A colorimetric / fluorescent dual-mode nano-enzyme aptamer sensor, a preparation method thereof and application thereof in bisphenol a detection

Abstract

This invention provides a colorimetric / fluorescence dual-mode nanozyme aptamer sensor, its preparation method, and its application in bisphenol A (BPA) detection. The sensor comprises a metal-organic framework nanozyme loaded with a BPA aptamer, hydrogen peroxide, and a chromogenic substrate, TMB. Cu / Zr-MOF oxidizes the colorless TMB to blue oxTMB, producing a UV absorption peak at 652 nm and a fluorescence emission peak at 415 nm. Modification with the BPA aptamer enhances the specific recognition ability of BPA, while simultaneously inhibiting the enzyme activity of Cu / Zr-MOF, leading to weakened colorimetric and fluorescence signals. In the presence of BPA, the aptamer specifically binds to the target and dissociates from the nanozyme surface, restoring enzyme activity and enhancing colorimetric and fluorescence signals. This invention integrates the excellent catalytic ability of nanozymes with the specific recognition ability of aptamers into a single system, achieving highly sensitive BPA detection.

Description

A colorimetric / fluorescence dual-mode nanozyme aptamer sensor, its preparation method, and its application in bisphenol A detection. Technical Field

[0001] This invention belongs to the field of biosensing technology, and particularly relates to a colorimetric / fluorescence dual-mode nanoenzyme aptamer sensor, its preparation method, and its application in bisphenol A detection. Background Technology

[0002] Bisphenol A (BPA) is a typical endocrine disruptor with a chemical structure similar to estrogen. During actual production and daily use, it is released into the environment and food chain, migrating through packaging materials to food, beverages, or water, and then entering the human body through ingestion, inhalation, or skin contact, leading to health risks such as endocrine and immune disorders, cardiovascular disease, and breast cancer. Medical research shows that even at extremely low concentrations (ng / L-μg / L), BPA exhibits estrogen-mimicking, anti-androgen, and thyroid-interfering biotoxicity, and is significantly associated with reproductive system diseases, metabolic diseases, neurodevelopmental abnormalities, childhood behavioral problems, and the risk of various cancers. Therefore, establishing rapid, sensitive, and reliable BPA detection methods is of paramount medical and public health significance for protecting public health, assessing environmental risks, and strengthening the safety supervision of medical supplies and food packaging.

[0003] Natural enzymes are considered environmentally friendly biocatalysts due to their high selectivity and efficient catalysis. However, their extraction and purification processes are complex and costly, and they are easily inactivated under harsh conditions such as strong acids / bases, high temperatures, or organic solvents, exhibiting poor stability, short lifespan, and difficult recovery. These inherent defects collectively limit their industrial-scale application. Compared with natural enzymes, nanozymes have significant advantages such as simple preparation, low cost, high stability, large-scale production, and easy surface functionalization. Aptamers are short single-stranded DNA or RNA segments obtained through in vitro screening technology (SELEX). They can bind to target molecules with high affinity and specificity and are known as "chemical antibodies." However, aptamers are superior to traditional antibodies in terms of stability, synthesis cost, and modification flexibility, making them ideal recognition elements for constructing biosensors. Combining nanozymes with aptamers to construct nanozyme-aptamer sensors allows for the modulation of the catalytic activity of nanozymes through target-aptamer binding, thereby converting biorecognition signals into easily detectable catalytic signals (such as colorimetric or fluorescent signals), enabling highly sensitive detection of targets.

[0004] Currently, conventional methods for detecting bisphenol A (BPA) mainly include high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), and liquid chromatography-mass spectrometry (LC-MS). While these methods offer high sensitivity and accuracy, they suffer from drawbacks such as expensive equipment, complex operation, cumbersome sample pretreatment, and long analysis cycles, making them unsuitable for rapid, real-time detection. Colorimetric and fluorescence methods, on the other hand, have attracted significant attention from researchers due to their accurate results and ease of operation. Therefore, utilizing a dual-mode colorimetric / fluorescence approach combined with aptamer sensing is of positive significance for improving the practical application performance of BPA detection methods. Summary of the Invention

[0005] To address the above technical problems, this invention provides a colorimetric / fluorescence dual-mode nanozyme aptamer sensor, its preparation method, and its application in bisphenol A detection. This colorimetric / fluorescence dual-mode nanozyme aptamer sensor can quantitatively detect bisphenol A in real time / on-site, with high sensitivity. The detection limits for colorimetric and fluorescence methods are 0.084 ng / mL and 0.053 ng / mL, respectively. The detection process is convenient, rapid, and low-cost.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a colorimetric / fluorescence dual-mode nanozyme aptamer sensor, characterized in that it comprises a metal-organic framework nanozyme (Cu / Zr-MOF@Apt) loaded with a bisphenol A aptamer, hydrogen peroxide, and a chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB); wherein the Cu / Zr-MOF@Apt is a copper / zirconium-based bimetallic organic framework nanozyme (Cu / Zr-MOF) material loaded with a bisphenol A aptamer, wherein the Cu / Zr-MOF is formed by the self-assembly of copper (Cu) metal ions, zirconium (Zr) metal ions, and the organic ligand 2,6-naphthalenedicarboxylic acid.

[0008] Metal-organic frameworks (MOFs) are self-assembled from organic ligands and transition metal ions through coordination. They are highly ordered porous crystalline frameworks that can immobilize aptamers on MOFs supports to form stable aptamer-MOFs complexes. This significantly improves the structural stability of aptamers and their recognition ability in complex media, and can be applied to aptamer biosensors with high stability and high sensitivity.

[0009] According to the present invention, copper (Cu) metal ions, zirconium (Zr) metal ions, and the organic ligand 2,6-naphthalenedicarboxylic acid are self-assembled to form Cu / Zr-MOF nanozyme materials. Compared with other single-metal MOF materials, the introduced copper metal center possesses excellent peroxidase-mimicking activity, effectively catalyzing the reaction of hydrogen peroxide with the chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB), thereby endowing the Cu / Zr-MOF material with significant enzyme-like catalytic performance. Furthermore, this material has a more stable framework structure and tunable electron transfer capabilities, which are beneficial for enhancing its catalytic activity and recyclability.

[0010] According to the present invention, a Cu / Zr-MOF nanozyme material is formed by the self-assembly of copper (Cu) metal ions, zirconium (Zr) metal ions, and the organic ligand 2,6-naphthalenedicarboxylic acid. A bisphenol A aptamer is then modified onto the surface of this Cu / Zr-MOF material to construct Cu / Zr-MOF@Apt. When mixed with hydrogen peroxide and the chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB), the Cu / Zr-MOF nanozyme catalyzes the oxidation of colorless TMB to blue oxTMB, producing an ultraviolet absorption peak at 652 ± 5 nm and a strong fluorescence emission peak at 415 ± 5 nm. The modification of the aptamer partially inhibits the peroxidase activity of Cu / Zr-MOF, resulting in a weakening of catalytic colorimetric and fluorescence signals. When bisphenol A is present in the system, the aptamer specifically recognizes and binds to it, detaches from its modification site, and restores the enzyme activity of Cu / Zr-MOF. The colorimetric and fluorescence signals are then restored, thus constructing a colorimetric / fluorescence dual-mode nanozyme aptamer sensor that can be used for highly sensitive detection of bisphenol A.

[0011] As a preferred embodiment, the particle size of the metal-organic framework nanozyme (Cu / Zr-MOF@Apt) loaded with bisphenol A aptamer is 120-200 nm.

[0012] The colorimetric / fluorescence dual-mode nanozyme aptamer sensor of the present invention has detection limits of 0.084 ng / mL and 0.053 ng / mL for bisphenol A, respectively. The detection process is rapid and inexpensive, meeting the needs of food safety and medical clinical testing.

[0013] Secondly, the present invention also provides a method for preparing the colorimetric / fluorescence dual-mode nanozyme aptamer sensor as described above, comprising the following steps:

[0014] Step (1), Preparation of Cu / Zr-MOF: Zirconium metal salt, copper metal salt and 2,6-naphthalenedicarboxylic acid are dissolved in N,N-dimethylformamide (DMF) solvent, and then trifluoroacetic acid is added as a regulator to carry out a solvothermal reaction. The obtained solution is then washed, filtered and dried to obtain Cu / Zr-MOF powder material.

[0015] Step (2), preparation of Cu / Zr-MOF@Apt: Dissolve the Cu / Zr-MOF obtained in step (1) in Tris-HCl buffer, mix with an equal volume of aptamer solution, and incubate with shaking for a period of time; in order to remove unbound excess Cu / Zr-MOF, wash, filter and dry the obtained mixture to obtain Cu / Zr-MOF@Apt;

[0016] Step (3), preparation of colorimetric / fluorescent dual-mode nanozyme aptamer sensor: The Cu / Zr-MOF@Apt obtained in step (2) is dispersed in Tris-HCl buffer, and mixed with TMB solution and hydrogen peroxide solution in NaAc-HAc buffer to obtain the colorimetric / fluorescent dual-mode nanozyme aptamer sensor.

[0017] In a preferred embodiment, in step (1), the copper metal salt is selected from copper nitrate, the zirconium metal salt is selected from zirconium oxychloride, the molar ratio of copper metal salt to 2,6-naphthalenedicarboxylic acid is 1:(0.4-0.8), and the molar ratio of zirconium metal salt to 2,6-naphthalenedicarboxylic acid is 1:(2.5-3.0); the solvothermal reaction temperature is 120℃, the reaction time is 12-24 h, the washing agent used is anhydrous ethanol, and the drying method is oven drying at a temperature of 60-75℃.

[0018] In a preferred embodiment, in step (2), the concentration of Cu / Zr-MOF dissolved in Tris-HCl buffer is 1-5 mg / mL; the aptamer solution is prepared by dissolving bisphenol A aptamer in Tris-HCl buffer, the concentration of the aptamer is 0.5-2 μM, and the molar ratio of Cu / Zr-MOF to aptamer is 1 g:(0.5-2) μmol, preferably 1 g:1 μmol; the reaction is carried out at 37°C. The washing agent used is Tris-HCl buffer, and the drying method is vacuum freeze-drying.

[0019] As a preferred embodiment, in step (3), the mass concentration of Cu / Zr-MOF@Apt dispersed in Tris-HCl buffer is 0.5-1 mg / mL, the molar concentration of TMB solution is 5 mM, and the molar concentration of hydrogen peroxide solution is 100 mM; 100 μL of Cu / Zr-MOF@Apt Tris-HCl dispersion, 50 μL of TMB solution and 100 μL of hydrogen peroxide solution are mixed, and the reaction system is adjusted to 2 mL using NaAc-HAc buffer.

[0020] Thirdly, the present invention also provides the application of the colorimetric / fluorescence dual-mode nanozyme aptamer sensor as described above and / or the colorimetric / fluorescence dual-mode nanozyme aptamer sensor prepared by the preparation method as described above in the detection of bisphenol A.

[0021] According to the present invention, the colorimetric / fluorescence dual-mode nanozyme aptamer sensor (Cu / Zr-MOF@Apt) prepared by the present invention can catalyze the oxidation of colorless TMB to blue oxTMB, producing a characteristic UV absorption peak at 652 ± 5 nm and a fluorescence emission peak at 415 ± 5 nm. In the presence of bisphenol A, the aptamer specifically recognizes and binds to bisphenol A, dissociates from the Cu / Zr-MOF surface, restores peroxidase activity, enhances catalytic ability, leading to an increase in the amount of oxTMB generated and a deeper blue color in the solution. The change in UV absorption intensity at 652 ± 5 nm is used as the colorimetric detection signal, and the change in fluorescence intensity at 415 ± 5 nm is used as the fluorescence detection signal, thereby realizing dual-mode quantitative analysis of bisphenol A. The detection limits of the colorimetric / fluorescence dual-mode nanozyme aptamer sensor of the present invention for bisphenol A are 0.084 ng / mL and 0.053 ng / mL, respectively. The detection process is rapid and low-cost, meeting the detection requirements for food safety.

[0022] Fourthly, the present invention also provides a method for detecting bisphenol A using the colorimetric / fluorescence dual-mode nanozyme aptamer sensor as described above, comprising the following steps:

[0023] Step S1: The colorimetric / fluorescence dual-mode nanozyme aptamer sensor is mixed with a series of bisphenol A solutions of different concentrations in a certain proportion to prepare a reaction system. After reacting at 25-30℃ for 20-30 min, the spectral detection is performed using a UV-Vis spectrophotometer and a fluorescence spectrometer. The changes in UV absorption at 652 nm and fluorescence intensity at 415 nm of the colorimetric / fluorescence dual-mode nanozyme aptamer sensor are used as the abscissa, and the UV peak at 652 nm and fluorescence intensity peak at 415 nm of the colorimetric / fluorescence dual-mode nanozyme aptamer sensor are used as the ordinate to plot the calibration curves.

[0024] Step S2: The colorimetric / fluorescence dual-mode nanozyme aptamer sensor is mixed with the bisphenol A solution to be tested in a certain proportion to prepare a reaction system. After reacting at 25-30℃ for 20-30 min, the spectral detection is performed using a UV-Vis spectrophotometer and a fluorescence spectrometer. By observing the changes in UV absorption at 652 nm and fluorescence intensity at 415 nm of the colorimetric / fluorescence dual-mode nanozyme aptamer sensor, the concentration of bisphenol A in the sample is calculated based on the calibration curve obtained in step S1, thus realizing the detection of bisphenol A.

[0025] As a preferred embodiment, the preparation method of the reaction system in steps S1 and S2 includes: adding 100 μL Cu / Zr-MOF@Apt solution, 100 μL bisphenol A solution to be tested, 50 μL TMB solution and 100 μL hydrogen peroxide solution, and adjusting the volume to 2 mL with NaAc-HAc buffer to prepare a 2 mL reaction system; the bisphenol A solution uses ethanol as the solvent; the TMB solution has a concentration of 5 mM and uses anhydrous ethanol as the solvent; the hydrogen peroxide solution has a concentration of 100 mM.

[0026] In a preferred embodiment, a UV-Vis spectrophotometer and a fluorescence spectrometer were used for spectral detection, and the absorbance value A at the UV absorption peak of 652 nm was recorded. 652 Fluorescence intensity F at 415 nm 415 Using the ultraviolet absorption peak A 652 and fluorescence intensity value F 415 A calibration curve was obtained by analyzing the functional relationship between the bisphenol A (BPA) concentration and the concentration of BPA to be measured. The concentration of BPA to be measured was calculated using the calibration curve. The colorimetric / fluorescence dual-mode nanozyme aptamer sensor exhibits a light blue color and emits light blue fluorescence in the absence of BPA. As the concentration of BPA increases, the ultraviolet absorption intensity at 652 nm increases, and the color changes from light blue to dark blue. The fluorescence intensity at 415 nm increases, and the fluorescence color changes from light blue to bright blue, thus realizing the colorimetric / fluorescence dual-mode detection of BPA content.

[0027] In a preferred embodiment, Cu / Zr-MOF@Apt catalyzes the oxidation of colorless TMB to blue oxTMB, producing a characteristic UV absorption peak at 652 ± 5 nm and a fluorescence emission peak at 415 ± 5 nm. Upon addition of bisphenol A, the aptamer specifically recognizes and binds to bisphenol A, dissociating from the Cu / Zr-MOF surface. This restores peroxidase activity, enhances catalytic ability, and leads to an increase in the amount of oxTMB generated, resulting in a deeper blue solution. The UV absorption intensity of the solution increases at 652 ± 5 nm, and the fluorescence intensity increases at 415 ± 5 nm. The UV absorption value at 652 nm and the fluorescence intensity at 415 nm, obtained by the colorimetric / fluorescence dual-mode nanozyme aptamer sensor, show a linear relationship with the concentration of the analyte, thus enabling the detection of bisphenol A concentration in the analyte. The UV-Vis spectral conditions include: an observation range of 500-750 nm; a fluorescence excitation wavelength of 325 nm; and a fluorescence emission spectrum observation range of 350-600 nm.

[0028] The technical principle of this invention is as follows: Cu / Zr-MOF@Apt can catalyze the oxidation of colorless TMB substrates to blue oxTMB. Before the addition of bisphenol A, the aptamer adheres to the surface of Cu / Zr-MOF through interaction, inhibiting its peroxidase activity, resulting in a lower amount of oxTMB generated, a lighter solution color, weaker UV absorption at 652 ± 5 nm, and lower fluorescence intensity at 415 ± 5 nm. When bisphenol A is present in the system, the aptamer can specifically recognize and bind to bisphenol A, dissociate from the binding site, restore the enzyme activity of Cu / Zr-MOF, enhance its catalytic ability, increase the amount of oxTMB generated, resulting in a deeper blue solution, enhanced UV absorption at 652 ± 5 nm, and increased fluorescence intensity at 415 ± 5 nm. Based on this, changes in bisphenol A concentration can cause quantitative changes in solution color, UV absorption intensity, and fluorescence intensity. By establishing a linear relationship between the above signal intensity and bisphenol A concentration, the quantitative detection of bisphenol A concentration in the analyte can be achieved.

[0029] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0030] 1. This invention uses Cu / Zr-MOF bimetallic nanozymes with excellent peroxidase activity as carriers and constructs novel nanozyme aptamer sensors through functional modification with aptamers. These sensors combine the high stability of nanozymes with the high specific recognition ability of aptamers, effectively overcoming the problems of high cost, poor stability, and complex antibody preparation of natural enzymes.

[0031] 2. This invention utilizes a colorimetric / fluorescence dual-mode sensor constructed based on Cu / Zr-MOF@Apt, ingeniously leveraging the reversible regulatory effect of aptamers on nanozyme activity (an "inhibition-recovery" mechanism) to integrate the catalytic performance of nanozymes with changes in fluorescence signals within the same system. This sensor is cleverly designed, easy to operate, exhibits high selectivity and good detection stability for bisphenol A, and simultaneously reduces detection time and cost.

[0032] 3. The bisphenol A detection method established in this invention has high detection sensitivity, with detection limits of 0.084 ng / mL for colorimetric method and 0.053 ng / mL for fluorescence method, which can meet the detection requirements of bisphenol A in complex real-world samples. Furthermore, this method exhibits good anti-interference and stability, and has broad application prospects in environmental monitoring and food safety fields. Attached Figure Description

[0033] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0034] Figure 1 is a SEM image of Cu / Zr-MOF in Example 1 and Cu / Zr-MOF@Apt in Example 2 of the present invention.

[0035] Figure 2 shows the particle size distribution of Cu / Zr-MOF in Example 1 and Cu / Zr-MOF@Apt in Example 2.

[0036] Figure 3 is a verification diagram of the catalytic ability of Cu / Zr-MOF@Apt in Verification Example 1 of this invention.

[0037] Figure 4 shows sunlight images of the colorimetric / fluorescence dual-mode nanozyme aptamer sensor solutions under different concentrations of bisphenol A in Example 4 of this invention.

[0038] Figure 5 shows fluorescence images of the colorimetric / fluorescence dual-mode nanozyme aptamer sensor solutions with different concentrations of bisphenol A in Example 4 of this invention.

[0039] Figure 6 shows the UV-Vis and fluorescence emission spectra of the colorimetric / fluorescence dual-mode nanozyme aptamer sensor solutions with different concentrations of bisphenol A in Example 4 of this invention.

[0040] Figure 7 shows the UV-Vis absorption peak and colorimetric detection curve of the colorimetric / fluorescence dual-mode nanozyme aptamer sensor solution under different concentrations of bisphenol A in Example 4 of this invention.

[0041] Figure 8 shows the fluorescence intensity values ​​and fluorescence detection curves of the colorimetric / fluorescence dual-mode nanozyme aptamer sensor solutions under different concentrations of bisphenol A in Example 4 of this invention.

[0042] Figure 9 is a selective analysis diagram of the colorimetric / fluorescence dual-mode nanozyme aptamer sensor investigated in Verification Example 2 of this invention.

[0043] Figure 10 is an analysis diagram of the anti-interference ability of the colorimetric / fluorescence dual-mode nanozyme aptamer sensor in verification example 3 of the present invention. Detailed implementation method:

[0044] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of this invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0045] Unless otherwise specified, all raw materials used in the embodiments are commercially available conventional raw materials, and the technical means used are conventional means well known to those skilled in the art.

[0046] Example 1:

[0047] This embodiment first provides a Cu / Zr-MOF material, the synthesis method of which includes the following steps:

[0048] 22 mg ZrOCl2·8H2O, 72.56 mg Cu(NO3)2·3H2O and 39.8 mg 2,6-naphthalenedicarboxylic acid were dissolved in 20 mL DMF and sonicated until completely dissolved.

[0049] Add 120 μL of trifluoroacetic acid as a regulator to the above DMF solution.

[0050] Place the mixture in silicone oil at 120°C, turn on the magnetic stirrer and stir at 200 rpm for 5 minutes, then turn off the magnetic stirrer and keep heating at 120°C for 24 hours.

[0051] After heating is complete, wait for the solution to cool to room temperature, then transfer the solution to equal portions into 50 mL centrifuge tubes, add 10 mL of anhydrous ethanol, and centrifuge at 12000 rpm, 4℃, for 20 min.

[0052] Remove the supernatant, add 10 mL of anhydrous ethanol, centrifuge, and repeat this step 3 times.

[0053] The resulting precipitate was placed in a 75°C oven until completely dry, yielding the Cu / Zr-MOF material.

[0054] The Cu / Zr-MOF was characterized. Figure 1a shows the SEM image of Cu / Zr-MOF. As can be seen from the figure, the synthesized Cu / Zr-MOF has a regular octahedral structure.

[0055] Example 2:

[0056] This embodiment further provides a method for synthesizing Cu / Zr-MOF@Apt, which specifically includes the following steps:

[0057] Take 5 mg of Cu / Zr-MOF into a 20 mL brown glass sample bottle, add 5 mL of Tris-HCl buffer, and sonicate for 1 h to completely disperse it, to obtain solution C.

[0058] Take 50 μL of 100 μM bisphenol A aptamer (sequence 5'-CCG GTG GGT GGT CAG GTG GGA TAGCGT TCC GCG TAT GGC CCA GCG CAT CAC GGG TTC GCA CCA-3') into a 10 mL centrifuge tube, add 4.95 mL of Tris-HCl buffer, and obtain solution D.

[0059] Add the above solution D to solution C in one go, and incubate at 37°C with shaking for 30 min.

[0060] After incubation, add 10 mL of Tris-HCl buffer solution and centrifuge at 12000 rpm, 4℃, for 10 min.

[0061] Remove the supernatant, add 10 mL of Tris-HCl buffer solution, centrifuge, and repeat this step 3 times.

[0062] The resulting precipitate was placed in a freeze dryer and freeze-dried for 2 days to obtain Cu / Zr-MOF@Apt material.

[0063] Characterization of Cu / Zr-MOF@Apt revealed that, as shown in Figure 1b, the prepared Cu / Zr-MOF@Apt retains a regular octahedral structure compared to Cu / Zr-MOF, but its surface is slightly rougher, confirming that Apt is immobilized on Cu / Zr-MOF. Furthermore, Figure 2 shows an increased particle size of Cu / Zr-MOF@Apt, further confirming the successful synthesis of Cu / Zr-MOF@Apt.

[0064] Verification Example 1:

[0065] To verify the catalytic activity of Cu / Zr-MOF@Apt, Cu / Zr-MOF@Apt was synthesized according to the method in Example 2, and then 5 mg of Cu / Zr-MOF@Apt was dissolved in 10 mL of Tris-HCl buffer.

[0066] Subsequently, 100 μL of Cu / Zr-MOF@Apt solution, 50 μL of TMB solution, and 100 μL of hydrogen peroxide solution were taken and diluted to 2 mL with NaAc-HAc buffer solution at pH 4.0. After reacting for 20-30 min, the UV absorption intensity at 652 nm was recorded using a UV-Vis spectrophotometer.

[0067] As a control 1: Take 100 μL of Cu / Zr-MOF solution, add 50 μL of TMB solution and 100 μL of hydrogen peroxide solution, and adjust the volume to 2 mL with NaAc-HAc buffer solution with a pH of 4.0. After reacting for 20-30 min, record the absorbance at 652 nm.

[0068] As a control 2: Take 100 μL of Cu / Zr-MOF@Apt solution, add 100 μL of bisphenol A solution, 50 μL of TMB solution and 100 μL of hydrogen peroxide solution, and adjust the volume to 2 mL with NaAc-HAc buffer solution with a pH of 4.0. After reacting for 20-30 min, record the absorbance at 652 nm.

[0069] As a control 3: Cu / Zr-MOF@Apt or Cu / Zr-MOF was not added to the reaction system, and all other conditions were the same. The absorbance at 652 nm was recorded.

[0070] As a control 4: only TMB solution was added to the reaction system, and all other conditions were the same. The absorbance at 652 nm was recorded.

[0071] As a control 5: only hydrogen peroxide solution was added to the reaction system, and all other conditions were the same. The absorbance at 652 nm was recorded.

[0072] As shown in Figure 3, Cu / Zr-MOF has high catalytic activity, while Cu / Zr-MOF@Apt has lower catalytic activity than Cu / Zr-MOF. The catalytic activity is restored after the addition of bisphenol A.

[0073] Example 3:

[0074] This embodiment further provides a method for preparing a colorimetric / fluorescence dual-mode nanozyme aptamer sensor, specifically including the following steps:

[0075] Cu / Zr-MOF@Apt was synthesized according to the method in Example 2. Then, 5 mg of Cu / Zr-MOF@Apt was dissolved in 10 mL of Tris-HCl buffer. Subsequently, 100 μL of Cu / Zr-MOF@Apt solution, 50 μL of TMB solution, and 100 μL of hydrogen peroxide solution were taken and diluted to 2 mL with NaAc-Hac solution at pH 4.0. The reaction was allowed to proceed for 20-30 min to obtain the colorimetric / fluorescent dual-mode nanozyme aptamer sensor.

[0076] The TMB solution concentration was 5 mM, and the solvent was anhydrous ethanol; the hydrogen peroxide solution concentration was 100 mM.

[0077] Example 4:

[0078] This embodiment provides a colorimetric / fluorescence dual-mode detection method for detecting bisphenol A, which specifically includes the following steps:

[0079] Add 100 μL of bisphenol A solution of different concentrations to the colorimetric / fluorescent dual-mode nanozyme aptamer sensor prepared in Example 3 to make a 2 mL reaction system, and observe the color change of the solution under sunlight and ultraviolet light.

[0080] Figure 4 shows daylight images of the colorimetric / fluorescence dual-mode nanozyme aptamer sensor solution under different concentrations of bisphenol A. As can be seen from the figure, the solution color changes from light blue to dark blue with increasing bisphenol A concentration.

[0081] Figure 5 shows the fluorescence images of the colorimetric / fluorescence dual-mode nanozyme aptamer sensor solution at different concentrations of bisphenol A. As can be seen from the figure, the fluorescence of the solution changes from light blue to bright blue with increasing bisphenol A concentration.

[0082] The UV-Vis absorption spectrum and fluorescence emission spectrum were measured using a UV-Vis spectrophotometer and a fluorescence spectrometer. The observation range of the UV-Vis spectrum was 500-750 nm; the fluorescence excitation wavelength was 325 nm, and the observation range of the fluorescence emission spectrum was 350-600 nm.

[0083] Figure 6 shows the UV-Vis and fluorescence emission spectra of the colorimetric / fluorescence dual-mode nanozyme aptamer sensor solutions at different concentrations of bisphenol A, where 6a in Figure 6 is the UV-Vis spectrum and 6b in Figure 6 is the fluorescence emission spectrum.

[0084] The functional relationship between different bisphenol A concentrations in the solution and the UV-Vis absorption peak and fluorescence intensity values ​​yields the corresponding bisphenol A concentration values ​​between the UV-Vis absorption peak and fluorescence intensity values ​​in the solution.

[0085] Figure 7 shows the UV-Vis absorbance and colorimetric detection curves of the colorimetric / fluorescence dual-mode nanozyme aptamer sensor solution at different concentrations of bisphenol A. In Figure 7, 7a represents the relationship between bisphenol A concentration and UV-Vis absorption peak, and 7b represents the colorimetric detection curve of the colorimetric / fluorescence dual-mode nanozyme aptamer sensor solution. The x-axis represents the logarithm of the bisphenol A concentration, and the y-axis represents the UV-Vis absorption peak.

[0086] The ultraviolet-visible absorption peak value is: A 652 The values ​​were obtained by monitoring the UV-Vis absorption peak around 652 ± 5 nm in multiple parallel experiments and calculating the average value.

[0087] As shown in Figure 7b, the functional relationship between the UV-Vis absorption peak and the bisphenol A concentration is: Y = 0.109X + 0.653(R) 2 =0.98), where Y represents the UV-Vis absorption peak at 652 nm, and X represents the logarithm of the bisphenol A concentration. Therefore, the concentration of bisphenol A can be measured using a colorimetric / fluorescence dual-mode sensor containing an unknown concentration of bisphenol A. 652 The concentration of bisphenol A can be calculated using the above formula, thus achieving quantitative analysis of bisphenol A.

[0088] According to the formula 3σ / S, where σ is the standard deviation of the blank response value and S is the slope of the detection curve, the detection limit for bisphenol A by the colorimetric / fluorescent dual-mode nanozyme aptamer sensor is calculated to be 0.084 ng / mL.

[0089] Figure 8 shows the fluorescence intensity values ​​and fluorescence detection curves of the colorimetric / fluorescence dual-mode nanozyme aptamer sensor solution under different concentrations of bisphenol A. In Figure 8a, 8a represents the relationship between bisphenol A concentration and fluorescence intensity values, and Figure 8b represents the fluorescence detection curve of the colorimetric / fluorescence dual-mode nanozyme aptamer sensor solution. The x-axis represents the logarithm of the bisphenol A concentration, and the y-axis represents the fluorescence intensity. 415 Fluorescence intensity value.

[0090] F 415 The fluorescence intensity values ​​near 415 nm were obtained by monitoring multiple parallel experiments and calculating the average value.

[0091] As shown in Figure 8b, the functional relationship between the fluorescence intensity value and the bisphenol A concentration is: Y = 545.75X + 4261.60 (R 2 =0.98), where Y represents the fluorescence intensity peak at 415 nm and X represents the logarithm of the bisphenol A concentration. Therefore, the F-value of a colorimetric / fluorescence dual-mode nanozyme aptamer sensor containing an unknown concentration of bisphenol A can be measured. 415 The concentration of bisphenol A can be calculated using the above formula, thus achieving quantitative analysis of bisphenol A.

[0092] The detection limit is calculated using the formula 3σ / S, where σ is the standard deviation of the blank response and S is the slope of the detection curve. Based on this linear relationship, the detection limit for bisphenol A using the colorimetric / fluorescence dual-mode nanozyme aptamer sensor via fluorescence detection is 0.053 ng / mL.

[0093] Example 5

[0094] This embodiment provides a colorimetric / fluorescence dual-mode detection method for detecting bisphenol A, specifically including:

[0095] Step (1): Referring to Example 4, the fluorescence detection curve and colorimetric detection curve of the colorimetric / fluorescence dual-mode nanozyme aptamer sensor solution were obtained, as well as the functional relationship between the UV-Vis absorption peak and the bisphenol A concentration, and the functional relationship between the fluorescence intensity value and the bisphenol A concentration.

[0096] Step (2): Add 100 μL of the bisphenol A solution to be tested (concentration unknown, prepared by diluting 2000 ng / mL bisphenol A solution several times) to the colorimetric / fluorescent dual-mode nanozyme aptamer sensor prepared in Example 3 to make a 2 mL reaction system. Use a UV-Vis spectrophotometer and a fluorescence spectrometer for spectral detection. Calculate the concentration of bisphenol A in the sample based on the detection curve and function relationship obtained in step (1) by observing the UV absorption change at 652 nm and the fluorescence intensity change at 415 nm of the colorimetric / fluorescent dual-mode nanozyme aptamer sensor. The average value is 376 ng / mL.

[0097] Example 6

[0098] This embodiment provides a colorimetric / fluorescence dual-mode nanozyme aptamer detection method for detecting bisphenol A, specifically including:

[0099] Step (1): Referring to Example 4, the fluorescence detection curve and colorimetric detection curve of the colorimetric / fluorescence dual-mode nanozyme aptamer sensor solution were obtained, as well as the functional relationship between the UV-Vis absorption peak and the bisphenol A concentration, and the functional relationship between the fluorescence intensity value and the bisphenol A concentration.

[0100] Step (2): Add 100 μL of the bisphenol A solution (concentration known, 300 ng / mL bisphenol A solution) to the colorimetric / fluorescent dual-mode nanozyme aptamer sensor prepared in Example 3 to make a 2 mL reaction system. Use a UV-Vis spectrophotometer and a fluorescence spectrometer for spectral detection. Calculate the concentration of bisphenol A in the sample based on the detection curve and function relationship obtained in step (1) by observing the UV absorption change at 652 nm and the fluorescence intensity change at 415 nm of the colorimetric / fluorescent dual-mode nanozyme aptamer sensor. The average value is 298 ng / mL.

[0101] Verification Example 2:

[0102] A colorimetric / fluorescence dual-mode nanozyme aptamer sensor was prepared according to Example 3. Bisphenol S (BPS), bisphenol F (BPF), hydroquinone (HQ), catechol (CC), and resorcinol (RES) were selected as interfering substances to investigate the selectivity of the colorimetric / fluorescence dual-mode nanozyme aptamer sensor in detecting bisphenol A. The concentration of the interfering substances was 100 times that of the bisphenol A solution. After reacting at 25-30℃ for 20-30 min, spectral detection was performed using a UV-Vis spectrophotometer and a fluorescence spectrometer. The changes in UV absorption at 652 nm and fluorescence intensity at 415 nm of the colorimetric / fluorescence dual-mode nanozyme aptamer sensor were analyzed.

[0103] Figure 9a shows the selectivity analysis in colorimetric mode, and Figure 9b shows the selectivity analysis in fluorescence mode. As can be seen from the figures, the UV absorbance and fluorescence intensity of the colorimetric / fluorescence dual-mode nanozyme aptamer sensor change significantly only in the presence of bisphenol A. The structural analogues do not cause changes in the sensor, indicating that the sensor has good selectivity for bisphenol A.

[0104] Verification Example 3:

[0105] A colorimetric / fluorescence dual-mode nanozyme aptamer sensor was prepared according to Example 3, selecting K... + Cl - Ca 2+ Mg 2+ CO3 2- Na + SO4 2- Plasma was used as an interfering ion to investigate the anti-interference ability of a colorimetric / fluorescent dual-mode nanozyme aptamer sensor in detecting bisphenol A. The concentration of the interfering ion was 100 times that of the bisphenol A solution. After reacting at 25-30℃ for 20-30 min, spectral detection was performed using a UV-Vis spectrophotometer and a fluorescence spectrometer. The changes in UV absorption at 652 nm and fluorescence intensity at 415 nm of the colorimetric / fluorescent dual-mode nanozyme aptamer sensor were observed.

[0106] Figure 10a shows the anti-interference analysis in colorimetric mode, and Figure 10b shows the anti-interference analysis in fluorescence mode. As can be seen from the figures, there is no significant difference in the effect of bisphenol A solution mixed with interfering ions and bisphenol A solution alone on the ultraviolet absorption value and fluorescence intensity of the colorimetric / fluorescence dual-mode nanozyme aptamer sensor. This indicates that the sensor has good anti-interference properties and can be used for the detection of bisphenol A in complex matrices.

[0107] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. A colorimetric / fluorescence dual-mode nanozyme aptamer sensor, characterized in that, The invention comprises a metal-organic framework nanozyme Cu / Zr-MOF@Apt loaded with a bisphenol A aptamer, hydrogen peroxide, and a chromogenic substrate 3,3',5,5'-tetramethylbenzidine (TMB). The Cu / Zr-MOF@Apt is a copper / zirconium-based bimetallic organic framework nanozyme Cu / Zr-MOF material loaded with a bisphenol A aptamer. The Cu / Zr-MOF is formed by the self-assembly of copper ions, zirconium ions, and the organic ligand 2,6-naphthalenedicarboxylic acid. The Cu / Zr-MOF catalyzes the oxidation of colorless TMB to blue oxTMB, exhibiting a UV absorption peak at 652 ± 5 nm and a peak at 415 ± 5 nm. It exhibits a strong fluorescence emission peak at nm; the modification of the aptamer partially inhibits the peroxidase activity of Cu / Zr-MOF, resulting in weakened catalytic color development and fluorescence signals; when bisphenol A is present in the system, the aptamer specifically recognizes and binds to it, detaches from its modification site, restores the enzyme activity of Cu / Zr-MOF, and the colorimetric and fluorescence signals are restored accordingly, thus constructing a colorimetric / fluorescence dual-mode nanozyme aptamer sensor that can be used for highly sensitive detection of bisphenol A.

2. The colorimetric / fluorescence dual-mode nanozyme aptamer sensor according to claim 1, characterized in that, The Cu / Zr-MOF@Apt has a particle size of 120-200 nm.

3. A method for preparing a colorimetric / fluorescence dual-mode nanozyme aptamer sensor as described in claim 1 or 2, characterized in that, The process includes the following steps: Step (1), preparation of Cu / Zr-MOF: Zirconium metal salt, copper metal salt and 2,6-naphthalenedicarboxylic acid are dissolved in N,N-dimethylformamide DMF solvent, and then trifluoroacetic acid is added as a regulator to carry out a solvothermal reaction. The obtained solution is then washed, filtered and dried to obtain Cu / Zr-MOF powder material; Step (2), preparation of Cu / Zr-MOF@Apt: Cu / Zr-MOF obtained in step (1) is dissolved in Tris-HCl buffer, mixed with an equal volume of aptamer solution, and shaken and incubated for a period of time. The obtained mixture is then washed, filtered and dried to obtain Cu / Zr-MOF@Apt; Step (3), preparation of colorimetric / fluorescent dual-mode nanozyme aptamer sensor: Cu / Zr-MOF@Apt obtained in step (2) is dispersed in Tris-HCl buffer, and mixed with TMB solution and hydrogen peroxide solution in NaAc-HAc buffer to obtain the colorimetric / fluorescent dual-mode nanozyme aptamer sensor.

4. The method for preparing the colorimetric / fluorescence dual-mode nanozyme aptamer sensor according to claim 3, characterized in that, In step (1), the copper metal salt is selected from copper nitrate, the zirconium metal salt is selected from zirconium oxychloride, the molar ratio of copper metal salt to 2,6-naphthalenedicarboxylic acid is 1:(0.4-0.8), and the molar ratio of zirconium metal salt to 2,6-naphthalenedicarboxylic acid is 1:(2.5-3.0); the solvothermal reaction temperature is 120℃, the reaction time is 12-24 h, the washing agent used is anhydrous ethanol, and the drying method is oven drying at a temperature of 60-75℃.

5. The preparation method according to claim 3, characterized in that, In step (2), the mass concentration of Cu / Zr-MOF dissolved in Tris-HCl buffer is 1-5 mg / mL; the aptamer solution is prepared by dissolving bisphenol A aptamer in Tris-HCl buffer, the concentration of the aptamer is 0.5-2 μM, and the mass molar ratio of Cu / Zr-MOF to aptamer is 1 g: (0.5-2) μmol; the reaction is carried out at 37°C, the washing agent used is Tris-HCl buffer, and the drying method is vacuum freeze drying.

6. The preparation method according to claim 3, characterized in that, In step (3), the mass concentration of Cu / Zr-MOF@Apt dispersed in Tris-HCl buffer is 0.5-1 mg / mL, the molar concentration of TMB solution is 5 mM, and the molar concentration of hydrogen peroxide solution is 100 mM. 100 μL of Cu / Zr-MOF@Apt Tris-HCl dispersion, 50 μL of TMB solution and 100 μL of hydrogen peroxide solution are mixed, and the reaction system is adjusted to 2 mL using NaAc-HAc buffer.

7. The application of a colorimetric / fluorescence dual-mode nanozyme aptamer sensor as described in claim 1 or 2 and / or a colorimetric / fluorescence dual-mode nanozyme aptamer sensor prepared by any one of claims 3-6 in the detection of bisphenol A.

8. The application according to claim 7, characterized in that, The detection limits for the colorimetric and fluorescence methods were 0.084 ng / mL and 0.053 ng / mL, respectively.

9. A method for detecting bisphenol A using a colorimetric / fluorescence dual-mode nanozyme aptamer sensor as described in claim 1 or 2, characterized in that, The process includes the following steps: Step S1: The colorimetric / fluorescence dual-mode nanozyme aptamer sensor is mixed with a series of bisphenol A solutions of different concentrations in a certain proportion to form a reaction system. After reacting at 25-30℃ for 20-30 min, the spectral detection is performed using a UV-Vis spectrophotometer and a fluorescence spectrometer. The changes in UV absorption at 652 nm and fluorescence intensity at 415 nm of the colorimetric / fluorescence dual-mode nanozyme aptamer sensor are used as the abscissa, and the UV peak at 652 nm and the fluorescence intensity peak at 415 nm of the colorimetric / fluorescence dual-mode nanozyme aptamer sensor are used as the ordinate to plot the calibration curves. Step S2: The colorimetric / fluorescence dual-mode nanozyme aptamer sensor is mixed with the bisphenol A solution to be tested in a certain proportion to prepare a reaction system. After reacting at 25-30℃ for 20-30 min, the spectral detection is performed using a UV-Vis spectrophotometer and a fluorescence spectrometer. By observing the changes in UV absorption at 652 nm and fluorescence intensity at 415 nm of the colorimetric / fluorescence dual-mode nanozyme aptamer sensor, the concentration of bisphenol A in the sample is calculated based on the calibration curve obtained in step S1, thus realizing the detection of bisphenol A.

10. The method for detecting bisphenol A using a colorimetric / fluorescence dual-mode nanozyme aptamer sensor according to claim 9, characterized in that, The preparation method of the reaction system in steps S1 and S2 includes: adding 100 μL Cu / Zr-MOF@Apt solution, 100 μL bisphenol A solution to be tested, 50 μL TMB solution and 100 μL hydrogen peroxide solution, and adjusting the volume to 2 mL with NaAc-HAc buffer to prepare a 2 mL reaction system; the bisphenol A solution uses ethanol as solvent, the TMB solution has a concentration of 5 mM and the solvent is anhydrous ethanol; the hydrogen peroxide solution has a concentration of 100 mM.

11. The method for detecting bisphenol A using a colorimetric / fluorescence dual-mode nanozyme aptamer sensor according to claim 9, characterized in that, Spectroscopic detection was performed using a UV-Vis spectrophotometer and a fluorescence spectrometer, and the absorbance value A at the UV absorption peak of 652 nm was recorded. 652 Fluorescence intensity F at 415 nm 415 Using the ultraviolet absorption peak A 652 and fluorescence intensity value F 415 A calibration curve was obtained by analyzing the functional relationship between the bisphenol A (BPA) concentration and the concentration of BPA to be measured. The concentration of BPA to be measured was calculated using the calibration curve. The colorimetric / fluorescence dual-mode nanozyme aptamer sensor exhibits a light blue color and emits light blue fluorescence in the absence of BPA. As the concentration of BPA increases, the ultraviolet absorption intensity at 652 nm increases, and the color changes from light blue to dark blue. The fluorescence intensity at 415 nm increases, and the fluorescence color changes from light blue to bright blue, thus realizing the colorimetric / fluorescence dual-mode detection of BPA content.

12. The method for detecting bisphenol A using a colorimetric / fluorescence dual-mode nanozyme aptamer sensor according to claim 9, characterized in that, Cu / Zr-MOF@Apt catalyzes the oxidation of colorless TMB to blue oxTMB, producing a characteristic UV absorption peak at 652 ± 5 nm and a fluorescence emission peak at 415 ± 5 nm. Upon addition of bisphenol A, the aptamer specifically recognizes and binds to bisphenol A, dissociating from the Cu / Zr-MOF surface. This restores peroxidase activity, enhances catalytic ability, and leads to increased oxTMB production and a deeper blue solution. The UV absorption intensity at 652 ± 5 nm and the fluorescence intensity at 415 ± 5 nm both increase. The colorimetric / fluorescence dual-mode nanozyme aptamer sensor demonstrates a linear relationship between the UV absorption at 652 nm and the fluorescence intensity at 415 nm and the concentration of the analyte, thus enabling the detection of bisphenol A concentration in the analyte. The UV-Vis spectral conditions include: an observation range of 500-750 nm; a fluorescence excitation wavelength of 325 nm; and a fluorescence emission spectrum observation range of 350-600 nm.

Images