present invention, preferably, the method for preparing the nanoparticles comprises the following steps:

[0057] S1. Performing a step-by-step co-precipitation reaction on 1-octadecene, oleic acid, gadolinium acetate hydrate, erbium acetate hydrate, ytterbium acetate hydrate, yttrium acetate hydrate, thulium acetate hydrate, NH4F, and NaOH. Subsequently, raising the system temperature to 290-310° C., more preferably 295-305° C., under an argon flowing atmosphere, and maintaining the temperature for 0.6-1.5 h, more preferably 0.8-1.2 h, to obtain upconversion nanoparticles.

[0058] S2, performing carboxyl modification on the upconversion nanoparticles described in step S1 to obtain carboxylated upconversion nanoparticles;

[0059] S3. Perform an amide reaction between the carboxyl-modified upconversion nanoparticles described in step S2 and the nucleic acid aptamer to obtain nanoparticles.

[0060] According to the present invention, preferably, in step S1, the volume ratio of the hydrated erbium acetate and the hydrated thulium acetate is 1:0.05-0.15, and more preferably 1:0.08-0.12. The inventors have found that by adopting this preferred embodiment to carry out a step-by-step co-precipitation reaction, the reaction order and proportion of each component can be accurately controlled to form a core (NaGdF4:Yb,Er), an intermediate layer (NaGdF4:Yb,Er@NaYF4:Yb,Tm) and a shell layer (NaGdF4:Yb,Er@NaYF4:Yb,Tm@NaYF4) in sequence. This precisely constructed core-shell structure greatly reduces the surface defects of the nanoparticles, reduces energy loss, and significantly improves the upconversion luminescence efficiency. In the subsequent detection of microcystin, the stable and efficient luminescence provides a reliable signal basis for ratiometric detection, which can keenly capture the fluorescence changes caused by the presence of microcystin, thereby enhancing the detection sensitivity.

[0061] According to the present invention, preferably, in step S2, the carboxyl modification step comprises: before the upconversion nanoparticles (UCNPs) are carboxyl modified, the upconversion nanoparticles are mixed with cyclohexane, octylphenol polyoxyethylene ether (Lgepal CO-520) is mixed with ammonia water and tetraethyl orthosilicate (TEOS solution) to carry out a mixing reaction I to obtain silica-coated upconversion nanoparticles (UCNPs@SiO2); the silica-coated upconversion nanoparticles (UCNPs@SiO2) are then mixed with ethanol and 3-aminopropyltriethoxysilane (APTES) to carry out a mixing reaction II to obtain amino-modified silica-coated upconversion nanoparticles (UCNPs@SiO2-NH2); finally, the amino-modified silica-coated upconversion nanoparticles (UCNPs@SiO2-NH2) are mixed with N,N-dimethylformamide solution DMF and a carboxylating agent to carry out a mixing reaction III to obtain carboxyl-modified conversion nanoparticles (UCNPs@SiO2-COOH). Preferably, the carboxylating agent is selected from at least one of succinic anhydride, succinic anhydride and glutaric anhydride; more preferably, succinic anhydride. The inventors have found that the nanoparticles modified with carboxyl groups according to the above preferred embodiment have better water solubility and biocompatibility, which facilitates subsequent connection with nucleic acid aptamers.

[0062] Exemplarily, the carboxyl modification step includes:

[0063] (1) Silica Coating: Dissolve the prepared upconversion nanoparticles (UCNPs) in cyclohexane solution, add Lgepal CO-520 and sonicate until completely dissolved. Transfer the mixture to a round-bottom flask, inject ammonia solution, and then add TEOS solution dropwise with vigorous stirring (the volume of TEOS can control the thickness of the silicon layer). React at 20°C-30°C for 10-15 hours, and wash with anhydrous ethanol by centrifugation to obtain UCNPs@SiO2.

[0064] (2) Amination modification: The UCNPs@SiO2 precipitate prepared in step (1) was dissolved in anhydrous ethanol, APTES was added and stirred evenly, and the mixture was reacted at room temperature for 1.5-3 hours. After the reaction, the mixture was centrifuged and washed with anhydrous ethanol to obtain a UCNPs@SiO2-NH2 precipitate;

[0065] (3) Carboxylation modification: The UCNPs@SiO2-NH2 precipitate prepared in step (2) was dispersed in DMF solution, succinic anhydride was weighed (the mass ratio of precipitate to succinic anhydride was 1:30) and ultrasonically dissolved in DMF, and then slowly added dropwise to the DMF solution of UCNPs@SiO2-NH2 at 20℃-30℃. After reacting for 20-30h, the product was separated with anhydrous ethanol to finally obtain carboxyl-modified upconversion nanoparticles (UCNPs@SiO2-COOH).

[0066] According to the present invention, preferably, in step S3, the amide reaction process includes: activating the carboxylated upconversion nanoparticles (UCNPs@SiO2-COOH) described in step S2, mixing them with a HEPES buffer solution and a nucleic acid aptamer, wherein the mixing reaction conditions include: a time of 10-14 hours and a temperature of 25-35°C. The inventors have found that adopting the above preferred embodiment can enhance the specific recognition of the nanocomposite probe, optimize the detection model, improve the detection sensitivity, further stabilize the performance of the nanocomposite probe, and ensure detection reliability.

[0067] Exemplarily, the process of the amide reaction includes:

[0068] (i) Activation: The carboxyl-modified upconversion nanoparticles (UCNPs@SiO2-COOH) prepared in step S2 were uniformly dispersed in 5 mg / mL 2-(N-morpholino)ethanesulfonic acid (MES solution). 5 mg / mL 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC solution) and N-hydroxysuccinimide (NHS solution) (volume ratio of 4:1) were added to the above carboxyl-modified upconversion nanoparticle dispersion, and the mixture was incubated at 20-40°C with slow shaking for 1-3 h. After activation, the precipitate was obtained by centrifugation.

[0069] (2) dissolving the precipitate obtained in step (1) in 10 mM HEPES buffer (pH 7.4), injecting MC-LR nucleic acid aptamer (10 μM) having the nucleotide sequence shown in SEQ ID NO. 1, and reacting the mixture at 25° C.-35° C. overnight;

[0070] (3) Centrifuge the mixture obtained in step (2) at 5000-7000 rpm for 4-6 minutes using a 30,000 MW ultrafiltration tube to remove unreacted aptamers, EDC, and NHS. After removing the lower phase, wash three times with ultrapure water. Dissolve the final product in ultrapure water and store at 2°C-5°C.

[0071] The second aspect of the present invention provides a method for preparing a nanocomposite probe, which includes the following steps: preparing nanoparticles; and then loading the nanoparticles on a MOF material; wherein the nanoparticles include the carboxyl-modified upconversion nanoparticles as described above and the nucleic acid aptamer as described above that is covalently linked to the upconversion nanoparticles via an amide bond, and the upconversion nanoparticles contain thulium and erbium elements.

[0072] The MOF material is PCN-224 nanoparticles, and the preparation method of the PCN-224 nanoparticles includes: mixing tetrakis(4-carboxyphenyl)porphine, zirconyl chloride octahydrate, benzoic acid, and N,N-dimethylformamide solution, followed by washing with N,N-dimethylformamide solution. Preferably, the mass ratio of tetrakis(4-carboxyphenyl)porphine, zirconyl chloride octahydrate, benzoic acid, and N,N-dimethylformamide solution (DMF) is 1:2-4:26-30:700-1400, more preferably 1:2.5-3.5:27-29:800-1200. Preferably, the mixing reaction conditions include: a temperature of 80-100°C and a reaction time of 4-6 hours.

[0073] Preferably, the method for preparing the nanoparticles comprises the following steps:

[0074] S1. Performing a stepwise coprecipitation reaction of 1-octadecene, oleic acid, gadolinium acetate hydrate, erbium acetate hydrate, ytterbium acetate hydrate, yttrium acetate hydrate, thulium acetate hydrate, NH4F, and NaOH. Subsequently, raising the system temperature to 290-310°C under an argon flowing atmosphere and maintaining the temperature for 0.6-1.5 hours to obtain upconversion nanoparticles.

[0075] S2, performing carboxyl modification on the upconversion nanoparticles described in step S1 to obtain carboxylated upconversion nanoparticles;

[0076] S3. Perform an amide reaction between the carboxyl-modified upconversion nanoparticles described in step S2 and the nucleic acid aptamer to obtain nanoparticles.

[0077] According to the present invention, preferably, in step S1, the volume ratio of the hydrated erbium acetate to the hydrated thulium acetate is 1:0.05-0.15, more preferably 1:0.08-0.12.

[0078] According to the present invention, preferably, in step S2, the carboxyl modification step comprises: before the upconversion nanoparticles (UCNPs) are carboxyl modified, the upconversion nanoparticles are mixed with cyclohexane, octylphenol polyoxyethylene ether (Lgepal CO-520) is mixed with ammonia water and tetraethyl orthosilicate (TEOS solution) to carry out a mixing reaction I to obtain silica-coated upconversion nanoparticles (UCNPs@SiO2); the silica-coated upconversion nanoparticles (UCNPs@SiO2) are then mixed with ethanol and 3-aminopropyltriethoxysilane (APTES) to carry out a mixing reaction II to obtain amino-modified silica-coated upconversion nanoparticles (UCNPs@SiO2-NH2); finally, the amino-modified silica-coated upconversion nanoparticles (UCNPs@SiO2-NH2) are mixed with N,N-dimethylformamide solution DMF and a carboxylating agent to carry out a mixing reaction III to obtain carboxyl-modified conversion nanoparticles (UCNPs@SiO2-COOH). Preferably, the carboxylating agent is selected from at least one of succinic anhydride, succinic anhydride and glutaric anhydride; more preferably, succinic anhydride.

[0079] According to the present invention, preferably, in step S3, the process of the amide reaction includes: activating the carboxyl-modified upconversion nanoparticles (UCNPs@SiO2-COOH) described in step S2 and mixing them with a HEPES buffer solution and a nucleic acid aptamer, and the conditions of the mixed reaction include: a time of 10-14 hours and a temperature of 25-35°C.

[0080] According to the present invention, preferably, the nucleic acid aptamer is a nucleic acid aptamer having the nucleotide sequence described in SEQ ID NO.1.

[0081] According to the present invention, preferably, the loading method comprises: mixing and incubating the aforementioned nanoparticles, the aforementioned MOF material, and a buffer solution. Preferably, the buffer solution is selected from any one of a sodium monohydrogen phosphate-potassium dihydrogen phosphate buffer solution, a potassium dihydrogen phosphate buffer solution, and a disodium hydrogen phosphate buffer solution; further preferably, the buffer solvent is a sodium monohydrogen phosphate-potassium dihydrogen phosphate buffer solution.

[0082] According to the present invention, preferably, the incubation conditions include: a temperature of 20-30°C, a time of 10-20 minutes, and a pH of 7.2-8. Furthermore, preferably, the mass ratio of the nanoparticles to the MOF material is 1:0.45-0.7, and even more preferably, 1:0.48-0.6. The inventors have discovered that employing this preferred embodiment can further enhance the detection sensitivity and efficiency of the nanocomposite probe, while ensuring its stability.

[0083] An exemplary method for preparing an assembled nanocomposite probe includes mixing and incubating the aforementioned nanoparticles, the aforementioned MOF material, and a sodium monohydrogen phosphate-potassium dihydrogen phosphate buffer solution, wherein the mass ratio of the nanoparticles to the MOF material is 1:0.5, incubating at 3500 rpm, a temperature of 25°C, an incubation time of 15 minutes, and a pH of 7.5. The resulting nanocomposite probe is provided by the present invention.

[0084] The third aspect of the present invention provides use of the aforementioned nanocomposite probe and / or the nanocomposite probe prepared by the aforementioned method in detecting microcystins in liquid.

[0085] A fourth aspect of the present invention provides a method for detecting microcystins in liquid, comprising: contacting a sample containing microcystins with the nanofluorescent probe as described above and / or the nanofluorescent probe prepared by the method as described above.

[0086] According to the present invention, preferably, the concentration of microcystin in the sample is 0.5-80 nM, specifically 0.5 nM, 1 nM, 20 nM, 60 nM, 80 nM, or any value therebetween.

[0087] According to the present invention, preferably, the contact conditions include: a temperature of 20-30°C, specifically 20°C, 22°C, 24°C, 28°C, 30°C, or any value between these values; a time of 15-35 min, specifically 15 min, 20 min, 25 min, 30 min, 35 min, or any value between these values.

[0088] The inventors have found that the above-mentioned preferred implementation method has significant beneficial effects in terms of detection accuracy, efficiency, applicability and stability, can meet actual detection needs, and provide strong technical support for related fields.

[0089] The present invention will be described in detail below through examples.

[0090] In the following examples, microcystin subtype MC-LR was purchased from Hefei Ruijie Biological Co., Ltd., China; gadolinium (III) acetate hydrate, erbium (III) acetate hydrate, ytterbium (III) acetate hydrate, thulium (III) acetate hydrate, yttrium (III) acetate hydrate, Lgepal CO-520, tetraethyl orthosilicate (TEOS, ≥99.0%), succinic anhydride / succinic anhydride (≥99.0%), and tetrakis(4-carboxyphenyl)porphyrin (H2TCPP) were purchased from Sigma-Aldrich Reagent Company. 1-Octadecene (ODE, 90.0%), ammonium fluoride (NH4F, AR), sodium hydroxide (NaOH, 99.9%), morpholineethanesulfonic acid (MES, 99%), oleic acid (OA, AR), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 98.0%), N-hydroxysuccinimide (NHS, 98%), and 3-aminopropyltriethoxysilane (APTES) were purchased from Aladdin Reagent Company; other reagents and raw materials were conventional commercial products.

[0091] Example 1-1

[0092] The process of preparing the nanocomposite probe is as follows:

[0093] (1) Synthesis of NaGdF4:Yb,Er@NaYF4:Yb,Tm@NaYF4 (upconversion nanoparticles):

[0094] I first synthesized the core NaGdF4:Yb,Er: 0.99 mL of 0.2M ytterbium acetate hydrate, 0.99 mL of 0.2M gadolinium acetate hydrate, 20 μL of 0.2M erbium acetate hydrate, 4 mL of oleic acid, and 6 mL of 1-octadecene solution were added to a 50 mL round-bottom, double-necked flask at room temperature. The system temperature was slowly raised to 150°C in an oil bath while stirring continuously. After 40 minutes, the system was cooled to 50°C. Next, 4.3 mL of a thoroughly mixed precipitant (a mixture of 1M NaOH-CH3OH and 0.4M NH4F-CH3OH in a volume ratio of 1:3.3) was quickly added to the round-bottom flask. Stirring was continued and the temperature was maintained at 50°C for 30 minutes. Afterwards, the temperature was raised to 100°C and the flask was connected to a double-row tube. Under an argon flowing atmosphere, it was connected to liquid nitrogen for degassing to remove impurities such as residual water, oxygen, and methanol in the system, thereby achieving an oxygen-free state. Subsequently, the system temperature was raised to 300°C under an argon flowing atmosphere and maintained at this temperature for 1 hour. Finally, the resulting product was evenly distributed into a centrifuge tube, 1 mL of cyclohexane solution was added as a solvent, and 1 mL of ethanol was added to precipitate the upconversion nanoparticles. The centrifuge tube was placed in a centrifuge and centrifuged at 8000 rpm / min for 4 minutes. This was repeated three times to obtain the core NaYF4:Yb,Er, which was stored in the cyclohexane solution.

[0095] II. Then synthesize the intermediate layer NaGdF4:Yb,Er@NaYF4:Yb,Tm: prepare it according to the method used in step I, except that 20 μL of erbium acetate hydrate in step I is adjusted to 2 μL of thulium acetate hydrate; and adjust "quickly add 4.3 mL of thoroughly mixed precipitant to the round-bottom flask" in step I to "quickly add 2 mL of the core NaYF4:Yb,Er prepared in step I and stored in cyclohexane and 4.3 mL of thoroughly mixed precipitant to the round-bottom flask" to obtain the intermediate layer NaGdF4:Yb,Er@NaYF4:Yb,Tm, and store it in cyclohexane solution.

[0096] III The last product synthesized is core-shell NaGdF4:Yb,Er@NaYF4:Yb,Tm@NaYF4: it was prepared according to the method used in step I, except that the "0.2M ytterbium acetate hydrate 0.99mL, 0.2M gadolinium acetate hydrate 0.99mL, 0.2M erbium acetate hydrate 20μL" in step I was adjusted to "0.2M yttrium acetate hydrate 2mL"; the "quickly add 4.3mL of thoroughly mixed precipitant into the round-bottom flask" in step I was adjusted to "quickly add 2mL of core-shell NaGdF4:Yb,Er@NaYF4:Yb,Tm and 4.3mL of thoroughly mixed precipitant stored in cyclohexane into the round-bottom flask", thereby obtaining core-shell NaGdF4:Yb,Er@NaYF4:Yb,Tm@NaYF4 upconversion luminescent nanoparticles (UCNPs).

[0097] (II) Preparation of carboxyl-modified upconversion nanoparticles

[0098] I used the reverse microemulsion method to silanize the surface of upconversion nanoparticles. 40 mg of UCNPs prepared in step (1) was dispersed in 33 mL of cyclohexane solution to form a mixed solution I. Then, 1.6 g of Lgepal CO-520 was weighed in a 10 mL centrifuge tube. The mixed solution I was added in small amounts several times and ultrasonically treated to completely dissolve it and transferred to a round-bottom flask. Then, in a fume hood, 270 μL of ammonia water was evenly added dropwise to the above solution. Finally, 20 μL of TEOS solution was added dropwise to the solution and stirred vigorously. After reacting at room temperature for one day, a transparent and stable microemulsion system was formed. The alkyl-functionalized upconversion nanoparticles UCNPs@SiO2 were obtained by centrifugal washing (10,000 rpm, 10 min) three times with anhydrous ethanol.

[0099] Next, the surface of the upconversion nanoparticles was amino-functionalized. 31 mg of the UCNPs@SiO2 precipitate prepared in the previous step was redispersed in 25 mL of anhydrous ethanol. 0.8 mL of APTES was added and stirred thoroughly. The mixture was allowed to react at room temperature for 2 hours. After the reaction, the particles were centrifuged and washed three times with anhydrous ethanol (10,000 rpm for 10 minutes) to obtain amino-functionalized upconversion nanoparticles, UCNPs@SiO2-NH2. This step allowed the -NH2 groups to attach to the SiO2 surface through hydrolysis of the APTES.

[0100] Finally, the surface of the upconversion nanoparticles was functionalized with carboxyl groups. 25 mg of the UCNPs@SiO2-NH2 precipitate prepared in the above steps was dispersed in 5 mL of DMF solution. 750 mg of succinic anhydride was transferred to 10 mL of DMF and sonicated to completely dissolve it. The anhydride was then slowly added dropwise to the DMF solution containing UCNPs@SiO2-NH2. After reacting at room temperature for one day, the solution was centrifuged and washed three times with anhydrous ethanol (10,000 rpm, 10 min) to obtain carboxyl-functionalized upconversion nanoparticles UCNPs@SiO2-COOH. This step allows the carboxyl-functionalized upconversion nanoparticles to undergo a ring-opening reaction, generating covalent amide bonds and terminal carboxylic acid groups on the surface of the UCNPs. The final product was dispersed in deionized water for subsequent use.

[0101] (III) Preparation of aptamer-modified upconversion nanoparticles

[0102] IFirst, the carboxyl groups of UCNPs@SiO2-COOH were activated. 1.2 mg of the carboxyl-functionalized upconversion nanoparticles prepared in step (ii) were uniformly dispersed in 2 mL of 5 mg / mL MES (pH = 6, 10 mM) buffer solution. 20 μL of 5 mg / mL EDC solution and 5 μL of NHS solution were added to the MES solution of the above carboxyl-functionalized upconversion nanoparticles.

[0103] II. UCNPs@SiO2-COOH nanoparticles were incubated in an EDC / NHS solution with slow shaking for 2 h at room temperature. After activation, the solution was centrifuged at 10,000 rpm for 10 min to obtain a precipitate. The resulting precipitate was then dissolved in 2 mL of 10 mM HEPES buffer (pH 7.4), injected with MC-LR aptamer (40 μL, 10 μM), and reacted at 30°C for 12 h. Finally, the solution was centrifuged at 6,000 rpm for 5 min using a 30,000 MW ultrafiltration tube to remove unreacted aptamer, EDC, and NHS. After removing the lower phase, the solution was washed three times with ultrapure water. The final product (nanoparticles: UCNPs-aptamer) was dissolved in 1.2 mL of ultrapure water and stored in a refrigerator.

[0104] (IV) Preparation of MOF materials

[0105] First, 100 mg of tetrakis(4-carboxyphenyl)porphine, 300 mg of zirconyl chloride octahydrate, and 2.8 g of benzoic acid were completely dissolved in 100 mL of DMF. The mixture was then poured into a 250 mL round-bottom flask and stirred under reflux in a 90°C oil bath for 5 hours. After the reaction, the solution was evenly dispersed into a centrifuge tube and centrifuged at 10,000 rpm for 10 minutes to obtain a precipitate. The precipitate was then washed three times with DMF to obtain PCN-224 nanoparticles (MOF material).

[0106] (V) Preparation of nanocomposite probes

[0107] The UCNPs-aptamer (120 μL, 1 mg / mL) prepared in step (3) and the MOF material (30 μL, 2 mg / mL) prepared in step (4) were added to a 2 mL centrifuge tube, followed by the addition of 100 μL of sodium monohydrogen phosphate-potassium dihydrogen phosphate buffer solution (pH 7.6) and the reaction was shaken at 25 ° C for 15 min to obtain the UCNPs-aptamer-MOFs nanocomposite probe Z1.

[0108] Example 1-2

[0109] The process of preparing the nanocomposite probe is as follows:

[0110] (1) Synthesis of NaGdF4:Yb,Er@NaYF4:Yb,Tm@NaYF4 (upconversion nanoparticles):

[0111] I first synthesized the core NaGdF4:Yb,Er: 0.99 mL of 0.2M ytterbium acetate hydrate, 0.99 mL of 0.2M gadolinium acetate hydrate, 20 μL of 0.2M erbium acetate hydrate, 4 mL of oleic acid, and 6 mL of 1-octadecene solution were added to a 50 mL round-bottom, double-necked flask at room temperature. The system temperature was slowly raised to 150°C in an oil bath while stirring continuously. After 40 minutes, the system was cooled to 50°C. Next, 4.3 mL of a thoroughly mixed precipitant (a mixture of 1M NaOH-CH3OH and 0.4M NH4F-CH3OH in a volume ratio of 1:3.3) was quickly added to the round-bottom flask. Stirring was continued and the temperature was maintained at 50°C for 30 minutes. Afterwards, the temperature was raised to 100°C and the flask was connected to a double-row tube. Under an argon flowing atmosphere, it was connected to liquid nitrogen for degassing to remove impurities such as residual water, oxygen, and methanol in the system, thereby achieving an oxygen-free state. Subsequently, the system temperature was raised to 295°C under an argon flowing atmosphere and maintained at this temperature for 0.8 h. Finally, the resulting product was evenly distributed into a centrifuge tube, 1 mL of cyclohexane solution was added as a solvent, and 1 mL of ethanol was added to precipitate the upconversion nanoparticles. The centrifuge tube was placed in a centrifuge and centrifuged at 8000 rpm / min for 4 min. This was repeated three times to obtain the core NaYF4:Yb,Er, which was stored in a cyclohexane solution.

[0112] II. Then synthesize the intermediate layer NaGdF4:Yb,Er@NaYF4:Yb,Tms: prepare it according to the method used in step I, except that 20 μL of erbium acetate hydrate in step I is adjusted to 1.6 μL of thulium acetate hydrate; and adjust "quickly add 4.3 mL of thoroughly mixed precipitant to the round-bottom flask" in step I to "quickly add 2 mL of the core NaYF4:Yb,Er prepared in step I and stored in cyclohexane and 4.3 mL of thoroughly mixed precipitant to the round-bottom flask" to obtain the intermediate layer NaGdF4:Yb,Er@NaYF4:Yb,Tm, and store it in cyclohexane solution.

[0113] III The last product synthesized is core-shell NaGdF4:Yb,Er@NaYF4:Yb,Tm@NaYF4: it was prepared according to the method used in step I, except that the "0.2M ytterbium acetate hydrate 0.99mL, 0.2M gadolinium acetate hydrate 0.99mL, 0.2M erbium acetate hydrate 20μL" in step I was adjusted to "0.2M yttrium acetate hydrate 2mL"; the "quickly add 4.3mL of thoroughly mixed precipitant into the round-bottom flask" in step I was adjusted to "quickly add 2mL of core-shell NaGdF4:Yb,Er@NaYF4:Yb,Tm and 4.3mL of thoroughly mixed precipitant stored in cyclohexane into the round-bottom flask", thereby obtaining core-shell NaGdF4:Yb,Er@NaYF4:Yb,Tm@NaYF4 upconversion luminescent nanoparticles (UCNPs).

[0114] (II) Preparation of carboxyl-modified upconversion nanoparticles

[0115] I used the reverse microemulsion method to silanize the surface of upconversion nanoparticles. 40 mg of UCNPs prepared in step (I) was dispersed in 33 mL of cyclohexane solution to form a mixed solution I. Then, 1.6 g of Lgepal CO-520 was weighed in a 10 mL centrifuge tube. The mixed solution I was added in small amounts several times and ultrasonically treated to completely dissolve it and transferred to a round-bottom flask. Then, in a fume hood, 270 μL of ammonia water was evenly added to the above solution. Finally, 20 μL of TEOS solution was added dropwise to the solution and stirred vigorously. After reacting at room temperature for one day, a transparent and stable microemulsion system was formed. The alkyl-functionalized upconversion nanoparticles UCNPs@SiO2 were obtained by centrifugal washing (10,000 rpm, 10 min) three times with anhydrous ethanol. II

[0116] Next, the surface of the upconversion nanoparticles was amino-functionalized. 31 mg of the UCNPs@SiO2 precipitate prepared in the previous step was redispersed in 25 mL of anhydrous ethanol. 0.8 mL of APTES was added and stirred thoroughly. The mixture was allowed to react at room temperature for 2 hours. After the reaction, the particles were centrifuged and washed three times with anhydrous ethanol (10,000 rpm for 10 minutes) to obtain amino-functionalized upconversion nanoparticles, UCNPs@SiO2-NH2. This step allowed the -NH2 groups to attach to the SiO2 surface through hydrolysis of APTES.

[0117] III. Finally, the surface carboxyl functionalization of the upconversion nanoparticles was performed. 25 mg of the UCNPs@SiO2-NH2 precipitate prepared in the above steps was dispersed in 5 mL of DMF solution. 750 mg of succinic anhydride was weighed and transferred to 10 mL of DMF and sonicated to completely dissolve it. The anhydride was then slowly dripped into the DMF solution containing UCNPs@SiO2-NH2. After reacting at room temperature for one day, the solution was centrifuged and washed three times with anhydrous ethanol (10,000 rpm, 10 min) to obtain carboxyl-functionalized upconversion nanoparticles UCNPs@SiO2-COOH. This step allows the carboxyl-functionalized upconversion nanoparticles to undergo a ring-opening reaction, generating covalent amide bonds and terminal carboxylic acid groups on the surface of the UCNPs. The final product was dispersed in deionized water for subsequent use.

[0118] (III) Preparation of aptamer-modified upconversion nanoparticles

[0119] IFirst, the carboxyl groups of UCNPs@SiO2-COOH were activated. 1.2 mg of the carboxyl-functionalized upconversion nanoparticles prepared in step (ii) were uniformly dispersed in 2 mL of 5 mg / mL MES (pH = 6, 10 mM) buffer solution. 20 μL of 5 mg / mL EDC solution and 5 μL of NHS solution were added to the MES solution of the above carboxyl-functionalized upconversion nanoparticles.

[0120] II UCNPs@SiO2-COOH nanoparticles were incubated in an EDC / NHS solution with slow shaking for 2 h at room temperature. After activation, the solution was centrifuged at 10,000 rpm for 10 min to obtain a precipitate. The resulting precipitate was then dissolved in 2 mL of 10 mM HEPES buffer (pH 7.4), injected with MC-LR aptamer (40 μL, 10 μM), and reacted at 25°C for 10 h. Finally, the solution was centrifuged at 6,000 rpm for 5 min using a 30,000 MW ultrafiltration tube to remove unreacted aptamer, EDC, and NHS. After removing the lower phase, the solution was washed three times with ultrapure water. The final product (nanoparticles: UCNPs-aptamer) was dissolved in 1.2 mL of ultrapure water and stored in a refrigerator.

[0121] (IV) Preparation of MOF materials

[0122] First, 100 mg of tetrakis(4-carboxyphenyl)porphine, 250 mg of zirconyl chloride octahydrate, and 2.7 g of benzoic acid were completely dissolved in 80 mL of DMF. The mixture was then poured into a 250 mL round-bottom flask and stirred under reflux in an 80°C oil bath for 4 hours. After the reaction, the solution was evenly dispersed into a centrifuge tube and centrifuged at 10,000 rpm for 10 minutes to obtain a precipitate. The precipitate was then washed three times with DMF to obtain PCN-224 nanoparticles (MOF material).

[0123] (V) Preparation of nanocomposite probes

[0124] The UCNPs-aptamer (120 μL, 1 mg / mL) prepared in step (3) and the MOF material (28.8 μL, 2 mg / mL) prepared in step (4) were added to a 2 mL centrifuge tube, followed by the addition of 100 μL of sodium monohydrogen phosphate-potassium dihydrogen phosphate buffer solution (pH 7.6).

[0125] Oscillating reaction at 20°C 10 min To obtain UCNPs-aptamer-MOFs nanocomposite probe Z2.

[0126] Examples 1-3

[0127] The process of preparing the nanocomposite probe is as follows:

[0128] (1) Synthesis of NaGdF4:Yb,Er@NaYF4:Yb,Tm@NaYF4 (upconversion nanoparticles):

[0129] I first synthesized the core NaGdF4:Yb,Er: 0.99 mL of 0.2M ytterbium acetate hydrate, 0.99 mL of 0.2M gadolinium acetate hydrate, 20 μL of 0.2M erbium acetate hydrate, 4 mL of oleic acid, and 6 mL of 1-octadecene solution were added to a 50 mL round-bottom, double-necked flask at room temperature. The system temperature was slowly raised to 150°C in an oil bath while stirring continuously. After 40 minutes, the system was cooled to 50°C. Next, 4.3 mL of a thoroughly mixed precipitant (a mixture of 1M NaOH-CH3OH and 0.4M NH4F-CH3OH in a volume ratio of 1:3.3) was quickly added to the round-bottom flask. Stirring was continued and the temperature was maintained at 50°C for 30 minutes. Afterwards, the temperature was raised to 100°C and the flask was connected to a double-row tube. Under an argon flowing atmosphere, it was connected to liquid nitrogen for degassing to remove impurities such as residual water, oxygen, and methanol in the system, thereby achieving an oxygen-free state. Subsequently, the system temperature was raised to 305°C under an argon flowing atmosphere and maintained at this temperature for 1.2 hours. Finally, the resulting product was evenly distributed into a centrifuge tube, 1 mL of cyclohexane solution was added as a solvent, and 1 mL of ethanol was added to precipitate the upconversion nanoparticles. The centrifuge tube was placed in a centrifuge and centrifuged at 8000 rpm / min for 4 minutes. This was repeated three times to obtain the core NaYF4:Yb,Er, which was stored in a cyclohexane solution.

[0130] II. Then synthesize the intermediate layer NaGdF4:Yb,Er@NaYF4:Yb,Tm: prepare it according to the method used in step I, except that 20 μL of erbium acetate hydrate in step I is adjusted to 2.4 μL of thulium acetate hydrate; and adjust "quickly add 4.3 mL of thoroughly mixed precipitant to the round-bottom flask" in step I to "quickly add 2 mL of the core NaYF4:Yb,Er prepared in step I and stored in cyclohexane and 4.3 mL of thoroughly mixed precipitant to the round-bottom flask" to obtain the intermediate layer NaGdF4:Yb,Er@NaYF4:Yb,Tm, and store it in cyclohexane solution.

[0131] III The last product synthesized is core-shell NaGdF4:Yb,Er@NaYF4:Yb,Tm@NaYF4: it was prepared according to the method used in step I, except that the "0.2M ytterbium acetate hydrate 0.99mL, 0.2M gadolinium acetate hydrate 0.99mL, 0.2M erbium acetate hydrate 20μL" in step I was adjusted to "0.2M yttrium acetate hydrate 2mL"; the "quickly add 4.3mL of thoroughly mixed precipitant into the round-bottom flask" in step I was adjusted to "quickly add 2mL of core-shell NaGdF4:Yb,Er@NaYF4:Yb,Tm and 4.3mL of thoroughly mixed precipitant stored in cyclohexane into the round-bottom flask", thereby obtaining core-shell NaGdF4:Yb,Er@NaYF4:Yb,Tm@NaYF4 upconversion luminescent nanoparticles (UCNPs).

[0132] (II) Preparation of carboxyl-modified upconversion nanoparticles

[0133] I used the reverse microemulsion method to silanize the surface of upconversion nanoparticles. 40 mg of UCNPs prepared in step (1) was dispersed in 33 mL of cyclohexane solution to form a mixed solution I. Then, 1.6 g of Lgepal CO-520 was weighed in a 10 mL centrifuge tube. The mixed solution I was added in small amounts several times and ultrasonically treated to completely dissolve it and transferred to a round-bottom flask. Then, in a fume hood, 270 μL of ammonia water was evenly added dropwise to the above solution. Finally, 20 μL of TEOS solution was added dropwise to the solution and stirred vigorously. After reacting at room temperature for one day, a transparent and stable microemulsion system was formed. The alkyl-functionalized upconversion nanoparticles UCNPs@SiO2 were obtained by centrifugal washing (10,000 rpm, 10 min) three times with anhydrous ethanol.

[0134] Next, the surface of the upconversion nanoparticles was amino-functionalized. 31 mg of the UCNPs@SiO2 precipitate prepared in the previous step was redispersed in 25 mL of anhydrous ethanol. 0.8 mL of APTES was added and stirred thoroughly. The mixture was allowed to react at room temperature for 2 hours. After the reaction, the particles were centrifuged and washed three times with anhydrous ethanol (10,000 rpm for 10 minutes) to obtain amino-functionalized upconversion nanoparticles, UCNPs@SiO2-NH2. This step allowed the -NH2 groups to attach to the SiO2 surface through hydrolysis of the APTES.

[0135] Finally, the surface of the upconversion nanoparticles was functionalized with carboxyl groups. 25 mg of the UCNPs@SiO2-NH2 precipitate prepared in the above steps was dispersed in 5 mL of DMF solution. 750 mg of succinic anhydride was transferred to 10 mL of DMF and sonicated to completely dissolve it. The anhydride was then slowly added dropwise to the DMF solution containing UCNPs@SiO2-NH2. After reacting at room temperature for one day, the solution was centrifuged and washed three times with anhydrous ethanol (10,000 rpm, 10 min) to obtain carboxyl-functionalized upconversion nanoparticles UCNPs@SiO2-COOH. This step allows the carboxyl-functionalized upconversion nanoparticles to undergo a ring-opening reaction, generating covalent amide bonds and terminal carboxylic acid groups on the surface of the UCNPs. The final product was dispersed in deionized water for subsequent use.

[0136] (III) Preparation of aptamer-modified upconversion nanoparticles

[0137] IFirst, the carboxyl groups of UCNPs@SiO2-COOH were activated. 1.2 mg of the carboxyl-functionalized upconversion nanoparticles prepared in step (ii) were uniformly dispersed in 2 mL of 5 mg / mL MES (pH = 6, 10 mM) buffer solution. 20 μL of 5 mg / mL EDC solution and 5 μL of NHS solution were added to the MES solution of the above carboxyl-functionalized upconversion nanoparticles.

[0138] II UCNPs@SiO2-COOH nanoparticles were incubated in an EDC / NHS solution with slow shaking for 2 hours at room temperature. After activation, the solution was centrifuged at 10,000 rpm for 10 minutes to obtain a precipitate. The resulting precipitate was then dissolved in 2 mL of 10 mM HEPES buffer (pH 7.4), injected with MC-LR aptamer (40 μL, 10 μM), and reacted at 35°C for 14 hours. Finally, the solution was centrifuged at 6,000 rpm for 5 minutes using a 30,000 MW ultrafiltration tube to remove unreacted aptamer, EDC, and NHS. After removing the lower phase, the solution was washed three times with ultrapure water. The final product (nanoparticles: UCNPs-aptamer) was dissolved in 1.2 mL of ultrapure water and stored in a refrigerator.

[0139] (IV) Preparation of MOF materials

[0140] First, 100 mg of tetrakis(4-carboxyphenyl)porphine, 350 mg of zirconyl chloride octahydrate, and 2.9 g of benzoic acid were completely dissolved in 120 mL of DMF. The mixture was then poured into a 250 mL round-bottom flask and stirred under reflux in an oil bath at 100°C for 6 hours. After the reaction, the solution was evenly dispersed into a centrifuge tube and centrifuged at 10,000 rpm for 10 minutes to obtain a precipitate. The precipitate was then washed three times with DMF to obtain PCN-224 nanoparticles (MOF material).

[0141] (V) Preparation of nanocomposite probes

[0142] The UCNPs-aptamer (120 μL, 1 mg / mL) prepared in step (3) and the MOF material (36 μL, 2 mg / mL) prepared in step (4) were added to a 2 mL centrifuge tube, followed by the addition of 100 μL of sodium monohydrogen phosphate-potassium dihydrogen phosphate buffer solution (pH 7.6) and the reaction was shaken at 30°C for 20 min to obtain the UCNPs-aptamer-MOFs nanocomposite probe Z3.

[0143] Examples 1-4

[0144] (1) Synthesis of NaGdF4:Yb,Er@NaYF4:Yb,Tm@NaYF4 (upconversion nanoparticles):

[0145] I first synthesized the core NaGdF4:Yb,Er: 0.99 mL of 0.2M ytterbium acetate hydrate, 0.99 mL of 0.2M gadolinium acetate hydrate, 20 μL of 0.2M erbium acetate hydrate, 4 mL of oleic acid, and 6 mL of 1-octadecene solution were added to a 50 mL round-bottom, double-necked flask at room temperature. The system temperature was slowly raised to 150°C in an oil bath while stirring continuously. After 40 minutes, the system was cooled to 50°C. Next, 4.3 mL of a thoroughly mixed precipitant (a mixture of 1M NaOH-CH3OH and 0.4M NH4F-CH3OH in a volume ratio of 1:3.3) was quickly added to the round-bottom flask. Stirring was continued and the temperature was maintained at 50°C for 30 minutes. Afterwards, the temperature was raised to 100°C and the flask was connected to a double-row tube. Under an argon flowing atmosphere, it was connected to liquid nitrogen for degassing to remove impurities such as residual water, oxygen, and methanol in the system, thereby achieving an oxygen-free state. Subsequently, the system temperature was raised to 290°C under an argon flowing atmosphere and maintained at this temperature for 0.8 h. Finally, the resulting product was evenly distributed into a centrifuge tube, 1 mL of cyclohexane solution was added as a solvent, and 1 mL of ethanol was added to precipitate the upconversion nanoparticles. The centrifuge tube was placed in a centrifuge and centrifuged at 8000 rpm / min for 4 min. This was repeated three times to obtain the core NaYF4:Yb,Er, which was stored in a cyclohexane solution.

[0146] II. Then synthesize the intermediate layer NaGdF4:Yb,Er@NaYF4:Yb,Tm: prepare it according to the method used in step I, except that 20 μL of erbium acetate hydrate in step I is adjusted to 1 μL of thulium acetate hydrate; and adjust "quickly add 4.3 mL of thoroughly mixed precipitant to the round-bottom flask" in step I to "quickly add 2 mL of the core NaYF4:Yb,Er prepared in step I and stored in cyclohexane and 4.3 mL of thoroughly mixed precipitant to the round-bottom flask" to obtain the intermediate layer NaGdF4:Yb,Er@NaYF4:Yb,Tm, and store it in cyclohexane solution.

[0147] III The last product synthesized is core-shell NaGdF4:Yb,Er@NaYF4:Yb,Tm@NaYF4: it was prepared according to the method used in step I, except that the "0.2M ytterbium acetate hydrate 0.99mL, 0.2M gadolinium acetate hydrate 0.99mL, 0.2M erbium acetate hydrate 20μL" in step I was adjusted to "0.2M yttrium acetate hydrate 2mL"; the "quickly add 4.3mL of thoroughly mixed precipitant into the round-bottom flask" in step I was adjusted to "quickly add 2mL of core-shell NaGdF4:Yb,Er@NaYF4:Yb,Tm and 4.3mL of thoroughly mixed precipitant stored in cyclohexane into the round-bottom flask", thereby obtaining core-shell NaGdF4:Yb,Er@NaYF4:Yb,Tm@NaYF4 upconversion luminescent nanoparticles (UCNPs).

[0148] (II) Preparation of carboxyl-modified upconversion nanoparticles

[0149] I used the reverse microemulsion method to silanize the surface of upconversion nanoparticles. 40 mg of UCNPs prepared in step (1) was dispersed in 33 mL of cyclohexane solution to form a mixed solution I. Then, 1.6 g of Lgepal CO-520 was weighed in a 10 mL centrifuge tube. The mixed solution I was added in small amounts several times and ultrasonically treated to completely dissolve it and transferred to a round-bottom flask. Then, in a fume hood, 270 μL of ammonia water was evenly added dropwise to the above solution. Finally, 20 μL of TEOS solution was added dropwise to the solution and stirred vigorously. After reacting at room temperature for one day, a transparent and stable microemulsion system was formed. The alkyl-functionalized upconversion nanoparticles UCNPs@SiO2 were obtained by centrifugal washing (10,000 rpm, 10 min) three times with anhydrous ethanol.

[0150] Next, the surface of the upconversion nanoparticles was amino-functionalized. 31 mg of the UCNPs@SiO2 precipitate prepared in the previous step was redispersed in 25 mL of anhydrous ethanol. 0.8 mL of APTES was added and stirred thoroughly. The mixture was allowed to react at room temperature for 2 hours. After the reaction, the particles were centrifuged and washed three times with anhydrous ethanol (10,000 rpm for 10 minutes) to obtain amino-functionalized upconversion nanoparticles, UCNPs@SiO2-NH2. This step allowed the -NH2 groups to attach to the SiO2 surface through hydrolysis of the APTES.

[0151] Finally, the surface of the upconversion nanoparticles was functionalized with carboxyl groups. 25 mg of the UCNPs@SiO2-NH2 precipitate prepared in the above steps was dispersed in 5 mL of DMF solution. 750 mg of succinic anhydride was transferred to 10 mL of DMF and sonicated to completely dissolve it. The anhydride was then slowly added dropwise to the DMF solution containing UCNPs@SiO2-NH2. After reacting at room temperature for one day, the solution was centrifuged and washed three times with anhydrous ethanol (10,000 rpm, 10 min) to obtain carboxyl-functionalized upconversion nanoparticles UCNPs@SiO2-COOH. This step allows the carboxyl-functionalized upconversion nanoparticles to undergo a ring-opening reaction, generating covalent amide bonds and terminal carboxylic acid groups on the surface of the UCNPs. The final product was dispersed in deionized water for subsequent use.

[0152] (III) Preparation of aptamer-modified upconversion nanoparticles

[0153] IFirst, the carboxyl groups of UCNPs@SiO2-COOH were activated. 1.2 mg of the carboxyl-functionalized upconversion nanoparticles prepared in step (ii) were uniformly dispersed in 2 mL of 5 mg / mL MES (pH = 6, 10 mM) buffer solution. 20 μL of 5 mg / mL EDC solution and 5 μL of NHS solution were added to the MES solution of the above carboxyl-functionalized upconversion nanoparticles.

[0154] II UCNPs@SiO2-COOH nanoparticles were incubated in an EDC / NHS solution with slow shaking for 2 h at room temperature. After activation, the solution was centrifuged at 10,000 rpm for 10 min to obtain a precipitate. The resulting precipitate was then dissolved in 2 mL of 10 mM HEPES buffer (pH 7.4), injected with MC-LR aptamer (40 μL, 10 μM), and reacted at 25°C for 10 h. Finally, the solution was centrifuged at 6,000 rpm for 5 min using a 30,000 MW ultrafiltration tube to remove unreacted aptamer, EDC, and NHS. After removing the lower phase, the solution was washed three times with ultrapure water. The final product (nanoparticles: UCNPs-aptamer) was dissolved in 1.2 mL of ultrapure water and stored in a refrigerator.

[0155] (IV) Preparation of MOF materials

[0156] First, 100 mg of tetrakis(4-carboxyphenyl)porphine, 200 mg of zirconyl chloride octahydrate, and 2.6 g of benzoic acid were completely dissolved in 70 mL of DMF. The mixture was then poured into a 250 mL round-bottom flask and stirred under reflux in an 80°C oil bath for 4 hours. After the reaction, the solution was evenly dispersed into a centrifuge tube and centrifuged at 10,000 rpm for 10 minutes to obtain a precipitate. The precipitate was then washed three times with DMF to obtain PCN-224 nanoparticles (MOF material).

[0157] (V) Preparation of nanocomposite probes

[0158] The UCNPs-aptamer (120 μL, 1 mg / mL) prepared in step (3) and the MOF material (27 μL, 2 mg / mL) prepared in step (4) were added to a 2 mL centrifuge tube, followed by the addition of 100 μL of sodium monohydrogen phosphate-potassium dihydrogen phosphate buffer solution (pH 7.6) and the reaction was shaken at 25 °C for 15 min to obtain the UCNPs-aptamer-MOFs nanocomposite probe Z4.

[0159] Examples 1-5

[0160] The process of preparing the nanocomposite probe is as follows:

[0161] (1) Synthesis of NaGdF4:Yb,Er@NaYF4:Yb,Tm@NaYF4 (upconversion nanoparticles):

[0162] I first synthesized the core NaGdF4:Yb,Er UCNPs: To a 50-mL round-bottom, two-necked flask at room temperature were added 0.99 mL of 0.2 M ytterbium acetate hydrate, 0.99 mL of 0.2 M gadolinium acetate hydrate, 20 μL of 0.2 M erbium acetate hydrate, 4 mL of oleic acid, and 6 mL of 1-octadecene solution. The system temperature was slowly raised to 150°C in an oil bath with continuous stirring. After 40 minutes, the system was cooled to 50°C. Next, 4.3 mL of a thoroughly mixed precipitant (a mixture of 1 M NaOH-CH3OH and 0.4 M NH4F-CH3OH in a volume ratio of 1:3.3) was quickly added to the round-bottom flask, and the mixture was maintained at 50°C with continued stirring for 30 minutes. Afterwards, the temperature was raised to 100°C and the flask was connected to a double-row tube. Liquid nitrogen was then connected under an argon flow atmosphere for degassing to remove impurities such as residual water, oxygen, and methanol in the system, thereby achieving an oxygen-free state. Subsequently, the system temperature was raised to 310°C under an argon flow atmosphere and maintained at this temperature for 1.5 hours. Finally, the resulting product was evenly distributed into centrifuge tubes, 1 mL of cyclohexane solution was added as a solvent, and 1 mL of ethanol was added to precipitate the upconversion nanoparticles. The centrifuge tubes were placed in a centrifuge and centrifuged at 8000 rpm / min for 4 minutes. This was repeated three times to obtain the core NaYF4:Yb,Er, which was then stored in a cyclohexane solution.

[0163] II Then synthesize the intermediate layer NaGdF4:Yb,Er@NaYF4:Yb,Tm UCNPs: prepare them according to the method used in step I, except that 20 μL of erbium acetate hydrate in step I is adjusted to 3 μL of thulium acetate hydrate; and the "quickly add 4.3 mL of thoroughly mixed precipitant into the round-bottom flask" in step I is adjusted to "quickly add 2 mL of the core NaYF4:Yb,Er prepared in step I and stored in cyclohexane and 4.3 mL of thoroughly mixed precipitant into the round-bottom flask" to obtain the intermediate layer NaGdF4:Yb,Er@NaYF4:Yb,Tm, and store it in cyclohexane solution.

[0164] III The last product synthesized is core-shell NaGdF4:Yb,Er@NaYF4:Yb,Tm@NaYF4: it was prepared according to the method used in step I, except that the "0.2M ytterbium acetate hydrate 0.99mL, 0.2M gadolinium acetate hydrate 0.99mL, 0.2M erbium acetate hydrate 20μL" in step I was adjusted to "0.2M yttrium acetate hydrate 2mL"; the "quickly add 4.3mL of thoroughly mixed precipitant into the round-bottom flask" in step I was adjusted to "quickly add 2mL of core-shell NaGdF4:Yb,Er@NaYF4:Yb,Tm and 4.3mL of thoroughly mixed precipitant stored in cyclohexane into the round-bottom flask", thereby obtaining core-shell NaGdF4:Yb,Er@NaYF4:Yb,Tm@NaYF4 upconversion luminescent nanoparticles (UCNPs).

[0165] (II) Preparation of carboxyl-modified upconversion nanoparticles

[0166] I adopts the reverse microemulsion method to carry out the surface silanization of up-conversion nanoparticles. Take 40mg of UCNPs prepared in step (1) and disperse in 33mL of cyclohexane solution to form mixed solution I. Then weigh 1.6g of Lgepal CO-520 in a 10mL centrifuge tube, add mixed solution I repeatedly in small amounts and ultrasonicate it to dissolve it completely and transfer it to a round-bottom flask. Then, operate inside a fume hood and take 270L of ammonia water and evenly add it dropwise into the above-mentioned solution. Finally, 20L of TEOS solution is dropwise added to the solution and stirred vigorously. After reacting for one day at room temperature, a transparent and stable microemulsion system is formed. With anhydrous ethanol centrifugal washing (10000rpm, 10min) three times, obtain alkyl-functionalized up-conversion nanoparticles UCNPs@SiO2.

[0167] Next, the surface of the upconversion nanoparticles was amino-functionalized. 31 mg of the UCNPs@SiO2 precipitate prepared in the previous step was redispersed in 25 mL of anhydrous ethanol. 0.8 mL of APTES was added and stirred thoroughly. The mixture was allowed to react at room temperature for 2 hours. After the reaction, the particles were centrifuged and washed three times with anhydrous ethanol (10,000 rpm for 10 minutes) to obtain amino-functionalized upconversion nanoparticles, UCNPs@SiO2-NH2. This step allowed the -NH2 groups to attach to the SiO2 surface through hydrolysis of APTES.

[0168] Finally, the surface of the upconversion nanoparticles was functionalized with carboxyl groups. 25 mg of the UCNPs@SiO2-NH2 precipitate prepared in the above steps was dispersed in 5 mL of DMF solution. 750 mg of succinic anhydride was transferred to 10 mL of DMF and sonicated to completely dissolve it. The anhydride was then slowly added dropwise to the DMF solution containing UCNPs@SiO2-NH2. After reacting at room temperature for one day, the solution was centrifuged and washed three times with anhydrous ethanol (10,000 rpm, 10 min) to obtain carboxyl-functionalized upconversion nanoparticles UCNPs@SiO2-COOH. This step allows the carboxyl-functionalized upconversion nanoparticles to undergo a ring-opening reaction, generating covalent amide bonds and terminal carboxylic acid groups on the surface of the UCNPs. The final product was dispersed in deionized water for subsequent use.

[0169] (III) Preparation of aptamer-modified upconversion nanoparticles

[0170] IFirst, the carboxyl groups of UCNPs@SiO2-COOH were activated. 1.2 mg of the carboxyl-functionalized upconversion nanoparticles prepared in step (ii) were uniformly dispersed in 2 mL of 5 mg / mL MES (pH = 6, 10 mM) buffer solution. 20 μL of 5 mg / mL EDC solution and 5 μL of NHS solution were added to the MES solution of the above carboxyl-functionalized upconversion nanoparticles.

[0171] II UCNPs@SiO2-COOH nanoparticles were incubated in an EDC / NHS solution with slow shaking for 2 hours at room temperature. After activation, the solution was centrifuged at 10,000 rpm for 10 minutes to obtain a precipitate. The resulting precipitate was then dissolved in 2 mL of 10 mM HEPES buffer (pH 7.4), injected with MC-LR aptamer (40 μL, 10 μM), and reacted at 35°C for 14 hours. Finally, the solution was centrifuged at 6,000 rpm for 5 minutes using a 30,000 MW ultrafiltration tube to remove unreacted aptamer, EDC, and NHS. After removing the lower phase, the solution was washed three times with ultrapure water. The final product (nanoparticles: UCNPs-aptamer) was dissolved in 1.2 mL of ultrapure water and stored in a refrigerator.

[0172] (IV) Preparation of MOF materials

[0173] First, 100 mg of tetrakis(4-carboxyphenyl)porphine, 400 mg of zirconyl chloride octahydrate, and 3 g of benzoic acid were completely dissolved in 140 mL of DMF. The mixture was then poured into a 250 mL round-bottom flask and stirred under reflux in an oil bath at 100°C for 6 hours. After the reaction, the solution was evenly dispersed into a centrifuge tube and centrifuged at 10,000 rpm for 10 minutes to obtain a precipitate. The precipitate was then washed three times with DMF to obtain PCN-224 nanoparticles (MOF material).

[0174] (V) Preparation of nanocomposite probes

[0175] The UCNPs-aptamer (120 μL, 1 mg / mL) prepared in step (3) and the MOF material (42 μL, 2 mg / mL) prepared in step (4) were added to a 2 mL centrifuge tube, followed by the addition of 100 μL of sodium monohydrogen phosphate-potassium dihydrogen phosphate buffer solution (pH 7.6) and the reaction was shaken at 30°C for 20 min to obtain the UCNPs-aptamer-MOFs nanocomposite probe Z5.

[0176] Examples 1-6

[0177] The method of Example 1-1 was followed, except that the amount of thulium acetate hydrate in step (1) was adjusted to 0.8 μL to obtain UCNPs-aptamer-MOFs nanocomposite probe Z6.

[0178] Examples 1-7

[0179] The method of Example 1-1 was followed, except that the amount of thulium acetate hydrate in step (1) was adjusted to 3.5 μL to obtain UCNPs-aptamer-MOFs nanocomposite probe Z7.

[0180] Examples 1-8

[0181] The method of Example 1-1 was followed, except that the temperature of the system was adjusted to 250° C. under the argon flowing atmosphere in step (1), and the constant temperature time was adjusted to 0.4 h to obtain the UCNPs-aptamer-MOFs nanocomposite probe Z8.

[0182] Examples 1-9

[0183] The method of Example 1-1 was followed, except that the step (iii) of "reacting at 30°C for 12 h" was adjusted to "reacting at 20°C for 6 h" to obtain the UCNPs-aptamer-MOFs nanocomposite probe Z9.

[0184] Examples 1-10

[0185] The method of Example 1-1 was followed, except that the mass of zirconyl chloride octahydrate in step (iv) was adjusted to 150 mg to obtain UCNPs-aptamer-MOFs nanocomposite probe Z10.

[0186] Examples 1-11

[0187] The method of Example 1-1 was followed, except that the mass of benzoic acid in step (iv) was adjusted to 2.4 g, to obtain UCNPs-aptamer-MOFs nanocomposite probe Z11.

[0188] Examples 1-12

[0189] The method of Example 1-1 was followed, except that the temperature of the oil bath in step (iv) was adjusted to 70° C. and the stirring time was 3.5 h to obtain UCNPs-aptamer-MOFs nanocomposite probe Z12.

[0190] Examples 1-13

[0191] The method of Example 1-1 was followed, except that the amount of MOF material in step (V) was adjusted to 25 μL to obtain UCNPs-aptamer-MOFs nanocomposite probe Z13.

[0192] Examples 1-14

[0193] The method of Example 1-1 was followed, except that step (5) "oscillation reaction at 25°C for 15 min" was adjusted to "oscillation reaction at 15°C for 30 min" to obtain UCNPs-aptamer-MOFs nanocomposite probe Z14.

[0194] Examples 1-15

[0195] The method of Example 1-1 was followed, except that the nucleic acid aptamer in step (III) was adjusted to MC-LA (having the nucleotide sequence shown in SEQ ID NO. 2), purchased from Shanghai Bio-Technology Reagent Co., Ltd. UCNPs-aptamer-MOFs nanocomposite probe Z15 was obtained.

[0196] Comparative Example 1-1

[0197] The method of Example 1-1 was followed, except that the use of thulium acetate hydrate in step (1) was omitted to obtain the UCNPs-aptamer-MOFs nanocomposite probe ZA.

[0198] Comparative Example 1-2

[0199] The method of Example 1-1 was followed, except that the use of thulium acetate hydrate and erbium acetate hydrate in step (1) was omitted to obtain the UCNPs-aptamer-MOFs nanocomposite probe ZA.

[0200] Test Example 1

[0201] The core (NaYF4: Yb, Er), the intermediate layer (NaGdF 4: Yb,Er@NaYF4:Yb,Tm) and core-shell (NaGdF4:Yb,Er@NaYF 4: Yb, Tm@NaYF4) were used for morphological characterization, and the test results were as follows Figure 1 Figures A, B, and C show the characterization results for the core, intermediate layer, and core-shell structures, respectively. These core-shell upconversion nanoparticles exhibit consistent morphology. This uniform morphology helps ensure the stability and repeatability of the nanoparticles' performance in subsequent applications. For microcystin detection, this uniform morphology allows for more regular interactions between the nanoprobe and the toxin, improving detection accuracy and reliability. Figure 1 Figures D and E are transmission and scanning images of MOF. As can be seen from the figure, MOF materials are in the form of approximately round or elliptical particles with relatively uniform size and a scale of about 100 nm. MOF materials are also granular, three-dimensional, and regular in appearance. The particle size is within 1 μm. Smaller and more uniform particles can provide a larger specific surface area, which is conducive to combining with other substances such as UCNPs and aptamers to enhance the performance of nanoprobes; the regular appearance and uniform size help the material to be better dispersed in the solution, making the nanoprobe system more stable.

[0202] Test Example 2

[0203] The XRD spectra of the upconversion nanoparticles, carboxyl-modified upconversion nanoparticles, and nucleic acid aptamer-modified upconversion nanoparticles prepared in Example 1-1 were measured using a SmartLab 9kw X-ray diffractometer. Figure 2 As shown, Figure 2 It shows that the diffraction peak positions and intensities of the upconversion nanoparticles, carboxyl-modified upconversion nanoparticles, and nucleic acid aptamer-modified upconversion nanoparticles samples prepared in Example 1-1 are highly consistent with the characteristic peaks of the corresponding substances in the standard card, indicating that the synthesized core-shell (NaGdF4:Yb, Er@NaYF4:Yb, Tm@NaYF4) layers match the standard card well, that is, NaGdF4:Yb, Er@NaYF4:Yb, Tm@NaYF4 upconversion nanoparticles with a specific crystal structure are successfully synthesized.

[0204] Energy dispersive X-ray spectroscopy (EDS) was performed on the elements of NaGdF4:Yb,Er and NaGdF4:Yb,Er@NaYF4:Yb,Tm in Example 1-1 using a S-8100 scanning electron microscope. The results are as follows: Figure 3 As shown in Figures A, B, and C, the peak positions and intensities show that the characteristic peaks of the expected elements appear in the figure, which indicates that the synthesized nanoparticles contain these elements, further proving that the composition of the synthesized nanoparticles meets the design requirements.

[0205] The infrared spectra of upconversion nanoparticles in different modification states (unmodified upconversion nanoparticles a-UCNPs, silica-coated upconversion nanoparticles b-UCNPs@SiO, amino-modified upconversion nanoparticles c-UCNPs@SiO-NH, and carboxyl-modified upconversion nanoparticles d-UCNPs@SiO-COOH) prepared in Example 1-1) were displayed by Fourier transform infrared spectrometer. The results are as follows Figure 3 As shown in D, different absorption peaks correspond to different chemical bond vibrations. By comparison, it can be seen that as UCNPs are coated with silica and modified with amino and carboxyl groups, new characteristic absorption peaks appear in the infrared spectrum, indicating that the surface modification of the nanoparticles has been successful.

[0206] The surface potential of upconversion nanoparticles UCNPs and modified upconversion nanoparticles (prepared in Example 1-1) was measured using a ZS90 Zeta potential meter. Figure 3 As shown in E and F, Figure 3 Figure E shows the surface potentials of UCNPs, UCNPs@SiO, UCNPs@SiO-NH, and UCNPs@SiO-COOH. Different surface modifications lead to changes in the Zeta potential, such as UCNPs@SiO-COOH being negatively charged and UCNPs@SiO-NH being positively charged, which reflects the changes in the surface chemical properties and proves that the modification is successful. Figure 3 Figure F shows the surface potential of UCNPs-DNA, UCNPs-DNA-MOF and UCNPs-DNA-MOF-MC-LR (microcystin MC-LR was further connected on the basis of UCNPs-DNA-MOF). The change in potential shows that in the process of gradually constructing the nanoprobe, each step of assembly changes the surface properties of the nanoparticles, and it also indirectly proves that each component is successfully assembled on the nanoparticles. UCNPs-DNA-MOF is used as the basic nanoprobe component to identify specific substances; UCNPs-DNA-MOF-MC-LR may be used to detect microcystin, and sheep anti-mouse IgG enhances the specificity or signal output of the detection.

[0207] Based on the above analysis, from the characterization results of crystal structure, elemental composition, surface modification and surface potential, it can be concluded that Example 1-1 successfully prepared NaGdF4:Yb,Er@NaYF4:Yb,Tm@NaYF4 upconversion nanoparticles.

[0208] Test Example 3

[0209] The fluorescence intensity of the upconversion material (prepared in Example 1-1) was recorded using a Hitachi F-4600 fluorometer, and the UV-visible absorption spectrum was recorded using a Hitachi U-3900 spectrophotometer. The results are shown in FIG. Figure 4 As shown, Figure 4 is the spectral overlap between the absorption spectrum of MOF materials and the emission spectrum of upconversion nanoparticles. Figure 4 Curve a is the fluorescence curve of the upconversion nanomaterial, and curve b is the UV-visible absorption curve of the MOF material. The figure shows that the fluorescence and UV-visible absorption curves overlap significantly. This result demonstrates that the fluorescence intensity of the system can be quenched when the upconversion material and MOF material are present simultaneously.

[0210] Test Example 4

[0211] 50 μL of the nanocomposite probe Z1 prepared by the method of Example 1-1 was added with different concentrations of MC-LR standard solution (20, 40, 60, 80 nM), and then placed in a constant temperature shaker at 25°C for 15 minutes. The fluorescence was measured using a Hitachi F-4600 fluorometer and a working curve was plotted. The results are shown in Figure 2. Figure 5 In Figures A and B, there is a good linear relationship between the microcystin concentration and the fluorescence quenching intensity ΔI (ΔI=I-I0, I0 and I are the fluorescence intensity values of the system without and with microcystin, respectively).

[0212] Figure 5 Figure A shows fluorescence intensity spectra at different microcystin concentrations. The graph features multiple colored curves representing upconversion luminescence spectra measured using a Hitachi F-4600 fluorimeter at varying microcystin concentrations. The horizontal axis represents wavelength (nm), ranging from 500 to 900 nm, while the vertical axis represents fluorescence intensity. As the microcystin concentration increases, the fluorescence intensity at specific wavelengths (such as around 600 nm and 800 nm) gradually increases. This indicates that changes in microcystin concentration can cause changes in the fluorescence intensity of the system, a property that can be exploited for microcystin detection.

[0213] Figure 5Figure B shows a good linear relationship between microcystin concentration and fluorescence quenching intensity ΔI (ΔI = I - I0, where I0 and I are the fluorescence intensity values in the system without and with microcystin, respectively). This suggests that quantitative detection of microcystins can be achieved by monitoring changes in fluorescence intensity based on a linear relationship.

[0214] Test Example 5

[0215] 50 μL of the nanocomposite probe prepared by the method of Example 1-1 was added with 60 nM microcystin and interfering substances (MC-YR, MC-RR, MC-LA, Fe 2+ 、S 2- 、ONOO - 、H2O2、ClO - 、NO2 - 、S2O3 2- The sample was diluted to 1 mL with ultrapure water and then placed in a constant temperature shaker at 25°C for 15 min. The experimental data were recorded using a fluorescence spectrometer. Based on the obtained fluorescence intensity values, a histogram was drawn. 550 / 812 , the results are shown in Figure 6 It can be seen from the figure that various interferences have little effect on the system. It can be seen that the fluorescence intensity remains basically unchanged. The first bar graph is microcystin. It can be seen that the fluorescence intensity recovery effect is good, indicating that this fluorescent probe has good selectivity.

[0216] Example 2-1

[0217] Local lake water was taken as an actual sample. After filtering it three times with a 0.45 μm microporous membrane to remove impurities, 50 μL of Z1 prepared in Example 1-1 was added. Microcystin MC-LR was added thereto to make the concentration of MC-LR 60 nM to obtain a spiked sample. The sample was then placed in a constant temperature oscillator at 25°C and continuously shaken for 20 minutes. The principle diagram of the nanocomposite probe for detecting microcystin is shown in the figure. Figure 7 As shown, the obtained liquid is detection liquid 1.

[0218] Example 2-2

[0219] Local lake water was used as an actual sample. After filtering it three times with a 0.45 μm microporous membrane to remove impurities, 50 μL of Z2 prepared in Example 1-2 was added. Microcystin MC-LR was added thereto to a concentration of 10 nM to obtain a spiked sample. The sample was then placed in a constant temperature oscillator at 20°C and continuously shaken for 15 min. The resulting liquid was test liquid 2.

[0220] Example 2-3

[0221] Local lake water was taken as an actual sample. After filtering it three times with a 0.45 μm microporous membrane to remove impurities, 50 μL of Z3 prepared in Example 1-3 was added. Microcystin MC-LR was added thereto to give an MC-LR concentration of 70 nM to obtain a spiked sample. The sample was then placed in a constant temperature oscillator at 30°C and continuously shaken for 35 min. The resulting liquid was test liquid 3.

[0222] Examples 2-4

[0223] Local lake water was taken as an actual sample. After filtering it three times with a 0.45 μm microporous membrane to remove impurities, 50 μL of Z4 prepared in Example 1-4 was added. Microcystin MC-LR was added thereto to give an MC-LR concentration of 0.5 nM to obtain a spiked sample. The sample was then placed in a constant temperature oscillator at 20°C and continuously shaken for 15 min. The resulting liquid was test liquid 4.

[0224] Examples 2-5

[0225] Local lake water was taken as an actual sample. After filtering it three times with a 0.45 μm microporous filter membrane to remove impurities, 50 μL of Z5 prepared in Example 1-5 was added. Microcystin MC-LR was added thereto to give an MC-LR concentration of 80 nM to obtain a spiked sample. The sample was then placed in a constant temperature oscillator at 30°C and continuously shaken for 35 min. The resulting liquid was test liquid 5.

[0226] Examples 2-6 to 2-15 and Comparative Examples 2-1 and 2-2

[0227] The experiment was carried out according to the method of Example 2-1, except that Z1 prepared in Example 1-1 was replaced by Z6, Z7, Z8, Z9, Z10, Z11, Z12, Z13, Z14, Z15, ZA and ZB prepared in Examples 1-6 to 1-15 and Comparative Example 1-1 and Comparative Example 1-2, respectively, and the obtained liquids were detection liquid 6, detection liquid 7, detection liquid 8, detection liquid 9, detection liquid 10, detection liquid 11, detection liquid 12, detection liquid 13, detection liquid 14, detection liquid 15, detection liquid A and detection liquid B.

[0228] Measurement example 7

[0229] Fluorescence measurements were performed on the test liquids 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and 15 obtained in the examples and comparative examples, and on the test liquids A and B using a Hitachi F-4600 fluorometer. Figure 5The concentration of MC-LR was determined using the linear relationship diagram of B, and the relative deviation (%) between the measured concentration and the scalar concentration was calculated. The determination formula is shown in formula (I), and the test results are shown in Table 1;

[0230]

[0231] Table 1

[0232] serial number Spike amount (nM) Assay concentration (nM) Relative deviation (%) Detection liquid 1 60 60.01 0.0167 Detection liquid 2 10 10.02 0.02 Detection liquid 3 70 70.03 0.0429 Detection liquid 4 0.5 0.4996 0.08 Detection liquid 5 80 80.1 0.025 Detection liquid 6 60 60.06 0.1 Detection liquid 7 60 60.05 0.0833 Detection liquid 8 60 60.058 0.0967 Detection liquid 9 60 60.07 0.1167 Detection liquid 10 60 59.93 0.1167 Detection liquid 11 60 60.065 0.1083 Detection liquid 12 60 59.92 0.1333 Detection liquid 13 60 60.069 0.115 Detection liquid 14 60 60.08 0.1333 Detection liquid 15 60 60.092 0.1533 Detection liquid A 60 60.12 0.2 Detection liquid B 60 60.15 0.25

[0233] As can be seen from Table 1, the detection sensitivity of the nanocomposite probe prepared in the example is significantly higher than that of the nanocomposite probe prepared in the comparative example. Therefore, the detection effect of the nanocomposite probe prepared by the method provided by the present invention is better.

[0234] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, various simple variations of the technical solution of the present invention may be made, including combining the various technical features in any other appropriate manner. These simple variations and combinations should also be regarded as disclosed in the present invention and fall within the scope of protection of the present invention.

Claims

1. A nanocomposite probe, characterized in that: The nanocomposite probe includes an MOF material and nanoparticles loaded on the MOF material. The nanoparticles include carboxyl-modified upconversion nanoparticles and nucleic acid aptamers covalently linked to the upconversion nanoparticles via amide bonds. The upconversion nanoparticles contain thulium and erbium elements.

2. The nanocomposite probe according to claim 1, characterized in that The nucleic acid aptamer has a nucleotide sequence shown in SEQ ID NO.

1.

3. The nanocomposite probe according to claim 1 or 2, characterized in that: The preparation method of the MOF material comprises: mixing tetrakis (4-carboxyphenyl) porphine, zirconyl chloride octahydrate, benzoic acid and N, N-dimethylformamide solution for reaction, and then washing with N, N-dimethylformamide solution; Preferably, the mass ratio of tetrakis(4-carboxyphenyl)porphine, zirconyl chloride octahydrate, benzoic acid and N,N-dimethylformamide solution is 1:2-4:26-30:700-1400; Preferably, the mixing reaction conditions include: temperature of 80-100° C. and time of 4-6 h.

4. The nanocomposite probe according to any one of claims 1 to 3, characterized in that The method for preparing the nanoparticles comprises the following steps: S1. Performing a stepwise coprecipitation reaction of 1-octadecene, oleic acid, gadolinium acetate hydrate, erbium acetate hydrate, ytterbium acetate hydrate, yttrium acetate hydrate, thulium acetate hydrate, NH4F, and NaOH. Subsequently, raising the system temperature to 290-310°C under an argon flowing atmosphere and maintaining the temperature for 0.6-1.5 hours to obtain upconversion nanoparticles. S2, performing carboxyl modification on the upconversion nanoparticles described in step S1 to obtain carboxylated upconversion nanoparticles; S3, performing an amide reaction between the carboxyl-modified upconversion nanoparticles described in step S2 and the nucleic acid aptamer to obtain nanoparticles; Preferably, in step S1, the volume ratio of the hydrated erbium acetate to the hydrated thulium acetate is 1:0.05-0.15; Preferably, in step S2, the carboxylation modification step comprises: before the upconversion nanoparticles are carboxylated, mixing the upconversion nanoparticles with cyclohexane and octylphenol polyoxyethylene ether, and then performing a mixing reaction I with ammonia water and ethyl orthosilicate to obtain silica-coated upconversion nanoparticles; then performing a mixing reaction II with the silica-coated upconversion nanoparticles with ethanol and 3-aminopropyltriethoxysilane to obtain amino-modified silica-coated upconversion nanoparticles; and finally performing a mixing reaction III with the amino-modified silica-coated upconversion nanoparticles with N,N-dimethylformamide solution and a carboxylating agent to obtain carboxylated conversion nanoparticles. Preferably, the carboxylating agent is selected from at least one of succinic anhydride, succinic anhydride and glutaric anhydride; more preferably succinic anhydride; Preferably, in step S3, the amide reaction process comprises: activating the carboxyl-modified upconversion nanoparticles described in step S2 and then mixing the activated carboxyl-modified upconversion nanoparticles with a HEPES buffer solution and a nucleic acid aptamer for reaction; Preferably, the mixing reaction conditions include: time of 10-14 hours and temperature of 25-35°C.

5. A method for preparing a nanocomposite probe, characterized in that: The method comprises the following steps: preparing nanoparticles; and loading the nanoparticles on a MOF material; The nanoparticles include carboxyl-modified upconversion nanoparticles and nucleic acid aptamers covalently linked to the upconversion nanoparticles via amide bonds, and the upconversion nanoparticles contain thulium and erbium elements.

6. The method according to claim 5, characterized in that The MOF material is PCN-224 nanoparticles, and the preparation method of the PCN-224 nanoparticles comprises: mixing tetrakis (4-carboxyphenyl) porphine, zirconyl chloride octahydrate, benzoic acid and N, N-dimethylformamide solution for reaction, and then washing with N, N-dimethylformamide solution; Preferably, the mixing reaction conditions include: temperature of 80-100°C and time of 4-6h; Preferably, the method for preparing the nanoparticles comprises the following steps: S1. Performing a stepwise coprecipitation reaction of 1-octadecene, oleic acid, gadolinium acetate hydrate, erbium acetate hydrate, ytterbium acetate hydrate, yttrium acetate hydrate, thulium acetate hydrate, NH4F, and NaOH. Subsequently, raising the system temperature to 290-310°C under an argon flowing atmosphere and maintaining the temperature for 0.6-1.5 hours to obtain upconversion nanoparticles. S2, performing carboxyl modification on the upconversion nanoparticles described in step S1 to obtain carboxylated upconversion nanoparticles; S3, performing an amide reaction between the carboxyl-modified upconversion nanoparticles described in step S2 and the nucleic acid aptamer to obtain nanoparticles; Preferably, in step S1, the volume ratio of the hydrated erbium acetate to the hydrated thulium acetate is 1:0.05-0.15; Preferably, in step S2, the carboxylation modification step comprises: before the upconversion nanoparticles are carboxyl-modified, first mixing the upconversion nanoparticles with cyclohexane and octylphenol polyoxyethylene ether, and then performing a mixing reaction I with ammonia water and ethyl orthosilicate to obtain silica-coated upconversion nanoparticles, then performing a mixing reaction II with the silica-coated upconversion nanoparticles with ethanol and 3-aminopropyltriethoxysilane to obtain amino-modified silica-coated upconversion nanoparticles, and then performing a mixing reaction II with the amino-modified silica-coated upconversion nanoparticles with N,N-dimethylformamide solution and a carboxylating agent to obtain a supernatant as the carboxyl-modified upconversion nanoparticles; Preferably, the carboxylating agent comprises succinic anhydride, succinic anhydride and glutaric anhydride; More preferably succinic anhydride; Preferably, in step S3, the amide reaction process comprises: first activating the carboxyl-modified upconversion nanoparticles described in step S2 and then mixing the activated carboxyl-modified upconversion nanoparticles with a HEPES buffer solution and a nucleic acid aptamer for reaction; Preferably, the mixing reaction time is 10-14h and the temperature is 25-35°C; Preferably, the nucleic acid aptamer is a nucleic acid aptamer having the nucleotide sequence described in SEQ ID NO.

1.

7. The method according to claim 5 or 6, characterized in that The loading method comprises: mixing and incubating the nanoparticles, the MOF material and the buffer solution; Preferably, the buffer solution is selected from any one of sodium monohydrogen phosphate-potassium dihydrogen phosphate buffer solution, potassium dihydrogen phosphate buffer solution and sodium hydrogen phosphate buffer solution; Preferably, the buffer solvent is a sodium monohydrogen phosphate-potassium dihydrogen phosphate buffer solution; Preferably, the incubation conditions include: a temperature of 20-30° C. and a time of 10-20 min.

8. The method according to any one of claims 5 to 7, characterized in that: The mass ratio of the nanoparticles to the MOF material is 1:0.45-0.

7.

9. Use of the nanocomposite probe according to any one of claims 1 to 4 or the nanocomposite probe prepared by the method according to any one of claims 5 to 8 in detecting microcystins in liquids.

10. A method for detecting microcystins in a liquid, characterized in that: The method comprises: contacting a liquid sample containing microcystin with the nanofluorescent probe according to any one of claims 1 to 4 and / or the nanofluorescent probe prepared by the method according to any one of claims 5 to 8; Preferably, the concentration of microcystin in the sample is 0.5-80 nM; Preferably, the contact conditions include: temperature of 20-30° C. and time of 15-35 min.

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