Preparation method of nano fluorescent probe for detecting heavy metal ions in liquid sample

Abstract

The invention relates to a preparation method of a nano fluorescent probe for detecting heavy metal ions in a liquid sample, and belongs to the field of chemical detection. The preparation method is a chemical synthesis method and comprises the following steps: firstly, carrying out dehydration condensation on a tetradentate amine monomer (TBDT) and a linear dialdehyde monomer (BCBA) under the action of a catalyst, then carrying out ultrasonic treatment, degassing, vacuum sealing, high-temperature reaction and other series operations to prepare a covalent organic framework (TB-BA COF) material taking a solid terbium-benzoic acid complex as a construction unit, and finally, washing and extracting to obtain the covalent organic framework material. The purified nano fluorescent probe of the covalent organic framework (TB-BA COF) material with the artificial antibody terbium-benzoic acid complex as a construction unit for selectively recognizing and detecting the heavy metal ions is obtained. The nano shell layer on the surface of the nanosphere not only increases the specific surface area of the polymer, but also enables all recognition sites to be located on the surface of the nano shell layer, so that the number, binding quantity and selectivity of the recognition sites are increased.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of chemical detection, and particularly relates to a rapid detection method for heavy metal pollutants in food and cosmetics. BACKGROUND

[0002] With the rapid development of modern industry, the problem of heavy metal pollution is becoming increasingly serious. Heavy metal ions (such as lead (Pb 2+ ), mercury (Hg 2+ ), cadmium (Cd 2+ ), arsenic (As 3+ / 5 + ) and the like) are discharged into water bodies and the environment through mining, metallurgy, electroplating, chemical industry and other industries, and can be enriched in organisms through the food chain, causing serious threats to human health, such as carcinogenicity, teratogenicity, damage to the nervous system and the kidneys, etc. Therefore, the development of rapid, sensitive and selective heavy metal ion detection technology has great practical significance for environmental monitoring, food safety and disease prevention. At present, the methods for detecting heavy metal ions in liquid samples mainly include the following: 1. Atomic absorption spectrometry (AAS) and inductively coupled plasma mass spectrometry (ICP-MS); this method is the gold standard for laboratory detection of heavy metals, and has the advantages of high sensitivity and good accuracy, but the limitations are: the instrument equipment is expensive and bulky, requires a complex sample pretreatment process, and must be operated by professional personnel in a laboratory environment, which is difficult to use for on-site rapid detection, real-time monitoring and large-scale screening, and the application scene is greatly limited. 2. Electrochemical analysis method; this method (such as anodic stripping voltammetry) has relatively simple equipment and high sensitivity. However, its disadvantages are that the electrode surface is easily contaminated, frequent pretreatment and regeneration steps are required, and the reproducibility is sometimes poor. The detection process is also relatively cumbersome, and the operator has certain technical requirements. 3. Traditional colorimetric method and fluorescent probe method; this method is simple to operate and is expected to achieve rapid detection. However, traditional small organic molecule fluorescent dyes often have weak fluorescence intensity, poor light stability (easy to photobleach), small Stokes shift and other shortcomings. More importantly, they often have difficulty in balancing selectivity and sensitivity for heavy metal ions, and are easily disturbed by other coexisting ions in the sample, resulting in unreliable detection results.

[0003] In recent years, certain progress has been made in the rapid detection of trace substances using biological immunosensors. Immunosensors use antibodies to reversibly bind to their antigens with high specificity, enriching the antigen target in a complex matrix to determine the concentration of the target. Immunosensors have high specificity, sensitivity and stability.

[0004] Currently, artificial synthesis of nanoprobes has the following shortcomings: high cost of DNA template, difficult to control synthesis conditions, low selectivity and sensitivity to heavy metal elements, complicated preparation process, and mutual interference between ions, etc. Therefore, simplifying the process, improving performance, and reducing cost have become the focus of researchers. A DNA-silver nanocluster fluorescence probe for detecting mercury ions, a preparation method and application thereof discloses that gold nanoclusters are prepared by using small molecules such as glutathione (GSH) as ligands and reducing agents in one-pot method, and the affinity between Hg 2+ However, the preparation of nanoprobes still has the following problems: (1) insufficient selectivity of the probe: the prepared nanoprobes often respond to multiple metal ions, and lack high specific recognition ability to specific target heavy metal ions, which is mainly due to the poor selection or immobilization method of recognition elements (such as ligands, DNA aptamers); (2) stability problem: some nanoprobes are prone to aggregation or fluorescence quenching in complex actual sample matrix (such as river water, blood, urine), resulting in signal distortion and detection failure; (3) cost and environmental friendliness: expensive or toxic reagents may be used in the synthesis process, increasing the cost and causing environmental burden. SUMMARY

[0005] In order to simplify the preparation process, reduce the manufacturing cost, and at the same time, realize the preparation of nanoprobes with high sensitivity, high selectivity, and good stability, the present application provides a preparation method of a nanofluorescent probe for detecting heavy metal ions in a liquid sample.

[0006] The preparation operation of the nanofluorescent probe for detecting heavy metal ions in a liquid sample is as follows: First, add tetradentate amine monomers (TBDT), linear dialdehyde monomers (BCBA) and solvents to the ferric oxide nanometer powder under the action of a catalyst to perform dehydration condensation, the solvents are o-dichlorobenzene and n-butanol, and the catalyst is 1,8-diazabicyclo[5.4.0]undec-7-ene; Then, through a series of operations such as ultrasonic, degassing, vacuum sealing, and high-temperature reaction, a covalent organic framework material with terbium-benzoic acid complex as a building unit is prepared, Finally, through washing and extraction, a purified nanofluorescent probe with selective recognition and detection of heavy metal ions, i.e. a nanofluorescent probe for detecting heavy metal ions in a liquid sample, is obtained, which is a covalent organic framework with terbium-benzoic acid complex as a building unit; The nanofluorescent probe is a covalent organic framework crystal material with imine bond (-C=N-) as a basic connecting unit, which produces strong and stable fluorescence under a 510 nm wavelength light source, and the specific surface area is greater than 600 m 2 / g, the fluorescence quantum yield is greater than 15%, the fluorescence intensity decay rate is less than 5%, the detection limit is nM level, and it is stable in ethanol, acetone, dichloromethane organic reagents and high temperature conditions of 300 DEG C.

[0007] The specific preparation operation steps of the nano-fluorescent probe for detecting heavy metal ions in a liquid sample are as follows: (1) In a reaction container, the following substances are added: 10 mg of ferroferric oxide nano powder, 20-25 mg of tetradentate amine monomer (TBDT), 18-25 mg of linear dialdehyde monomer (BCBA), 0.5-2.0 mL of o-dichlorobenzene, 0.5-2.0 mL of n-butanol, and 0.1-0.5 mL of 1,8-diazabicyclo[5.4.0]undec-7-ene solution with a concentration of 4 M; ultrasonic treatment is performed to disperse uniformly, and a mixed solution with a nano-shell structure on the surface of the ferroferric oxide nano powder particles is obtained. Among them, o-dichlorobenzene and n-butanol are used as mixed solvents, and 1,8-diazabicyclo[5.4.0]undec-7-ene is used as a catalyst. The tetradentate amine monomer and the linear dialdehyde monomer form a nano-shell structure to wrap the ferroferric oxide nano powder particles. By changing the addition amount of the tetradentate amine monomer and the linear dialdehyde monomer, the thickness of the nano-shell on the surface of the ferroferric oxide nano powder particles can be adjusted.

[0008] (2) The mixed solution is subjected to degassing treatment under vacuum conditions, mechanical pump degassing, and thawing at room temperature. The degassing treatment is cycled more than twice, and the reaction container after the degassing treatment is sealed under vacuum conditions. The purpose is to remove oxygen in the solvent and promote the reaction to proceed in the direction of thermodynamic stability.

[0009] (3) The sealed reaction container is placed in an oven at 120-180 DEG C, and the reaction is carried out for 2-5 days to obtain a reaction product. During the reaction, the tetradentate amine monomer is activated by 1,8-diazabicyclo[5.4.0]undec-7-ene to form an amine anion, which reacts with the carbonyl carbon on the linear dialdehyde monomer to form an imine bond (-C=N-); then it is tightly combined with the ferroferric oxide nano powder magnetic core to give the covalent organic framework shell high selectivity.

[0010] (4) The reaction product is subjected to reduced pressure filtration to obtain a solid material, which is a covalent organic framework material with terbium-benzoic acid complex as a building block.

[0011] (5) The solid material is sequentially washed with acetone, ethanol, and dichloromethane (CH2Cl2) to obtain a washing product; the purpose is to wash away the impurities physically adsorbed on the outer surface and pores of the crystal.

[0012] (6) The washing product is subjected to Soxhlet extraction in dichloromethane solvent to obtain an extract.

[0013] (7) In a vacuum drying oven, the extract is vacuum dried to obtain a nanofluorescent probe of covalent organic framework of purified artificial antibody terbium-benzoic acid complex as a building unit, i.e. a nanofluorescent probe for detecting heavy metal ions in a liquid sample.

[0014] Further technical solutions are as follows: In step (1), the ultrasonic treatment time is 5-15 minutes.

[0015] In step (2), the freezing conditions are: the freezing medium is liquid nitrogen, the temperature is -20℃, and the vacuum degree is 10 -2 Torr; the pumping conditions are: the temperature is room temperature, and the vacuum pump speed is 500 L / s; the degassing treatment cycle number is 2-4 times.

[0016] In step (4), the filter membrane for the reduced pressure suction filtration is a polytetrafluoroethylene filter membrane with a pore size of 0.22 μm, and the vacuum degree is 0.05 MPa.

[0017] In step (5), the sequential washing operation is as follows: acetone is used for washing 2-4 times, and the amount used each time is 3-8 mL; ethanol is used for washing 2-4 times, and the amount used each time is 3-8 mL; dichloromethane (CH2Cl2) is used for washing 2-4 times, and the amount used each time is 3-8 mL.

[0018] In step (6), the Soxhlet extraction conditions are: the temperature is 48℃, and the time is 10-14 hours.

[0019] In step (7), the vacuum drying conditions are: the temperature is 30-60℃, the vacuum degree is 10 -2 Torr, and the time is 10-14 hours.

[0020] In order to truly apply the molecularly imprinted material to the selective separation of trace heavy metal residues in actual samples, rapid enrichment, and sensitive optical signal output, the molecularly imprinted material should have high affinity for target molecules, fast binding kinetics, ability to post-functionalize, and consistent material morphology. However, the imprinted polymers prepared by traditional methods currently face many difficulties that need to be overcome in the actual application of molecular recognition, which can be summarized as follows: (1) The template molecules inside the cross-linked network cannot be completely removed due to the high cross-linking density of the molecularly imprinted polymer; (2) The number of effective sites is small, and the affinity for target molecules is small; (3) The target molecules are difficult to diffuse into the imprinted points inside the network, so the binding kinetics for the target molecules is slow (Markowitz, M. A.; Kust, P. R.; Deng, G.; Schoen, P. E.; Gaber, B. P. Langmuir2000, 16,.1759. Rao, MS; Dave, BC J. Am. Chem. Soc. 1998, 120, 13270.); (4) Molecularly imprinted polymers are usually materials with irregular shapes, which have poor compatibility with sensor devices (Hayden, O.; Mann, KJ; Krassnig, S.; Dickert, FL Angew. Chem. Int. Ed. 2006, 45 , 2626.; Schmidt, RH; Mosbach, K.; Haupt, K. Adv. Mater. 2004, 16 , 719.). In view of this, the synthesis of molecularly imprinted polymer materials with high selectivity, high binding capacity, easily accessible sites, fast binding kinetics, and highly sensitive signal output with regular morphology is of great practical and theoretical significance for the development of highly selective, highly integrated and miniaturized chemical and biological sensor devices, and has very broad application prospects in trace substance detection, environmental monitoring and other fields.

[0021] In recent years, the development of nanomaterial preparation technology has brought new opportunities to solve the difficulties faced by molecularly imprinted materials. Compared with traditional imprinted materials, molecularly imprinted materials with nanostructures have the following obvious advantages: (1) Due to the huge specific surface area, template molecules are almost all located on or near the surface of the nanostructure, which makes it possible to remove the template molecules almost completely. (2) The complete removal of template molecules can generate a high density of effective imprinted sites, thus having a high affinity for target molecules (GUO ZH, FLOREA A, CRISTEA C, et al.). Sensors & Actuators B Chemical , 2015 , 207 ,960-966.). (3) The molecular imprinted points are all located on or near the surface of the nanostructure, with low diffusion resistance, thus resulting in fast binding kinetics. (4) The molecular imprinted materials of the nanostructure have regular shapes and can be assembled onto sensors in practical applications. (5) The surface of the molecular imprinted nanostructure is easy to be chemically modified and functionalized.

[0022] The beneficial technical effects of this invention are reflected in the following aspects:

[0023] (1). The present invention provides a nano-fluorescent probe with a covalent organic framework, using terbium-benzoic acid complexes as building blocks, to detect heavy metal ions. The imprinted heavy metal ions are suitable for lead ions (Pb). 2+ ), manganese ions (Mn)2 + ) and copper ions (Cu 2+ ) have certain universality. That is, the terbium-benzoic acid complex unit inherent in the covalent organic framework material and the imine bond together constitute an oxygen and nitrogen atom-rich recognition platform. Since lead ions (Pb 2+ ), manganese ions (Mn 2+ ) and copper ions (Cu 2+ ) have the ability to coordinate with nitrogen and oxygen atoms, when the fluorescent probe contacts the heavy metal ions, electron transfer can be induced, resulting in energy transfer of Tb 3+ , and the detection of heavy metal ions is realized.

[0024] (2) Compared with the traditional molecularly imprinted polymer, the artificial antibody ferroferric oxide nanoparticle surface of the present application is modified with a fluorescent probe, has a fluorescent recognition performance, high sensitivity, simple operation, fast response speed, and improves the molecular recognition performance and selectivity and recognition efficiency. That is, the covalent organic framework shell formed in step (3) is closely combined with the ferroferric oxide nanopowder magnetic core through a covalent bond to provide a fluorescent signal and a recognition skeleton through a rigid π conjugated structure, and a three-dimensional cavity is created through a molecular imprinting technology to endow the covalent organic framework material with high selectivity.

[0025] (3) Compared with the prior art, in step (1), the ferroferric oxide nanosphere surface is rich in hydroxyl groups (-OH), and a layer of nanoshell of tetradentate amine monomers (TBDT) and linear dialdehyde monomers (BCBA) is pre-adsorbed through electrostatic interaction or van der Waals force, which not only increases the specific surface area of the polymer, but also makes all the recognition sites located on the surface of the nanoshell, thereby increasing the number of recognition sites, the binding capacity and the selectivity.

[0026] (4) In the preparation method of the present application, in step (1), the thickness of the nanoshell on the surface of the ferroferric oxide nanopowder particles is adjusted by changing the addition amount of the tetradentate amine monomer and the linear dialdehyde monomer. That is, with a fixed number of ferroferric oxide nanopowder particles, increasing the amount of tetradentate amine monomer and linear dialdehyde monomer will increase the reactant concentration on the surface of the magnetic core, driving the nanoshell to grow outward continuously, thereby increasing the shell thickness. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 is a preparation schematic diagram of the nanofluorescent probe prepared by the present application.

[0028] Figure 2 is a Langmuir and Freundlich adsorption isotherm model curve diagram of the nanofluorescent probe prepared by the present application for Pb 2+ , Mn 2+ and Cu 2+ ions.

[0029] Figure 3 is the adsorption kinetics curve of the nano fluorescent probe prepared by the present application to Pb 2+ , Mn 2+ and Cu 2+ ions.

[0030] Figure 4 is the adsorption curve of the nano fluorescent probe prepared by the present application to Cu 2+ , Cd 2+ , Mn 2+ , Zn 2+ , Sr 2+ and Pb 2+ ions with selective recognition and sensitive detection.

[0031] Figure 5 is the fluorescence spectrum of the nano fluorescent probe prepared by the present application. DETAILED DESCRIPTION

[0032] The present application will be further described in detail below in combination with specific examples. Example 1

[0033] Referring to Figure 1 , the preparation operation steps of a nano fluorescent probe for detecting heavy metal ions in a liquid sample are as follows: (1). 10 mg of ferroferric oxide nano powder, 23 mg of tetradentate amine monomer (TBDT) and 20 mg of linear dialdehyde monomer (BCBA) are added into a 100 mL conical flask with a ground stopper, 0.5 mL of o-dichlorobenzene and 0.5 mL of n-butanol are added, and then 0.15 mL of 1,8-diazabicyclo[5.4.0]undec-7-ene solution with a concentration of 4 M is added. In an ultrasonic instrument, ultrasonic treatment is performed for 10 min to uniformly disperse, so as to obtain a mixed solution with a nano shell structure on the surface of ferroferric oxide nano powder particles. The tetradentate amine monomer and the linear dialdehyde monomer form a nano shell structure to wrap the ferroferric oxide nano powder particles; the addition amount of the tetradentate amine monomer and the linear dialdehyde monomer realizes the adjustment of the thickness of the nano shell on the surface of the nano ferroferric oxide particles.

[0034] (2). Degassing treatment is performed in a freeze dryer, liquid nitrogen is used as a refrigerant, the mixed solution is frozen at -20℃, a mechanical vacuum pump is used to perform air exhaust at a pump speed of 500 L / s at room temperature so that the reaction container is under high vacuum of 10 -2 Torr, and thawing is performed at room temperature. The degassing treatment cycle of freezing, air exhaust and thawing is performed for 3 times; the reaction container after the degassing treatment is sealed under vacuum. The purpose is to create an oxygen-free, water-free inert reaction environment to lay a foundation for the formation of a covalent organic framework.

[0035] (3) Put the sealed reaction vessel into an oven at 150°C for 3 days to obtain the reactant. During the reaction, the tetradentate amine monomer is activated by 1,8-diazabicyclo[5.4.0]undec-7-ene to form an amine anion, and the amine aldehyde condensation reaction occurs between the amine anion and the carbonyl carbon on the linear dialdehyde monomer under high temperature and high pressure conditions to form an imine bond (-C=N-); then, the covalent organic framework shell is tightly combined with the magnetic core of ferroferric oxide nano powder to give the covalent organic framework shell high selectivity.

[0036] (4) Filter the reactant. Transfer all the reactants to a Buchner funnel with a polytetrafluoroethylene filter membrane with a pore size of 0.22 μm, connect a suction filter bottle and a diaphragm vacuum pump, and perform reduced pressure suction filtration under the condition of a vacuum degree of 0.05 Mpa in a fume hood to obtain a solid material, i.e., a covalent organic framework material with terbium-benzoic acid complexes as building units. The purpose is to separate the solid material of the synthesized covalent organic framework from the reaction solution and soluble by-products.

[0037] (5) Wash the solid material with acetone, ethanol, and dichloromethane (CH2Cl2) in sequence to obtain a washed material. The specific washing operation is as follows: First, wash with acetone for 3 times, each time with a dosage of 5 mL; then wash with ethanol for 3 times, each time with a dosage of 5 mL; finally, wash with dichloromethane (CH2Cl2) for 3 times, each time with a dosage of 5 mL.

[0038] (6) Perform Soxhlet extraction of the washed material in excess dichloromethane solvent at 48°C for 12 hours to obtain a solid extract. This step can improve the specific surface area, porosity, and availability of recognition sites of the covalent organic framework material.

[0039] (7) Dry the solid extract in a vacuum drying oven at 40°C for 12 hours to obtain a purified artificial antibody, i.e., a nanofluorescent probe of a covalent organic framework with terbium-benzoic acid complexes as building units, which is used for detecting heavy metal ions in a detected liquid sample. The purpose of step (7) is to remove solvent molecules in the pores under a negative pressure environment to obtain an activated material with a high specific surface area and open active sites.

[0040] The nanofluorescent probe prepared in Example 1 is a covalent organic framework crystal material with an imine bond (-C=N-) as a basic connecting unit, as shown in Figure 5 , which produces strong and stable fluorescence under a 510 nm wavelength light source. The specific surface area is 658 m 2 / g, the fluorescence quantum yield is 19%, the fluorescence intensity decay rate is 2.2%, the detection limit is nM level, and it remains stable in ethanol, acetone, dichloromethane organic reagents, and under high temperature conditions of 300°C.

[0041] The nano fluorescent probe prepared in this embodiment 1 can be found by scanning electron microscopy that the nano shell layer thickness is 65 nm; at a temperature of 298 K, the experimental results of lead ions (Pb 2+ ), manganese ions (Mn 2 + ) and copper ions (Cu 2+ ) are further fitted by using Langmuir and Freundlich adsorption isotherm models, see a in Figure 2 , b in Figure 2 , c in Figure 2 , the maximum adsorption capacity of lead ions, manganese ions and copper ions is calculated as 1820.447 mg / g, 538.956 mg / g and 460.552 mg / g respectively; the chemical adsorption of lead ions, manganese ions and copper ions is carried out, see c2 in Figure 3 , b2 in Figure 3 , a2 in Figure 3 , it can be found that the rapid metal ion absorption occurs in the first 5 minutes, and then reaches a platform at about 10 minutes, see c1 in Figure 3 , b1 in Figure 3 , a1 in Figure 3 , it is found that the adsorption of the nano fluorescent probe to lead ions, manganese ions and copper ions conforms to the pseudo-second-order kinetics model; see a in Figure 4 , it is found that the adsorption capacity of lead ions is more than 82%, which is much higher than that of other metal ions, see b in Figure 4 , it is found that the nano fluorescent probe also has a certain adsorption to manganese ions and copper ions; see Figure 5 , when the lead ion ion concentration is respectively: 0, 1×10 -9 , 1×10 -8 , 1×10 -7 , 1×10 -6 , 1×10 -5 , 1×10 -4 and 1×10 -3 mol·L-1, it is found that with the increase of metal ion concentration, the fluorescence intensity of the nano fluorescent probe gradually decreases, and further experiments find that when 1×10 -13 mol·L-1 and 1×10 -12 mol·L-1 of lead ion solution is added, the fluorescence intensity of the nano fluorescent probe decreases by 3.284% and 8.483%, and when 1×10 -12 mol·L-1 of lead ion solution is added, the fluorescence intensity decreases by more than 5%, which exceeds the minimum standard that can be perceived by the naked eye, so the minimum detection limit of the nano fluorescent probe prepared in this embodiment 1 for lead ions is 1×10 -12 mol·L-1. Embodiment 2

[0042] A preparation operation procedure of a nano-fluorescent probe for detecting heavy metal ions in a liquid sample is as follows: (1). 10 mg of ferroferric oxide nano powder, 20 mg of tetradentate amine monomer (TBDT), and 18 mg of linear dialdehyde monomer (BCBA) are added into a 100 mL conical flask with a ground stopper, 1.0 mL of o-dichlorobenzene and 1.0 mL of n-butanol are added, and then 0.3 mL of 1,8-diazabicyclo[5.4.0]undec-7-ene solution with a concentration of 4 M is added. Ultrasonic treatment is performed in an ultrasonic instrument for 5 min to uniformly disperse to obtain a mixed solution.

[0043] (2). The degassing treatment cycle of freezing, air extraction, and thawing is performed twice; other operations are the same as step (2) of Example 1. The reaction container after the degassing treatment is sealed under vacuum conditions.

[0044] (3). The sealed reaction container is placed in an oven at 120°C, and reaction is performed for 2 days to obtain a reaction product.

[0045] (4). The reaction product is filtered to obtain a solid material. The specific operation is the same as step (4) of Example 1.

[0046] (5). The solid material is sequentially washed with acetone, ethanol, and dichloromethane (CH2Cl2) to obtain a washed product. The specific washing operation is as follows: First, the washing is performed twice with acetone, each time with a dosage of 3 mL; then the washing is performed twice with ethanol, each time with a dosage of 3 mL; finally, the washing is performed twice with dichloromethane (CH2Cl2), each time with a dosage of 3 mL.

[0047] (6). The washed product is subjected to Soxhlet extraction in an excess amount of dichloromethane solvent at 48°C for 10 hours to obtain a solid extract.

[0048] (7). The solid extract is dried in a vacuum drying box at 30°C for 10 hours to obtain a nano-fluorescent probe of a covalent organic framework of a purified artificial antibody terbium-benzoic acid complex as a building unit, that is, a nano-fluorescent probe for detecting heavy metal ions in a detected liquid sample.

[0049] The nano-fluorescent probe prepared in Example 2 is a covalent organic framework crystal material with an imine bond (-C=N-) as a basic connecting unit, as shown in Figure 5 2 It produces strong and stable fluorescence under a 510 nm wavelength light source, has a specific surface area of 619 m2 / g, a fluorescence quantum yield of 17%, a fluorescence intensity decay rate of 4.1%, a detection limit of nM level, and is stable in ethanol, acetone, dichloromethane organic reagents, and at a high temperature of 300°C.​

[0050] The nano fluorescent probe prepared in this embodiment 2 can be found by scanning electron microscopy that the nano shell layer thickness is 57 nm; at a temperature of 298 K, the experimental results of lead ions (Pb 2+ ), manganese ions (Mn 2 + ) and copper ions (Cu 2+ ) are further fitted by using Langmuir and Freundlich adsorption isotherm model, and the maximum adsorption capacities of lead ions, manganese ions and copper ions are calculated as 1538.314 mg / g, 499.837 mg / g and 411.534 mg / g respectively; for the chemical adsorption of lead ions, manganese ions and copper ions, it can be found that the rapid metal ion absorption occurs in the first 10 minutes, and then reaches a platform at about 13 minutes; the adsorption capacity of lead ions is more than 74%; when the concentrations of lead ions are 0, 1×10 -9 , 1×10 -8 , 1×10 -7 , 1×10 -6 , 1×10 -5 , 1×10 -4 and 1×10 -3 mol·L-1, it can be found that the fluorescence intensity of the nano fluorescent probe gradually decreases with the increase of the concentration of metal ions, and further experiments show that when 1×10 -13 mol·L-1 and 1×10 -12 mol·L-1 of lead ion solution is added, the fluorescence intensity of the nano fluorescent probe decreases by 5.265% and 9.343%, and when 1×10 -11 mol·L-1 of lead ion solution is added, the fluorescence intensity decreases by more than 5%, which exceeds the minimum standard that can be perceived by the naked eye, so the minimum detection limit of the nano fluorescent probe prepared in this embodiment 2 for lead ions is 1×10 -11 mol·L-1. Embodiment 3

[0051] The preparation operation steps of a nano fluorescent probe for detecting heavy metal ions in a liquid sample are as follows: (1). Put 10 mg of ferroferric oxide nano powder, 25 mg of tetradentate amine monomer (TBDT) and 25 mg of linear dialdehyde monomer (BCBA) into a 100 mL conical flask with a ground stopper, add 2.0 mL of o-dichlorobenzene and 2.0 mL of n-butanol, and then add 0.5 mL of 1,8-diazabicyclo[5.4.0]undec-7-ene solution with a concentration of 4 M. In the ultrasonic instrument, ultrasonic treatment for 15 min, uniform dispersion, to obtain a mixed solution.

[0052] (2). The degassing treatment cycle of freezing, vacuumizing, and thawing was performed 4 times; other operations were the same as step (2) of Example 1. The reaction vessel after the degassing treatment was sealed under vacuum.

[0053] (3). The sealed reaction vessel was placed in an oven at 180°C for 5 days to obtain a reaction product.

[0054] (4). The reaction product was filtered to obtain a solid material. The specific operation was the same as step (4) of Example 1.

[0055] (5). The solid material was washed with acetone, ethanol, and dichloromethane (CH2Cl2) in sequence to obtain a washed product. The specific washing operation was as follows: First, the solid material was washed with acetone 4 times, each time with 8 mL of acetone; then, the solid material was washed with ethanol 4 times, each time with 8 mL of ethanol; finally, the solid material was washed with dichloromethane (CH2Cl2) 4 times, each time with 8 mL of dichloromethane (CH2Cl2).

[0056] (6). The washed product was subjected to Soxhlet extraction in an excess amount of dichloromethane solvent at 48°C for 14 hours to obtain a solid extract.

[0057] (7). The solid extract was dried in a vacuum drying oven at 60°C for 14 hours to obtain a nanofluorescent probe of a covalent organic framework of a purified artificial antibody terbium-benzoic acid complex as a building unit, i.e., a nanofluorescent probe for detecting heavy metal ions in a detected liquid sample.

[0058] The nanofluorescent probe prepared in Example 3 is a covalent organic framework crystal material with an imine bond (-C=N-) as a basic connecting unit, as shown in Figure 5 , which produces strong and stable fluorescence under a 510 nm wavelength light source, has a specific surface area of 633 m 2 / g, a fluorescence quantum yield of 18%, a fluorescence intensity decay rate of 3.4%, a detection limit of nM level, and is stable in ethanol, acetone, dichloromethane organic reagents, and at a high temperature of 300°C.

[0059] The nanofluorescent probe prepared in Example 3 can be found by electron microscope scanning that the nanoshell layer has a thickness of 73 nm; at a temperature of 298 K, the Langmuir and Freundlich adsorption isotherm models are used to detect lead ions (Pb 2+ ), manganese ions (Mn 2 + ), and copper ions (Cu 2+The experimental results are further fitted, and the maximum adsorption capacities of lead ions, manganese ions and copper ions are calculated as 1647.339 mg / g, 511.256 mg / g and 434.187 mg / g, respectively; the chemical adsorption of lead ions, manganese ions and copper ions can be found that the rapid metal ion absorption occurs in the first 8 minutes, and then reaches a platform at about 12 minutes; the adsorption capacity of lead ions is more than 78%; when the concentrations of lead ions are 0, 1×10 -9 , 1×10 -8 , 1×10 -7 , 1×10 -6 , 1×10 -5 , 1×10 -4 and 1×10 -3 mol·L-1, it can be found that the fluorescence intensity of the nano-fluorescent probe gradually decreases with the increase of the concentration of metal ions, and further experiments show that when 1×10 -13 mol·L-1 and 1×10 -12 mol·L-1 of lead ion solution is added, the fluorescence intensity of the nano-fluorescent probe decreases by 4.889% and 9.167%, and when 1×10 -11 mol·L-1 of lead ion solution is added, the fluorescence intensity decreases by more than 5%, which exceeds the minimum standard that can be perceived by the naked eye, so the minimum detection limit of the nano-fluorescent probe prepared in this embodiment 3 for lead ions is 1×10 -11 mol·L-1. Embodiment 4

[0060] The application of the nano-fluorescent probe in the detection of heavy metals in tea beverages is as follows: (1) Sample pretreatment: 10 mL of tea beverage is taken, 10 mL of methanol is added, vortexed and oscillated, and then centrifuged at 8000 rpm at 4℃ for 10 min, and the supernatant is taken; the pH value of the supernatant is adjusted to 6.0 with 1% nitric acid solution, and then the adjusted supernatant is filtered with a 0.22 μm water-based microporous filter membrane to obtain a test solution.

[0061] (2) Establishing a standard curve: a blank tea beverage solution without heavy metal ions and after the same pretreatment is used as a matrix, and the concentrations of lead ion standard solution are 1×10 -12 mol·L -1 , 1×10 -11 mol·L -1 , 5×10 -11 mol·L -1 , 1×10 -10 mol·L -1 , 5×10 -10 mol·L -1The fluorescence intensity was measured. The standard curve was plotted with the lead ion concentration as the horizontal coordinate and the fluorescence quenching efficiency as the vertical coordinate.

[0062] (3) 2 mL of the solution to be measured in step (1) was taken in a quartz cuvette, 0.2 mg of the nano-fluorescent probe was added, and after vortex mixing, the solution was reacted at room temperature for 15 minutes in the dark. Then, the fluorescence intensity was measured using a fluorescence spectrometer (excitation wavelength: 340 nm, emission wavelength: 510 nm). According to the degree of fluorescence quenching, the concentration of lead ions in the tea beverage was calculated through the standard curve.

[0063] Those skilled in the art will readily understand that the above embodiments 1-3 are merely preferred embodiments of the present application and are not intended to limit the present application, and any modifications, equivalent replacements and improvements made within the spirit and principles of the present application shall be included in the protection scope of the present application.

Claims

1. A method for preparing a nanofluorescent probe for detecting heavy metal ions in a liquid sample, characterized in that: firstly, tetraamine monomers, linear dialdehyde monomers and solvents are added to ferriferrous oxide nanoparticles to perform dehydration condensation under the action of a catalyst, the solvents are o-dichlorobenzene and n-butanol, and the catalyst is 1,8-diazabicyclo[5.4.0]undec-7-ene; secondly, through a series of operations of ultrasonic treatment, degassing, vacuum sealing and high-temperature reaction, a solid-state terbium-benzoic acid complex covalent organic framework material is prepared; and finally, through washing and extraction, a purified nanofluorescent probe of the artificial antibody terbium-benzoic acid complex covalent organic framework material with selective recognition and detection of heavy metal ions is obtained, i.e., a nanofluorescent probe for detecting heavy metal ions in a liquid sample; the operation steps are as follows: (1) adding the following substances to a reaction container: 10 mg of ferriferrous oxide nanoparticles, 20-25 mg of tetraamine monomers, 18-25 mg of linear dialdehyde monomers, 0.5-2.0 mL of o-dichlorobenzene, 0.5-2.0 mL of n-butanol and 0.1-0.5 mL of 1,8-diazabicyclo[5.4.0]undec-7-ene solution with a concentration of 4 M; performing ultrasonic treatment to obtain a mixed solution with a nanoshell structure on the surface of the ferriferrous oxide nanoparticle particles; (2) performing degassing treatment on the mixed solution under vacuum conditions, including freezing, mechanical pump degassing and thawing at room temperature, and the degassing treatment is repeated more than twice; sealing the reaction container after the degassing treatment under vacuum conditions; (3) placing the sealed reaction container in an oven at 120-180℃ for 2-5 days to obtain a reaction product; (4) performing reduced-pressure filtration on the reaction product to obtain a solid-state material, i.e., a terbium-benzoic acid complex covalent organic framework material; (5) sequentially washing the solid-state material with acetone, ethanol and dichloromethane to obtain a washed product; (6) performing Soxhlet extraction on the washed product in dichloromethane to obtain an extracted product; and (7) vacuum drying the extracted product in a vacuum drying box to obtain a purified nanofluorescent probe of the artificial antibody terbium-benzoic acid complex covalent organic framework material, i.e., a nanofluorescent probe for detecting heavy metal ions in a liquid sample. In step (1), the ultrasonic treatment time is 5-15 minutes. In step (4), the filter membrane for the reduced-pressure filtration is a polytetrafluoroethylene filter membrane with a pore size of 0.22 μm, and the vacuum degree is 0.05 MPa. In step (5), the sequential washing operation is as follows: washing with acetone for 2-4 times, each time using 3-8 mL; washing with ethanol for 2-4 times, each time using 3-8 mL; and washing with dichloromethane for 2-4 times, each time using 3-8 mL. In step (6), the Soxhlet extraction conditions are as follows: a temperature of 48℃ and a time of 10-14 hours. ​ ​ ​ The nano fluorescent probe is a covalent organic framework crystal material with imine bond as a basic connecting unit, generates strong and stable fluorescence under a 510nm wavelength light source, has a specific surface area greater than 600m 2 / g, a fluorescence quantum yield greater than 15%, a fluorescence intensity decay rate less than 5%, a detection limit of nM level, and is stable in ethanol, acetone, dichloromethane organic reagents and 300℃ high temperature conditions.

2. The method according to claim 1, wherein the method for preparing a nano-fluorescent probe for detecting heavy metal ions in a liquid sample is characterized in that, ​ ​ ​ ​ ​ ​ ​ ​ 3. The method according to claim 2, wherein the method is characterized by: ​ 4. The method as claimed in claim 2, wherein the method for preparing a nano-fluorescent probe for detecting heavy metal ions in a liquid sample is characterized by: In step (2), the freezing condition is: the freezing medium is liquid nitrogen, the temperature is -20°C, the vacuum degree is 10 -2 Torr; the pumping condition is: the temperature is room temperature, the vacuum pump speed is 500 L / s; the number of circulating degassing treatment is 2-4 times.

5. The method for preparing a nano-fluorescent probe for detecting heavy metal ions in liquid samples according to claim 2, characterized in that: ​ 6. The method as claimed in claim 2, wherein the method for the preparation of nano fluorescent probes for the detection of heavy metal ions in liquid samples is characterized by: ​ 7. The method as claimed in claim 2, wherein the method for the preparation of nano fluorescent probes for the detection of heavy metal ions in liquid samples is characterized by: ​ 8. The method as claimed in claim 2, wherein the method for the preparation of nano fluorescent probes for the detection of heavy metal ions in liquid samples is characterized by: In step (7), the vacuum drying conditions are: temperature 30-60°C, vacuum degree 10 -2 Torr, time 10-14 hours.

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