Tb-CeMOF (at) ATP (adenosine triphosphate) fluorescent probe as well as preparation method and application thereof

Abstract

The invention relates to the technical field of chemical hazardous substance detection, in particular to a Tb-CeMOF (at) ATP fluorescent probe as well as a preparation method and application thereof. The fluorescent probe is obtained by taking a terbium / cerium bimetal organic framework Tb-CeMOF as a matrix and an aptamer as a recognition element and modifying the Tb-CeMOF framework with the aptamer through synthesis. The detection mechanism is as follows: when mercury does not exist, the aptamer enables the characteristic fluorescence of terbium to be excited. When a target object exists, the aptamer is specifically combined with mercury, and conformational change causes change of an energy transfer path: the characteristic fluorescence of terbium is quenched; characteristic fluorescence of cerium is obviously enhanced, and an'on-off-on 'type ratio fluorescence signal is formed. A standard curve is established by monitoring the linear relation between the ratio change of the fluorescence intensity and the mercury ion concentration, and accurate quantification of the mercury ion residual quantity in the sample is achieved. By means of simple operation and excellent sensitivity and selectivity, a reliable and efficient new strategy is provided for trace detection of mercury ions.

Description

Technical Field

[0001] This invention relates to the field of chemical hazard detection technology, specifically to a Tb-CeMOF@ATP fluorescent probe, its preparation method, and its application. Background Technology

[0002] Mercury pollution is persistent, bioaccumulative, and non-degradable, posing a serious threat to ecosystems and public health. Even trace exposure to mercury can cause severe damage to the human body. Therefore, regulatory agencies worldwide have established strict limits on mercury levels in drinking water, food, and the environment. Atomic absorption spectrometry (AAS), atomic fluorescence spectrometry (AFS), and inductively coupled plasma mass spectrometry (ICP-MS) are currently the commonly used standard methods for detecting heavy metal residues. However, these methods are complex to operate, have long detection cycles, and require expensive instruments, failing to meet the need for rapid on-site detection of heavy metal residues. Therefore, there is an urgent need to develop a rapid, portable, and highly sensitive method for detecting mercury ions.

[0003] Lanthanide coordination polymers (Ln-CPs) have become ideal materials for constructing fluorescent probes due to their large Stokes shift, long fluorescence lifetime, and sharp emission peaks. Meanwhile, aptamers, with their high affinity and specificity for targets, ease of synthesis and modification, and low cost, are widely used recognition elements. Modifying Ln-CPs with aptamers can construct lanthanide coordination polymers for mercury ion detection that integrate interface recognition and signal transduction. However, while current mercury ion aptamers exhibit good recognition performance, their assembly into terbium / cerium bimetallic lanthanide coordination polymers often fails to effectively sensitize and excite lanthanide ions, causing them to emit light. Furthermore, traditional single-signal "on-off" fluorescent probes are highly susceptible to interference from factors such as excitation source fluctuations, changes in environmental conditions, and background fluorescence, affecting the accuracy and reliability of the detection results. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a Tb-CeMOF@ATP fluorescent probe, its preparation method, and its applications. Using a terbium / cerium bimetallic organic framework as the matrix and an aptamer as the recognition element, the aptamer is synthesized and modified onto the Tb-CeMOF framework. The matrix and aptamer are combined through electrostatic attraction and coordination to construct the Tb-CeMOF@ATP fluorescent probe. The G base in the aptamer enables the Tb-CeMOF to recognize the terbium content of the terbium bimetallic organic framework. 3+ The characteristic fluorescence of Ce is excited, exhibiting an "on" state. 4+ The characteristic fluorescence is suppressed; when the T base of the aptamer specifically binds to the analyte, forming T-Hg... 2+ -T mismatch structure leads to Tb of the terbium / cerium bimetallic organic framework 3+ Excitation is inhibited, Tb3+ The characteristic fluorescence of Ce is quenched, exhibiting an "off" state, while Ce 4+ The characteristic fluorescence is enhanced, exhibiting an "on" state, forming an "on-off-on" ratiometric fluorescence signal. This solves the problems that current mercury ion aptamers cannot effectively excite lanthanide ions to emit light, and that traditional single-signal "on-off" fluorescent probes are easily affected by multiple factors.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: The first objective of this invention is to provide a Tb-CeMOF@ATP fluorescent probe, wherein the Tb-CeMOF@ATP fluorescent probe uses a terbium / cerium bimetallic organic framework as a matrix and an aptamer as a recognition site. The terbium / cerium bimetallic organic framework is modified with an aptamer, wherein the matrix and the aptamer are combined through electrostatic attraction and coordination to construct the Tb-CeMOF@ATP fluorescent probe; wherein the G base in the aptamer enables the Tb of the terbium / cerium bimetallic organic framework to be recognized. 3+ The characteristic fluorescence of Ce is excited, exhibiting an "on" state. 4+ The characteristic fluorescence is suppressed; when the T base of the aptamer specifically binds to the analyte, forming T-Hg... 2+ -T mismatch structure leads to Tb of the terbium / cerium bimetallic organic framework 3+ Excitation is inhibited, Tb 3+ The characteristic fluorescence of Ce is quenched, exhibiting an "off" state, while Ce 4+ The characteristic fluorescence is enhanced, presenting an "on" state, thus forming an "on-off-on" ratio fluorescence signal.

[0006] A second objective of this invention is to provide a method for preparing the above-mentioned Tb-CeMOF@ATP fluorescent probe, comprising the following steps: S1. Using soluble terbium and soluble cerium salts as metal sources and 5-boron-isophthalic acid as an organic ligand, a coordination reaction is carried out at room temperature in a mixed solvent system. The 5-boron-isophthalic acid is deprotonated, exposing the carboxylic acid coordination sites and Tb. 3+ / Ce 4+ Coordination self-assembly occurs to form a stable bimetallic organic framework structure, resulting in a terbium / cerium bimetallic organic framework.

[0007] S2. Using aptamers and terbium / cerium bimetallic organic frameworks as raw materials, incubation was carried out in a solvent reaction system to obtain the Tb-CeMOF@ATP fluorescent probe.

[0008] In a preferred embodiment of the present invention, the nucleotide sequence of the aptamer is TTGGTTGGGGGGGGTTGGTT.

[0009] In a preferred embodiment of the present invention, the molar ratio of terbium in the soluble terbium salt to cerium in the soluble cerium salt is 3~5:5~7, the soluble terbium salt is terbium nitrate hexahydrate or terbium chloride, and the soluble cerium salt is cerium nitrate; the molar ratio of the metal source to the organic ligand is 1:5.

[0010] In a preferred embodiment of the present invention, the mass-to-volume ratio of the organic ligand to the mixed solvent is 105g:15mL~20mL, and the mixed solvent is obtained by mixing N,N-dimethylformamide and water in a volume ratio of 7:3; the coordination reaction time is 10h~15h.

[0011] In a preferred embodiment of the present invention, the mass-to-volume ratio of the terbium / cerium bimetallic organic framework to the aptamer is 2 mg:10 μL, the mass-to-volume ratio of the terbium / cerium bimetallic organic framework to the solvent is 2 mg:5 mL, and the solvent is water; the incubation temperature is 30°C, and the incubation time is 3 h to 5 h.

[0012] A third objective of this invention is to provide an application of the above-described Tb-CeMOF@ATP fluorescent probe in the detection of mercury ions.

[0013] In a preferred embodiment of the present invention, the detection process includes the following steps: S1. Dilute the Tb-CeMOF@ATP fluorescent probe to obtain a diluted solution.

[0014] S2. Mix the diluent with Hg containing different concentrations respectively. 2+ The standard solutions were mixed, incubated, and then Tb was measured. 3+ Characteristic fluorescence intensity and Ce 4+ The characteristic fluorescence intensity is used to calculate the fluorescence intensity ratio.

[0015] S3, with Hg 2+ A linear regression equation was established with concentration on the x-axis and fluorescence intensity ratio on the y-axis.

[0016] S4. Mix the sample to be tested with the diluent, incubate, and then measure the Tb in the test system. 3+ Characteristic fluorescence intensity and Ce 4+ The characteristic fluorescence intensity is used to calculate the fluorescence intensity ratio.

[0017] S5. The mercury ion content is obtained based on the linear regression equation and the fluorescence intensity ratio.

[0018] In a preferred embodiment of the present invention, the linear regression equation is y = 0.00204x + 0.918, R0 2 =0.994; the detection range of the linear regression equation is 10ppb~500ppb, and the detection limit is 5.69ppb.

[0019] In a preferred embodiment of the present invention, the incubation time is 25 min to 35 min.

[0020] Compared with the prior art, the beneficial effects of the present invention are: 1. This invention provides a Tb-CeMOF@ATP fluorescent probe, using a terbium / cerium bimetallic organic framework as the matrix and an aptamer as the recognition element. The probe is obtained by synthesizing an aptamer modified onto the terbium / cerium bimetallic organic framework. The matrix contains Tb... 3+ Electrostatic adsorption occurs between Tb and the negatively charged groups of the nucleic acid aptamer phosphate backbone. 3+ It can coordinate with electron-rich atoms such as nitrogen and phosphate atoms in the heterocyclic bases of nucleic acid aptamers to form stable bonds, thus constructing a Tb-CeMOF@ATP fluorescent probe; wherein, when no detection is performed, the G base in the aptamer enables the Tb of the terbium / cerium bimetallic organic framework to coordinate with electron-rich atoms such as nitrogen and oxygen atoms in the base heterocyclic bases to form stable bonds. 3+ The characteristic fluorescence of Ce is excited, exhibiting an "on" state, while Ce 4+ The characteristic fluorescence is suppressed; when detection is performed, the T base of the aptamer specifically binds to the analyte, forming T-Hg. 2+ -T mismatch structures cause conformational changes in the aptamer, leading to changes in the Tb of the terbium / cerium bimetallic organic framework. 3+ Excitation is inhibited, Tb 3+ The characteristic fluorescence of Ce is quenched, exhibiting an "off" state; while Ce 4+ The characteristic fluorescence is enhanced, exhibiting an "on" state, thus forming an "on-off-on" ratiometric fluorescence signal. This fluorescent probe solves the problems of current mercury ion aptamers failing to effectively excite lanthanide ions to emit light and the susceptibility of traditional single-signal "on-off" fluorescent probes to interference from multiple factors. The Tb-CeMOF@ATP fluorescent probe possesses advantages such as specific recognition ability, strong environmental tolerance, and ease of large-scale preparation, making it an important recognition material.

[0021] 2. This invention synthesizes a novel bifunctional aptamer through rational design. The aptamer's nucleotide sequence is TTGGTTGGGGGGGGTTGGTT, which not only specifically recognizes mercury ions but also acts as an "antenna" to efficiently sensitize the luminescence of lanthanide ions. Furthermore, by simultaneously combining it with a self-constructed terbium / cerium bimetallic organic framework, a novel "on-off-on" ratiometric fluorescence sensor was successfully constructed, achieving signal self-calibration and highly sensitive rapid detection of trace mercury ions.

[0022] 3. This invention provides the application of the Tb-CeMOF@ATP fluorescent probe in the detection of mercury ions, namely, a novel method for detecting residual mercury ion content. A mercury ion standard solution diluted with deionized water is placed in a black centrifuge tube. A standard curve is established based on the relationship between the change in material fluorescence intensity and the mercury ion concentration. The residual mercury ion content is calculated based on the standard curve. This method can specifically identify adsorbed mercury ions from the sample solution. The adsorbed mercury ions bind to the aptamer in Tb-CeMOF@ATP, causing a conformational change in the aptamer, interfering with the antenna effect between the base and terbium. This results in a weakening of the terbium fluorescence characteristic peak and an enhancement of the cerium fluorescence characteristic peak, forming ratiometric fluorescence for mercury ion detection. This method is simple and rapid to operate, highly sensitive, and selective, enabling the quantitative detection of residual mercury ion content. Attached Figure Description

[0023] Figure 1 This is a fluorescence intensity diagram of the Tb-CeMOF@ATP fluorescent probe of Example 1 with different concentrations of mercury ions in this invention.

[0024] Figure 2 This is the standard curve diagram of the present invention.

[0025] Figure 3 This is a fluorescence intensity diagram of the Tb-CeMOF@ATP fluorescent probe of Example 2 with different concentrations of mercury ions in this invention.

[0026] Figure 4 These are physical images of the Tb-CeMOF of the present invention under natural light and under ultraviolet light. Figure 4 Image a shows the actual object under natural light, and image b shows the actual object under ultraviolet light. Detailed Implementation

[0027] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0028] It should be noted that the technical terms used in this invention are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of this invention. Unless otherwise specified, all raw materials, reagents, instruments and equipment used in the following embodiments of this invention can be purchased from the market or prepared by existing methods.

[0029] Modifying Ln-CP with an aptamer allows for the construction of lanthanide coordination polymers for mercury ion detection that integrate interface recognition and signal transduction. However, while current mercury ion aptamers exhibit good recognition performance, their assembly into terbium / cerium bimetallic lanthanide coordination polymers often fails to effectively sensitize and excite lanthanide ions, enabling them to emit light. Furthermore, traditional single-signal "on-off" fluorescent probes are highly susceptible to interference from various factors such as excitation source fluctuations, changes in environmental conditions, and background fluorescence, affecting the accuracy and reliability of detection results. Therefore, this invention rationally designs and synthesizes a novel bifunctional aptamer that not only specifically recognizes mercury ions but also acts as an "antenna" to efficiently sensitize the luminescence of lanthanide ions. Combined with a self-constructed terbium / cerium bimetallic lanthanide coordination polymer, a novel "on-off-on" ratiometric fluorescence sensor is successfully constructed, achieving signal self-calibration and highly sensitive rapid detection of trace mercury ions.

[0030] Based on this, firstly, this invention provides a Tb-CeMOF@ATP fluorescent probe, which uses a terbium / cerium bimetallic organic framework as the matrix and an aptamer as the recognition site. The terbium / cerium bimetallic organic framework is modified by the aptamer, wherein the matrix and the aptamer combine through electrostatic attraction and coordination to construct the Tb-CeMOF@ATP fluorescent probe. The G base in the aptamer enables the Tb of the terbium / cerium bimetallic organic framework to... 3+ The characteristic fluorescence of Ce is excited, exhibiting an "on" state. 4+ The characteristic fluorescence is suppressed; when the T base of the aptamer specifically binds to the analyte, forming T-Hg... 2+ -T mismatch structure leads to Tb of the terbium / cerium bimetallic organic framework 3+ Excitation is inhibited, Tb 3+ The characteristic fluorescence of Ce is quenched, exhibiting an "off" state, while Ce 4+ The characteristic fluorescence is enhanced, presenting an "on" state, thus forming an "on-off-on" ratio fluorescence signal.

[0031] In the Tb-CeMOF@ATP fluorescent probe, the binding of the aptamer to Tb-CeMOF is a synergistic result of "electrostatic binding + coordination". The phosphate backbone of the aptamer is negatively charged, while the metal nodes of the lanthanide MOF are positively charged. Therefore, there is an electrostatic attraction between the aptamer and the lanthanide MOF, which is one of the common driving forces for the initial binding of the two. Subsequently, the lanthanide ion, as a "hard acid", tends to coordinate with "hard base" atoms such as O and N. Through the phosphate group and base in the aptamer, it forms a coordination bond with the metal node of the lanthanide MOF to form a stable binding.

[0032] It should be noted that the detection mechanism of the fluorescent probe is as follows: when Hg is not present... 2+ At that time, the aptamer makes terbium (Tb)3+ The characteristic fluorescence of Hg is excited. 2+ When present, the aptamer specifically binds to it, causing a conformational change that alters the energy transfer pathway: on the one hand, Tb 3+ The characteristic fluorescence was effectively quenched; on the other hand, cerium (Ce) 4+ The characteristic fluorescence of Hg was significantly enhanced, thus forming a stable "on-off-on" ratiometric fluorescence signal. This was achieved by monitoring the ratio of fluorescence intensity changes to Hg. 2+ Establishing a standard curve based on the linear relationship between concentrations allows for the determination of Hg levels in samples. 2+ Precise quantification of residual amounts.

[0033] Secondly, this invention provides a method for preparing the above-mentioned Tb-CeMOF@ATP fluorescent probe, comprising the following steps: S1. Using soluble terbium and soluble cerium salts as metal sources and 5-boron-isophthalic acid as an organic ligand, a coordination reaction is carried out at room temperature in a mixed solvent system. In the reaction system, 5-boron-isophthalic acid is first deprotonated to expose the carboxylic acid coordination site, and then reacts with Tb. 3+ / Ce 4+ Coordinate bonds are formed, and the structures spontaneously assemble into stable bimetallic organic frameworks, resulting in the terbium / cerium bimetallic organic framework Tb-CeMOF.

[0034] S2. Using aptamers and terbium / cerium bimetallic organic frameworks as raw materials, incubation is carried out in a solvent reaction system. There is an electrostatic attraction between the negatively charged phosphate backbone of the aptamer and the metal nodes of the positively charged lanthanide MOF. Then, the lanthanide ions, as "hard acids", tend to coordinate with "hard base" atoms such as O and N. Through the phosphate groups and bases in the aptamer, coordinate bonds are formed with the metal nodes of the lanthanide MOF, thus forming a stable bond, and obtaining the Tb-CeMOF@ATP fluorescent probe.

[0035] The aptamer was designed through multiple rounds, and its sequence was determined to be TTGGTTGGGGGGGGTTGGTT. This design places thymine (T bases) at both ends of the DNA sequence, providing binding sites for mercury ions. Simultaneously, it avoids the steric hindrance effect that might result from excessive stacking of T bases on both sides, thus hindering T-Hg binding. 2+ The formation of the -T structure involves concentrating the guanine (G) bases in the middle of the sequence, making it an energy transfer "antenna" for efficiently absorbing energy and transferring it to terbium ions (Tb). 3+ This significantly enhances its fluorescence intensity.

[0036] The molar ratio of terbium in the soluble terbium salt to cerium in the soluble cerium salt is 3-5:5-7, preferably 3:7. The soluble terbium salt is terbium nitrate hexahydrate or terbium chloride, preferably terbium nitrate hexahydrate, and the soluble cerium salt is cerium nitrate. This bimetallic molar ratio can directly regulate the coordination structure and morphology of the MOF, thereby affecting the fluorescence energy transfer efficiency and Hg. 2+ Sensitivity and linear range are key parameters that determine the core function of a material.

[0037] The molar ratio of the metal source to the organic ligand is 1:5.

[0038] The mass-to-volume ratio of the organic ligand to the mixed solvent is 105g:15mL~20mL, preferably 105g:20mL, and the mixed solvent is obtained by mixing N,N-dimethylformamide and water in a volume ratio of 7:3.

[0039] The coordination reaction time is 10h to 15h, preferably 12h.

[0040] The mass-to-volume ratio of the terbium / cerium bimetallic organic framework to the aptamer is 2 mg: 10 μL.

[0041] The aptamer solution contained 150 μM aptamer at a concentration of 150 μM, and the solvent was pure water. The aptamer was a solid powder purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0042] The mass-to-volume ratio of the terbium / cerium bimetallic organic framework to the solvent is 2 mg: 5 mL.

[0043] The incubation temperature is 30°C, and the incubation time is 3 to 5 hours, preferably 3 hours.

[0044] In the preparation of Tb-CeMOF, after the coordination reaction, washing and conversion are required to obtain Tb-CeMOF. Washing consists of DMF washing and ethanol washing. DMF washing involves adding DMF to the centrifuge tube to fully disperse the precipitate, centrifuging again to discard the supernatant, and removing uncoordinated free ligands; this process is repeated three times. Ethanol washing involves adding ethanol to the centrifuge tube to fully disperse the precipitate, centrifuging again to discard the supernatant, and removing residual DMF and impurities; this process is repeated three times. The phase transition involves adding pure water to the washed centrifuge tube and allowing it to stand at room temperature. The high polarity of water competes for the carboxyl coordination sites of the 5-bop ligand, inducing the transformation of the 3D amorphous complex into 2D ultrathin nanosheets.

[0045] Finally, this invention provides an application of the above-mentioned Tb-CeMOF@ATP fluorescent probe in the detection of mercury ions.

[0046] The application includes the following steps: S1. Dilute the Tb-CeMOF@ATP fluorescent probe to obtain a diluted solution.

[0047] S2, Mix the diluent with Hg of different concentrations. 2+ The standard solutions were mixed and incubated, followed by determination of Tb in the standard solution system. 3+ Characteristic fluorescence intensity and Ce 4+ Characteristic fluorescence intensity; calculate fluorescence intensity ratio.

[0048] S3, with Hg 2+ A linear regression equation was established with concentration on the x-axis and fluorescence intensity ratio on the y-axis.

[0049] S4. Mix the sample to be tested with the diluent, incubate, and then measure the Tb in the test system. 3+ Characteristic fluorescence intensity and Ce 4+ The characteristic fluorescence intensity is used to calculate the fluorescence intensity ratio.

[0050] S5. Based on the linear regression equation and the fluorescence intensity ratio, the residual mercury ion content is obtained.

[0051] The linear regression equation is y = 0.00204x + 0.918, R0 2 =0.994; the detection range of the linear regression equation is 10ppb~500ppb, and the detection limit is 5.69ppb.

[0052] The incubation time is 25 min to 35 min, preferably 30 min.

[0053] The contents contain different concentrations of Hg 2+ Hg in standard solution 2+ The concentrations were 10 ppb, 50 ppb, 150 ppb, 250 ppb and 500 ppb.

[0054] The diluent contains Hg at different concentrations. 2+ The volume ratio of the standard solutions is 1:1.

[0055] The method for detecting mercury ions using the above-mentioned Tb-CeMOF@ATP fluorescent probe is characterized by its simple and rapid operation, high sensitivity, and good selectivity, enabling quantitative detection of residual mercury ion content.

[0056] The following specific examples will provide further explanation.

[0057] In this invention, the English abbreviation for N,N-dimethylformamide is DMF, and the English abbreviation for 5-boronyl isophthalic acid is 5-bop.

[0058] Example 1 A method for preparing a Tb-CeMOF@ATP fluorescent probe includes the following steps: Preparation of S1 and Tb-CeMOF: Weigh 105 mg of 5-bop, add 7 mL of DMF and 3 mL of ultrapure water, then add triethylamine dropwise until the pH stabilizes at 8.0. Weigh 22.7 mg of terbium nitrate hexahydrate (Tb(NO3)3·6H2O) and 19.4 mg of cerium nitrate (Ce(NO3)4), dissolve them in a mixed solvent of 7 mL of DMF and 3 mL of ultrapure water, mix thoroughly, and stir magnetically for 12 h at room temperature and in the dark. After the reaction is complete, transfer the mixture to a 50 mL centrifuge tube, centrifuge at 8000 rpm for 15 min, and collect the white precipitate at the bottom of the tube.

[0059] S2. Washing: After the reaction is complete, allow the mixture to cool naturally to room temperature. Centrifuge at 8000 rpm for 5 min to collect the product. Add 10 mL of DMF to the centrifuge tube to fully disperse the precipitate. Centrifuge again to discard the supernatant. Wash three times. Then add 10 mL of ethanol to the centrifuge tube to fully disperse the precipitate. Centrifuge again to discard the supernatant to remove residual DMF and impurities. Wash three times.

[0060] S3. Phase transformation and drying: Add 5 mL of ultrapure water to the washed centrifuge tube, sonicate for 5 min to uniformly suspend the precipitate, and let it stand at room temperature for 2 h. Collect the white precipitate by centrifugation at 8000 rpm for 15 min, place it in a vacuum drying oven at 40℃ to dry, and grind it to obtain white powdery nanosheets, namely Tb-CeMOF.

[0061] S4. Material and Aptamer Incubation: Dissolve 2 mg of MOF powder in 5 mL of ultrapure water and sonicate for 30 min to obtain a MOF dispersion with a concentration of 0.1 mg / mL. Prepare a 150 μmol aptamer solution with the aptamer sequence TTGGTTGGGGGGGGTTGGTT. Mix 1 mL of the MOF dispersion with 10 μL of the 150 μM aptamer solution and incubate at 30 °C for 3 h to obtain the material dilution solution, i.e., the Tb-CeMOF@ATP fluorescent probe.

[0062] Example 2 A method for preparing a Tb-CeMOF@ATP fluorescent probe includes the following steps: Preparation of S1 and Tb-CeMOF: Weigh 105 mg of 5-bop, add 7 mL of DMF and 3 mL of ultrapure water, then add triethylamine dropwise until the pH stabilizes at 8.0. Weigh 13.6 mg of terbium nitrate hexahydrate and 27.2 mg of cerium nitrate, dissolve them in a mixed solvent of 7 mL of DMF and 3 mL of ultrapure water, mix thoroughly, and stir magnetically for 12 h at room temperature in the dark. After the reaction is complete, transfer the mixture to a 50 mL centrifuge tube, centrifuge at 8000 rpm for 15 min, and collect the white precipitate at the bottom of the tube.

[0063] S2. Washing: After the reaction is complete, allow the mixture to cool naturally to room temperature. Centrifuge at 8000 rpm for 5 min to collect the product. Add 10 mL of DMF to the centrifuge tube to fully disperse the precipitate. Centrifuge again to discard the supernatant. Wash three times. Then add 10 mL of ethanol to the centrifuge tube to fully disperse the precipitate. Centrifuge again to discard the supernatant to remove residual DMF and impurities. Wash three times.

[0064] S3. Phase transformation and drying: Add 5 mL of ultrapure water to the washed centrifuge tube, sonicate for 5 min to uniformly suspend the precipitate, and let it stand at room temperature for 2 h. Collect the white precipitate by centrifugation at 8000 rpm for 15 min, place it in a vacuum drying oven at 40℃ to dry, and grind it to obtain white powdery nanosheets, namely Tb-CeMOF.

[0065] S4. Material and Aptamer Incubation: Dissolve 2 mg of MOF powder in 5 mL of ultrapure water and sonicate for 30 min to obtain a MOF dispersion with a concentration of 0.1 mg / mL. Prepare a 150 μmol aptamer solution with the aptamer sequence TTGGTTGGGGGGGGTTGGTT. Mix 1 mL of the MOF dispersion with 10 μL of the 150 μM aptamer solution and incubate at 30 °C for 3 h to obtain the material dilution solution, i.e., the Tb-CeMOF@ATP fluorescent probe.

[0066] The structure and performance of the fluorescent probes in Examples 1 and 2 were tested.

[0067] Next, this invention demonstrates the application of the Tb-CeMOF@ATP fluorescent probe in the detection of residual mercury ions. Specifically, mercury ion solutions with final concentrations of 10 ppb, 50 ppb, 150 ppb, 250 ppb, and 500 ppb were tested. 100 μL of the target was added to 100 μL of the material dilution solution from Example 1, and after incubation for 30 min, fluorescence changes were detected. Figure 1 and Figure 3 As shown. A standard curve was established using the relationship between the change in Tb-CeMOF@ATP fluorescence intensity and mercury ion concentration, as shown. Figure 2As shown. The residual mercury ion content was calculated based on the standard curve.

[0068] Figure 1 This image shows the fluorescence intensity of the Tb-CeMOF@ATP fluorescent probe of Example 1 for different concentrations of mercury ions in this invention. Figure 1 It is known that Tb-CeMOF@ATP can highly selectively identify mercury ions from sample solutions. Utilizing the thymine-mercury ion-thymine (T-Hg-T) mismatch site in the DNA double helix, a stable base pair is formed. The adsorbed mercury ions bind to the aptamer in Tb-CeMOF@ATP, causing a conformational change in the aptamer. This interferes with the antenna effect between the base and terbium, resulting in a weakening of the terbium fluorescence characteristic peak and an enhancement of the cerium fluorescence characteristic peak, forming ratiometric fluorescence for mercury ion detection. This method is simple, rapid, highly sensitive, and selective, enabling the quantitative detection of residual mercury ions.

[0069] Figure 2 This is a standard curve diagram of the present invention. (From...) Figure 2 It can be seen that the concentration of Hg(II) in the range of 10 ppb to 500 ppb is similar to that of I. 680 / I 525 The response values ​​exhibited a good linear correlation, and the regression equation obtained through linear fitting was y = 0.00204x + 0.91, with a correlation coefficient (R²). 2 =0.994 (close to 1), indicating excellent fit. This standard curve has a wide linear range, high fit, and good reproducibility, and can be used for the quantitative detection of Hg(II) in real samples.

[0070] Figure 3 This image shows the fluorescence intensity of the Tb-CeMOF@ATP fluorescent probe of Example 2 for different concentrations of mercury ions in this invention. Figure 3 It can be seen that, with Hg 2+ With increasing Hg(II) concentration, the fluorescence intensity of the Tb-CeMOF@ATP fluorescent probe decreased accordingly, exhibiting a typical fluorescence quenching phenomenon. When the Hg(II) concentration was in the range of 250 ppb to 500 ppb, the quenching rate of the probe was 15.63% to 19.76%, preliminarily confirming that this probe can detect Hg. 2+ Its potential.

[0071] Figure 4 These are physical images of the Tb-CeMOF of the present invention under natural light and under ultraviolet light. Figure 4 Image a shows the object under natural light, and image b shows the object under ultraviolet light. Figure 4It can be seen that Tb-CeMOF emits bright green fluorescence under ultraviolet light excitation. This fluorescence is due to the characteristic transition of terbium ions, indicating that Tb-CeMOF has been successfully synthesized and that terbium ions have been effectively doped into the metal-organic framework structure.

[0072] It should be noted that when numerical ranges are involved in this invention, it should be understood that both endpoints of each numerical range, as well as any value between the two endpoints, can be selected. Since the steps and methods used are the same as in the embodiments, preferred embodiments are described here to avoid redundancy. Although preferred embodiments of this invention have been described, those skilled in the art, once they understand the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended scope of protection is intended to be interpreted as including the preferred embodiments and all changes and modifications falling within the scope of this invention.

[0073] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of protection of this invention and its equivalents, this invention also intends to include these modifications and variations.

Claims

1. A Tb-CeMOF@ATP fluorescent probe, characterized in that, The Tb-CeMOF@ATP fluorescent probe is constructed using a terbium / cerium bimetallic organic framework as the matrix and an aptamer as the recognition site. The terbium / cerium bimetallic organic framework is modified by the aptamer. The matrix and the aptamer are combined through electrostatic attraction and coordination to construct the Tb-CeMOF@ATP fluorescent probe. Among them, the G base in the aptamer enables the Tb of the terbium / cerium bimetallic organic framework to be reduced. 3+ The characteristic fluorescence of Ce is excited, exhibiting an "on" state. 4+ The characteristic fluorescence is suppressed; when the T base of the aptamer specifically binds to the analyte, forming T-Hg... 2+ -T mismatch structure leads to Tb of the terbium / cerium bimetallic organic framework 3+ Excitation is inhibited, Tb 3+ The characteristic fluorescence of Ce is quenched, exhibiting an "off" state, while Ce 4+ The characteristic fluorescence is enhanced, presenting an "on" state, thus forming an "on-off-on" ratio fluorescence signal.

2. A method for preparing the Tb-CeMOF@ATP fluorescent probe according to claim 1, characterized in that, Includes the following steps: Using soluble terbium and soluble cerium salts as metal sources and 5-boron-isophthalic acid as an organic ligand, a coordination reaction was carried out at room temperature in a mixed solvent system. The 5-boron-isophthalic acid was deprotonated, exposing the carboxylic acid coordination sites and Tb. 3+ / Ce 4+ Coordination self-assembly occurs to form a stable bimetallic organic framework structure, resulting in a terbium / cerium bimetallic organic framework; Using aptamers and terbium / cerium bimetallic organic frameworks as raw materials, Tb-CeMOF@ATP fluorescent probes were obtained by incubation in a solvent reaction system.

3. The method for preparing the Tb-CeMOF@ATP fluorescent probe according to claim 2, characterized in that, The nucleotide sequence of the aptamer is TTGGTTGGGGGGGGTTGGTT.

4. The method for preparing the Tb-CeMOF@ATP fluorescent probe according to claim 2, characterized in that, The molar ratio of terbium in soluble terbium salts to cerium in soluble cerium salts is 3~5:5~7. The soluble terbium salts are terbium nitrate hexahydrate or terbium chloride, and the soluble cerium salts are cerium nitrate. The molar ratio of the metal source to the organic ligand is 1:

5.

5. The method for preparing the Tb-CeMOF@ATP fluorescent probe according to claim 2, characterized in that, The mass-to-volume ratio of the organic ligand to the mixed solvent was 105 g: 15 mL to 20 mL. The mixed solvent was obtained by mixing N,N-dimethylformamide and water in a volume ratio of 7:

3. The coordination reaction time was 10 h to 15 h.

6. The method for preparing the Tb-CeMOF@ATP fluorescent probe according to claim 2, characterized in that, The mass-to-volume ratio of terbium / cerium bimetallic organic framework to aptamer was 2 mg:10 μL, and the mass-to-volume ratio of terbium / cerium bimetallic organic framework to solvent was 2 mg:5 mL, with water as the solvent; the incubation temperature was 30 °C, and the incubation time was 3 h to 5 h.

7. The application of the Tb-CeMOF@ATP fluorescent probe according to claim 1 in the detection of mercury ions.

8. The application of the Tb-CeMOF@ATP fluorescent probe according to claim 7 in the detection of mercury ions, characterized in that, The testing process includes the following steps: The Tb-CeMOF@ATP fluorescent probe was diluted to obtain a diluent; The diluent was mixed with Hg of different concentrations. 2+ The standard solutions were mixed, incubated, and then Tb was measured. 3+ Characteristic fluorescence intensity and Ce 4+ The characteristic fluorescence intensity is used to calculate the fluorescence intensity ratio; With Hg 2+ A linear regression equation was established with concentration on the x-axis and fluorescence intensity ratio on the y-axis. The sample to be tested was mixed with the diluent and incubated, followed by the determination of Tb in the test system. 3+ Characteristic fluorescence intensity and Ce 4+ The characteristic fluorescence intensity is used to calculate the fluorescence intensity ratio; The mercury ion content was obtained based on the linear regression equation and the fluorescence intensity ratio.

9. The application of the Tb-CeMOF@ATP fluorescent probe according to claim 7 in the detection of mercury ions, characterized in that, The linear regression equation is y = 0.00204x + 0.918, R0 2 =0.994; the detection range of the linear regression equation is 10ppb~500ppb, and the detection limit is 5.69ppb.

10. The application of the Tb-CeMOF@ATP fluorescent probe according to claim 7 in the detection of mercury ions, wherein the incubation time is 25 min to 35 min.

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