A MOF-based electrochemical aptasensor and its preparation method and application

Abstract

The present invention relates to a MOF-based electrochemical aptasensor and its preparation method and application. The sensor includes: NH2-MIL-88(Fe) as a carrier for immobilizing a specific aptamer; Hg 2+ for connecting the nucleic acid aptamer; the DAP aptamer and Hg 2+ aptamer form complementary pairing; the MXene-modified electrode serves as a signal amplification platform. The sensor is used for the quantitative detection of DAP, achieving high selectivity and sensitivity in the detection of DAP. Under optimized conditions, the electrochemical aptasensor exhibits a robust linear relationship between the response current (ΔI) and the DAP concentration. It shows a low detection limit, a wide linear range, and excellent reproducibility and stability.

Description

Technical Field

[0001] The present invention relates to an electrochemical aptamer sensor, in particular to a MOF-based electrochemical aptamer sensor, its preparation method and application in the detection of PAEs. Background Art

[0002] Phthalic acid esters (PAEs) are compounds widely used in various industries, mainly used as plasticizers to improve the durability and elasticity of products. PAEs are also known food contaminants that can cause reproductive problems, respiratory diseases, childhood obesity and neuropsychological disorders. Although the food industry prohibits the addition of PAEs to food, they are usually incorporated into polymer materials used in food manufacturing, processing and packaging. Therefore, it is crucial to develop effective methods to monitor and evaluate the presence of PAE residues in food.

[0003] Currently, methods commonly used for detecting PAEs include high performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS) and high performance liquid chromatography-mass spectrometry (HPLC-MS). However, these traditional methods usually have some limitations, such as the need for expensive instrument equipment, complex sample pretreatment procedures, large amounts of harmful organic solvents and specialized treatment schemes. On the contrary, electrochemical methods have the characteristics of simple operation, miniaturization of equipment and less time consumption, making them very suitable for on-site analysis of target pollutants. The integration of electrochemical properties and aptamers has been proven to be a sensitive target detection method, which consists of an electrode and an electrochemically active recognition molecule. After the aptamer is immobilized on the electrode surface and binds to the target, the concentration of the target can be detected by changes in electrochemical signals.

[0004] In order to solve the problems such as the lack of effective, rapid and inexpensive detection methods for PAEs. Taking DAP, one of the common PAEs in food, as an example, the present invention hopes to provide a novel electrochemical aptamer sensor for detecting DAP, which has stability, low price and strong specificity for the detection of DAP. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides a MOF-based electrochemical aptamer sensor, including:

[0006] NH2-MIL-88(Fe), as a carrier for immobilizing a specific aptamer;

[0007] Hg 2+ , for connecting the nucleic acid aptamer;

[0008] DAP aptamer (Apt1) and Hg 2+ aptamer (Apt2), DAP aptamer and Hg 2+The aptamers form complementary pairs;

[0009] The MXene-modified electrode serves as a signal amplification platform.

[0010] The preparation method of an MOF-based electrochemical aptamer sensing according to the present invention comprises the following steps:

[0011] (1) Preparation of NH2-MIL-88(Fe):

[0012] First, 2-aminoterephthalic acid (BDC-NH2) and FeCl3·6H2O are dissolved in a certain amount of dimethylformamide (DMF) at a molar ratio of 1:1; after the obtained mixture is vigorously and fully stirred, the substrate mixture is heated in a hot press reactor under convection oven conditions to promote crystallization; cooled to ambient temperature, the solid brown product is collected; subsequently, washed with DMF and acetic acid to remove the excess reactants; finally, the obtained particles, namely NH2-MIL-88(Fe), are dried overnight at 50-70 °C.

[0013] (2) Preparation of NH2-MIL-88(Fe) / Apt1-Hg 2+ -Apt2

[0014] First, a certain amount of NH2-MIL-88(Fe) powder is weighed and dissolved in a certain amount of water to obtain a 0.1% (mass concentration) NH2-MIL-88(Fe) solution, then a certain volume of 25% glutaraldehyde and a certain volume of 500 nM Apt1 are added to the solution, and the mixed solution is incubated at room temperature for at least 1 hour. Preferably, the addition amount of glutaraldehyde is in a volume ratio of 1:10 to the NH2-MIL-88(Fe) solution volume, and the addition amount of Apt1 is in a volume ratio of 1:100 to the NH2-MIL-88(Fe) solution volume; then the mixed solution is centrifuged to collect the precipitate, washed with water, and then a certain amount of water is added to suspend the precipitate to obtain the NH2-MIL-88(Fe) / Apt1 solution; next, a certain volume of 500 nM Apt2 is added to the NH2-MIL-88(Fe) / Apt1 solution and incubated at room temperature for 35-40 minutes. Preferably, the addition volume of Apt2 is the same as the addition volume of Apt1; again, the mixed solution is centrifuged to collect the precipitate, washed with water, and a certain amount of water is added to suspend the precipitate to obtain the NH2-MIL-88(Fe) / Apt1-Apt2 solution; finally, a 1 mg / mL HgSO4 solution is added and incubated at room temperature for 35 minutes to finally obtain NH2-MIL-88(Fe) / Apt1-Hg 2+-Apt2 solution, store at room temperature for later use; preferably, the volume ratio of the addition amount of HgSO4 solution to NH2-MIL-88(Fe) / Apt1-Apt2 solution is 1:10.

[0015] The Apt1 is a DAP aptamer, and the sequence is:

[0016] 5'-CTTTCTGTCCCCGTCACATCCCACGCATTCTCCACAT-3'

[0017] The Apt2 is a Hg 2+ aptamer, and the sequence is:

[0018] 5'-TTGTGGTGTTTGCGTGGGTTGTGTCGGTTGGTCTGTTTG-3'

[0019] (3) Preparation of MXene / GCE

[0020] First, weigh a certain amount of Ti3AlC2 (MAX) and add it to a 50 mg / mL LiF solution. Stir magnetically at 40 - 50 °C for etching to obtain Ti3C2T x MXene; obtain multi-layered Ti3C2T by centrifugal separation x , and wash it thoroughly with distilled water. Then, after ultrasonic treatment of the multi-layered Ti3C2T x in an inert gas atmosphere, centrifuge the solution of multi-layered Ti3C2T x to collect the supernatant and freeze-dry it; next, polish the GCE electrode with an α-aluminum suspension, wash it with water to remove the residual α-aluminum, and then detect the performance of the GCE electrode by cyclic voltammetry at a scanning rate of 100 mV / s until the voltammetric response is stable; finally, drop and deposit 1 mg / mL Ti3C2T x MXene dispersion on the surface of the pre-prepared GCE electrode, and then dry it in an oven to obtain MXene / GCE.

[0021] (4) Construction of NH2-MIL-88(Fe) / Apt1-Hg 2+ -Apt2 / MXene / GCE sensor

[0022] Add PAEs solution to the NH2-MIL-88(Fe) / Apt1-Hg 2+ -Apt2 solution, incubate at 35 - 40 °C for 40 - 60 minutes. The addition amount of PAEs solution to NH2-MIL-88(Fe) / Apt1-Hg 2+- The volume ratio of the Apt2 solution is 1:100; any excess substances are removed by centrifugation, and an appropriate amount of 10 mM PBS (pH = 7.4) is added to suspend the precipitate to obtain a solution containing the target PAEs; subsequently, the above solution is taken in a beaker, and the MXene / GCE is immersed therein to construct the NH2-MIL-88(Fe) / Apt1-Hg 2+ - Apt2 / MXene / GCE sensor, namely the MOF-based electrochemical aptamer sensing described in the present invention. The PAEs can be diallyl phthalate (DAP), dioctyl phthalate (DOP), diisodecyl phthalate (DIDP), dibutyl phthalate (DIBP), etc.

[0023] An MOF-based electrochemical aptamer sensor prepared by the present invention, namely NH2-MIL-88(Fe) / Apt1-Hg 2 + - Apt2 / MXene / GCE sensor can be used for the detection of PAEs. The PAEs can be diallyl phthalate (DAP), dioctyl phthalate (DOP), diisodecyl phthalate (DIDP), dibutyl phthalate (DIBP), etc.

[0024] Detection method

[0025] The competitive binding of DAP and Apt2 destroys the rigid structure originally formed by the complete pairing of Apt1 and Apt2, resulting in the release of Hg 2+ After washing the solution, chronoamperometry (i-t) is used to deposit for 50 - 60 seconds at an applied voltage of -1.2 mV to enrich Hg 2+ to achieve signal amplification; finally, square wave voltammetry (SWV) is used to measure the Hg 2+ current in the solution for 10 seconds; the scanning range of square wave voltammetry (SWV) is 0.2 to 0.6 V, and the pulse amplitude is 0.025 V.

[0026] Advantages of the present invention:

[0027] In order to achieve the specific recognition ability for PAEs, the present invention adopts the aptamer-regulated thymine-Hg 2+ - thymine strategy, and combines metal-organic frameworks (MOFs) to develop an electrochemical aptamer sensor based on NH2-MIL-88(Fe) / Apt1-Hg 2 + - Apt2 / MXene / GCE. NH2-MIL-88(Fe) is used as a carrier for immobilizing specific aptamers. After introducing Hg 2+ utilize Hg 2+The nucleic acid aptamer, DAP aptamer (Apt1) and Hg 2+ aptamer (Apt2) form complementary pairs, and an MXene-modified electrode is used as a nanoamplification material. The complete pairing of Apt1 and Apt2 forms a rigid structure. This structure is disrupted, leading to the dispersion and release of Hg 2+ and causing a change in the electrical signal. This sensor is used for the quantitative detection of DAP, achieving high selectivity and sensitivity in the detection of DAP. Under optimized conditions, the electrochemical aptamer sensor exhibits a robust linear relationship between the response current (ΔI) and the DAP concentration. It shows a low detection limit, a wide linear range, and excellent reproducibility and stability. In addition, this sensor effectively detects DAP in real samples, with a recovery rate between 94.87% and 103.09%. This innovation represents a novel electrochemical aptamer technology for simple, cost-effective, selective, and sensitive DAP detection. Therefore, this developed sensor has the potential for expansion to detect other PAEs in food or environmental samples. Description of the Drawings

[0028] Figure 1 This is the TEM photograph of NH2-MIL-88(Fe) of the present invention and the HAADF-STEM image of NH2-MIL-88(Fe);

[0029] Among them: (A) and (B) are the TEM photographs of NH2-MIL-88(Fe); (C-G) are the HAADF-STEM images of NH2-MIL-88(Fe), and the corresponding elemental mappings of Fe, O, N, and C.

[0030] Figure 2 This is the FTIR spectrogram of NH2-MIL-88(Fe) of the present invention.

[0031] Figure 3 This is the schematic diagram of the characterization of MXene of the present invention;

[0032] Among them, (A) is the SEM image of MXene; (B) is the BET surface area of MXene.

[0033] Figure 4 This is the Zeta potential diagram of NH2-MIL-88(Fe), NH2-MIL-88(Fe) / Apt1, and NH2-MIL-88(Fe) / Apt1-Hg 2 + -Apt2;

[0034] Among them Figure 4In A: Zeta potential diagrams of NH2-MIL-88(Fe)(a), NH2-MIL-88(Fe) / Apt1(b) and NH2-MIL-88(Fe) / Apt1-Hg 2+ -Apt2(c); Figure 4 Figure B is a schematic diagram of the current response before and after DAP incubation.

[0035] Figure 5 It is a schematic diagram of the optimization effect of the detection conditions of the present invention;

[0036] Among them: (A) is the incubation time of Apt2, (B) is the incubation time of DAP, (C) is the i-t deposition voltage, and (D) is the i-t deposition time.

[0037] Figure 6 It is the schematic diagram of the sensitivity test structure of the NH2-MIL-88(Fe) / Apt1-Hg 2+ -Apt2 sensor of the present invention;

[0038] Among them: (A) is the square wave voltammetry (SWV) of NH2-MIL-88(Fe) / Apt1-Hg 2+ -Apt2 / MXene / GCE in 10 mM PBS with different DAP concentrations; (B) is the linear relationship between the logarithm of the DAP concentration and ΔI (error bars represent standard deviation, n = 3).

[0039] Figure 7 It is the schematic diagram of the index for evaluating the recognition ability of the sensor of the present invention;

[0040] Among them: (A) is a histogram showing the differences in the current responses of different interfering molecules: DAP (0.0003 mg / mL), DOP (0.0003 mg / mL), DIDP, DIBP, curcumin, Glu, glucose to NH2-MIL-88(Fe) / Apt1-Hg 2+ -Apt2 / MXene / GCE, sodium benzoate (SB), cholesterol (0.003 mg / mL); (B) is the reproducibility of the aptasensor incubated with the same batch of 0.0003 mg / mL DAP; (C) is the storage stability of the aptasensor incubated with 0.0003 mg / mL DAP. Detailed implementation manners

[0041] An MOF-based electrochemical aptasensing provided in this example, the preparation method includes the following steps:

[0042] (1) Preparation of NH2-MIL-88(Fe):

[0043] First, 0.126 g (0.692 mmol) of 2-aminoterephthalic acid (BDC-NH2) and 187 g (0.692 mmol) of FeCl3·6H2O were dissolved in 15 mL of dimethylformamide (DMF); after the resulting mixture was vigorously stirred for at least one hour, the substrate mixture was heated in a hot press reactor at 110 °C under convection oven conditions for 24 hours to promote crystallization; cooled to ambient temperature, the solid brown product was collected; subsequently, it was washed with DMF and acetic acid to remove excess reactants; finally, the resulting NH2-MIL-88(Fe) particles were dried overnight at 60 °C.

[0044] (2) Preparation of NH2-MIL-88(Fe) / Apt1-Hg 2+ -Apt2

[0045] First, 0.02 g of NH2-MIL-88(Fe) powder was weighed and dissolved in 20 mL of water to obtain an NH2-MIL-88(Fe) solution. Then, 2 mL of 25% glutaraldehyde and 200 μL of 500 nM Apt1 were added to the solution, and it was incubated at room temperature for 1 hour. After that, the mixed solution was centrifuged at 11000 rpm for 10 min to collect the precipitate. After washing 2 - 3 times with water, 20 mL of water was added to resuspend the precipitate to obtain an NH2-MIL-88(Fe) / Apt1 solution; Next, 200 μL of 500 nM Apt2 was added to the NH2-MIL-88(Fe) / Apt1 solution, and it was incubated at room temperature for 40 minutes. The mixed solution was centrifuged at 11000 rpm for 10 min again to collect the precipitate, washed 2 - 3 times, and 20 mL of water was added to resuspend the precipitate to obtain an NH2-MIL-88(Fe) / Apt1-Apt2 solution; Finally, 2 mL of 1 mg / mL HgSO4 solution was added and incubated at room temperature for 35 minutes to finally obtain an NH2-MIL-88(Fe) / Apt1-Hg 2+ -Apt2 solution, stored at room temperature for later use.

[0046] The Apt1 is a DAP aptamer, and the sequence is:

[0047] 5'-CTTTCTGTCCCCGTCACATCCCACGCATTCTCCACAT-3'

[0048] The Apt2 is an Hg 2+ aptamer, and the sequence is:

[0049] 5'-TTGTGGTGTTTGCGTGGGTTGTGTCGGTTGGTCTGTTTG-3'

[0050] (3) Preparation of MXene / GCE

[0051] First, weigh 100 mg of Ti3AlC2 (MAX) and add it to 1.8 mL of a 50 mg / mL LiF solution (a 1:1 ratio of concentrated HCl and water). Stir magnetically at 40 °C for 24 h to obtain Ti3C2T x MXene; obtain multilayer Ti3C2T by centrifugal separation x , wash it with water 2 - 3 times, and then ultrasonically treat the multilayer Ti3C2T x in an inert gas atmosphere for 1 h; subsequently, centrifuge the solution of multilayer Ti3C2T x at 3500 rpm for 40 min, collect the supernatant and freeze-dry it; next, polish the GCE electrode with an α-aluminum (0.30 μm) suspension, wash it with water to remove the residual α-aluminum, and then detect the performance of the GCE electrode using cyclic voltammetry at a scanning rate of 100 mV / s until the voltammetric response is stable; finally, dropwise deposit 5 μL of a 1 mg / mL Ti3C2T x MXene dispersion onto the surface of the pre-prepared GCE electrode, and then dry it in an oven at 37 °C for 20 min to obtain MXene / GCE.

[0052] (4) Construction of the NH2-MIL-88(Fe) / Apt1-Hg 2+ -Apt2 / MXene / GCE sensor

[0053] Add 200 μL of a DAP solution with a certain concentration to 20 mL of the NH2-MIL-88(Fe) / Apt1-Hg 2+ -Apt2 solution, incubate at 37 °C for 40 min, centrifuge at 11000 rpm for 10 min to remove any excess substances, and add 20 mL of 10 mM PBS (pH = 7.4) to suspend the precipitate to obtain a solution containing the target DAP; subsequently, take 4 mL of the above solution in a beaker, immerse the MXene / GCE in it to construct the NH2-MIL-88(Fe) / Apt1-Hg 2+ -Apt2 / MXene / GCE sensor, which is the MOF-based electrochemical aptasensor described in the present invention.

[0054] Place the solution under an applied voltage of -1.2 V for 60 s using electrochemical i-t, and measure the Hg 2+ current in the solution using square wave voltammetry (SWV) technology for a duration of 10 s; the initial and final voltages of the SWV are 0.2 V and 0.6 V, respectively.

[0055] Characterization of the nanocomposite

[0056] Characterization of NH2-MIL-88(Fe)

[0057] The morphology and structural characteristics of NH2-MIL-88(Fe) were investigated by transmission electron microscopy. Figure 1 Figure A shows a well-prepared NH2-MIL-88(Fe) sample, showing excellent dispersion and typical octahedral morphology, with particle size of about 150 nm. In addition, TEM characterization confirmed different polyhedral morphologies ( Figure 1 Figure B). In addition, high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) analysis combined with energy-dispersive X-ray spectroscopy (EDX) mapping showed the uniform distribution of carbon (red), oxygen (purple), iron (yellow), and nitrogen (green) elements on the entire nanoscale polyhedral framework ( Figure 1 Figures C-G).

[0058] To comprehensively analyze the chemical structure of the NH2-MIL-88(Fe) sample and characterize its functional groups, Fourier transform infrared spectroscopy ( Figure 2 ) was used. The bands observed at 3456.57 cm -1 represented the asymmetric and symmetric stretching vibrations of the N-H bond, while the band at 1658.89 cm -1 corresponded to the bending vibration of the N-H bond. These observations confirmed the presence of amino groups in the synthesized NH2-MIL-88(Fe). In addition, the strong characteristic bands at 1586.38 cm -1 and 1385.95 cm -1 indicated the asymmetric and symmetric stretching vibration modes of the coordinated carboxyl group, elucidating the presence of the dicarboxylic acid linker in the obtained framework structure. At the same time, the peaks at 1258.31 cm -1 and 1339.34 cm -1 represented the characteristic C-N stretching, indicating the presence of aromatic amines. In addition, the peak at 770.68 cm -1 corresponded to the C-H bending vibration of the aromatic ring. Notably, the prominent peak at approximately 524.55 cm -1 was related to the Fe-O vibration.

[0059] Characterization of MXene

[0060] Figure 3 The SEM image of Figure A shows the unique accordion-like morphology of the synthesized MXene, showing good exfoliation and thin nanosheets. Figure 3 Figure B shows the N2 adsorption-desorption isotherm of MXene. At a relative pressure of 0.45 - 1.0, there is an H3-type hysteresis loop between adsorption and desorption, indicating the presence of a mesoporous structure.

[0061] NH2-MIL-88(Fe) / Apt1-Hg 2+Characterization of Apt2 Aptamer Complex

[0062] NH2-MIL-88(Fe), NH2-MIL-88(Fe) / Apt1, and NH2-MIL-88(Fe) / Apt1-Hg 2+ The Zeta potentials of -Apt2 were 30.9 mV, 6.98 mV, and 14.2 mV, respectively ( Figure 4 A). Data analysis showed that the Zeta potential of NH2-MIL-88(Fe) / Apt1 was significantly lower than that of NH2-MIL-88(Fe). This decrease was attributed to the introduction of negatively charged oligonucleotide chains on the surface of NH2-MIL-88(Fe).

[0063] NH2-MIL-88(Fe) / Apt1-Hg 2+ The Zeta potential of -Apt2 exceeded that of NH2-MIL-88(Fe) / Apt1. This increase might be attributed to the positively charged mercury ions in the NH2-MIL-88(Fe) / Apt1 solution, which might lead to a potential recovery. Therefore, these changes in the Zeta potential further confirmed the successful assembly of the aptamer electrochemical sensor.

[0064] In addition, the target was introduced into the NH2-MIL-88(Fe) / Apt1-Hg 2+ -Apt2 solution for electrochemical characterization ( Figure 4 B). The results showed that the current response decreased significantly after adding DAP to the solution. This decrease indicated a decrease in the Hg 2+ concentration, thus affirming the feasibility of the aptamer sensor.

[0065] Optimization of Detection Conditions

[0066] To achieve the best sensing ability of the DAP aptamer electrochemical sensor, several experimental parameters such as the Apt2 incubation time, DAP incubation time, i-t deposition voltage, and i-t deposition time were fine-tuned.

[0067] First, the effect of the Apt2 incubation time on the sensor detection performance was studied. As Figure 5 shown in A, the current signal gradually increased after Apt2 binding, which was related to the increase in the Hg 2+ binding concentration. However, after 35 seconds, the current signal weakened, and the H + ions in the solution affected the structure of the deoxyribonucleic acid chain. Therefore, the optimal Apt2 incubation time was determined to be 35 minutes.

[0068] The incubation time of DAP significantly affected the ability of the sensor to detect substances. In Figure 5 B, the present invention optimized the DAP incubation time for Hg2+ Effect of current signal. The longer the incubation time, the weaker the current signal. This indicates that Apt2 chain competes for the binding site, resulting in the Hg involved 2+ to be saturated. However, after a long incubation time, the signal partially recovers. This rebound may be due to Apt2 reattaching to the remaining mercury ions. Therefore, the present invention finds that 40 minutes is the optimal latency time for DAP.

[0069] To further improve the sensitivity of the sensor, the effects of different deposition voltages on the Hg 2+ current signal intensity were tested, as Figure 5 shown in C. At -1.2 mV, the current signal intensity reaches the maximum value and then gradually decreases. Similarly, the effect of deposition time on the current signal was also tested. As Figure 5 shown in D, the maximum current signal appears at 50 seconds. It decreases again at 70 seconds, which may be due to the reduction of mercury on the electrode to Hg 2+ and falling into the solution after a long deposition time. Therefore, -1.2 mV was selected as the optimal deposition voltage, and 50 seconds was selected as the optimal deposition time for subsequent experiments.

[0070] NH2-MIL-88(Fe) / Apt1-Hg 2+ -Apt2 sensor sensitivity

[0071] To evaluate the performance of the sensor, under optimized experimental conditions, SWV measurements were performed using NH2-MIL-88(Fe) / Apt1-Hg 2+ -Apt2 / MXene / GCE in 10 mM PBS solution. Various concentrations of DAP (ranging from 1.65 ng / mL to 3 μg / mL) were introduced ( Figure 6 A). Notably, the aptasensor showed a linear response corresponding to the DAP concentration. The current signal recorded when the electrode was inserted into the blank solution was labeled as I1, while the signal after incubation with the target was labeled as I2. The difference ΔI = I1 - I2 was used as an index to evaluate the linear response. The DAP calibration plot is as Figure 6 shown in B, showing the relationship between ΔI and the DAP concentration, revealing a linear regression equation of ΔI = 5.44601X + 5.63448 and an R 2 value of 0.95798. In addition, the detection range of this sensor is 3×10 -5 mg / ml to 3×10 -2 mg / ml, and the calculated detection limit is 8.94×10 -6 mg / ml. Compared with previously reported PAEs detection methods, this work performs well.

[0072] Verification of the Aptasensor

[0073] To evaluate the tolerance of the aptasensor of the present invention to structurally similar plasticizers and other potential interfering substances found in foods, diallyl phthalate (DAP), dioctyl phthalate (DOP), diisodecyl phthalate (DIDP), dibutyl phthalate (DIBP), glucose, glutamic acid (Glu), sodium benzoate (SB), cholesterol, and curcumin (each 25 μL, 0.003 mg / mL) were selected. The difference ΔI = I1 - I2 was used as an index to evaluate the recognition ability of the sensor. Figure 7 A shows that except for some plasticizers showing weak correlations, probably due to the structural differences of substances leading to unstable binding with the aptamer, the current signals of the interfering substances were significantly lower than those of the plasticizers. This observation highlights the strong selectivity of the sensor. Subsequently, the reproducibility of the sensor was studied by examining the current responses of five NH2-MIL-88(Fe) / Apt1-Hg 2+ -Apt2 / MXene / GCE sensors after incubation with 0.0003 mg / mL DAP, and the SWV was recorded ( Figure 7 B). No significant change in its performance was observed, confirming the excellent reproducibility of the aptasensor. Finally, the stability of the sensor was evaluated. Over 8 days, 0.0003 mg / mL DAP was detected daily using the aptasensor, and no significant change in the reaction was observed ( Figure 7 C). These findings indicate that the proposed aptasensor has good storage stability.

[0074] Application of the Aptasensor in the Detection of DAP in Water Samples

[0075] Further studies were carried out using the standard recovery method. Different concentrations of DAP were added to pure water, and the NH2-MIL-88(Fe) / Apt1-Hg 2+ -Apt2 / MXene / GCE sensor was used for detection. The performance of the aptasensor was evaluated according to the recovery rate and relative standard deviation (RSD). Table 1 shows the RSD values of the DAP samples, all ≤ 2.18%, and the recovery rates were between 94.87% and 103.09%. These preliminary findings verified the accurate and consistent determination ability of the aptasensor, demonstrating its suitability for analyzing real samples.

[0076] Table 1. Determination Results of NH2-MIL-88(Fe) / Apt1-Hg 2+ -Apt2 / MXene / GCE for DAP Solution in Pure Water

[0077]

[0078] In summary, an electrochemical aptasensor based on NH2-MIL-88(Fe) / Apt1-Hg 2+ -Apt2 / MXene / GCE was developed in the present invention for the quantitative detection of DAP. This sensor utilizes Hg 2+ to link nucleic acid aptamers and employs MXene as a nanoamplification material, achieving high selectivity and sensitivity in the detection of DAP. Under optimized conditions, the electrochemical aptasensor exhibits a robust linear relationship between the response current (ΔI) and the DAP concentration. It shows a low detection limit, a wide linear range, and excellent reproducibility and stability. In addition, this sensor effectively detects DAP in real samples, with a recovery rate between 94.87% and 103.09%. This innovation represents a novel electrochemical aptamer technology for simple, cost-effective, selective, and sensitive DAP detection. Therefore, this developed sensor has the potential for expansion to detect other PAEs in food or environmental samples.

[0079] In the embodiment of the present invention, Apt1: DAP aptamer (5'-CTTTCTGTCCCCGTCACATCCCACGCATTCTCCACAT-3') and Apt2: Hg 2+ aptamer (5'-TTGTGGTGTTTGCGTGGGTTGTGTCGGTTGGTCTGTTTG-3') were provided by Shanghai Shenggong Biotechnology Engineering Technology Service Company. The morphology and elemental composition of this material were characterized by Thermo Scientific Quattro scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FT-IR), Brunauer-Emmett-Teller (BET), and Zeta potential analysis. All electrochemical performance tests were carried out on a Shanghai Chenhua CHI 760E electrochemical workstation, using a conventional three-electrode system, including a modified glassy carbon electrode (GCE, Φ = 3 mm), a platinum wire (counter electrode), and a silver / silver chloride (Ag / AgCl) reference electrode. Square wave voltammetry (SWV) was directly recorded in 0.01 M PBS (pH = 7.4), with a scanning range from 0.2 to 0.6 V and a pulse amplitude of 0.025 V.

Claims

1. An MOF-based electrochemical aptasensor, characterized in that: Comprising: NH2-MIL-88(Fe), as a carrier for immobilizing aptamers; Hg 2+ , for connecting an aptamer; Diallyl phthalate (DAP) aptamer and Hg 2+ aptamer, DAP aptamer and Hg 2+ The aptamers form complementary pairs; MXene-modified electrode, as a signal amplification platform; The DAP aptamer described above, with the sequence: 5 '-CTTTCTGTCCCCGTCACATCCCACGCATTCTCCACAT-3 ' The described Hg 2+ aptamer, with the sequence: 5 '-TTGTGGTGTTTGCGTGGGTTGTGTCGGTTGGTCTGTTTG-3 '.

2. The preparation method of an MOF-based electrochemical aptasensor according to claim 1, characterized in that: Including the following steps: (1) Preparation of NH2-MIL-88(Fe): First, 2-aminoterephthalic acid (BDC-NH2) and FeCl3·6H2O are completely dissolved in a certain amount of dimethylformamide (DMF) at a molar ratio of 1:1; after the resulting mixture is vigorously and fully stirred, the substrate mixture is heated in a hot press reactor under convection oven conditions to promote crystallization; Cool to ambient temperature and collect the solid brown product; Subsequently, wash with DMF and acetic acid to remove excess reactants; finally, dry the resulting particles, i.e., NH2-MIL-88(Fe), overnight at 50-70 °C; (2) Preparation of NH2-MIL-88(Fe) / Apt1-Hg 2+ -Apt2 First, weigh a certain amount of NH2-MIL-88(Fe) powder and dissolve it in a certain amount of water to obtain a 0.1% (mass concentration) NH2-MIL-88(Fe) solution. Then, add a certain volume of 25% (mass concentration) glutaraldehyde and a certain volume of 500 nM Apt1 to the solution. The mixed solution is incubated at room temperature for at least 1 hour. After that, centrifuge the mixed solution to collect the precipitate. After washing with water, add a certain amount of water to suspend the precipitate to obtain the NH2-MIL-88(Fe) / Apt1 solution. Next, add a certain volume of 500 nM Apt2 to the NH2-MIL-88(Fe) / Apt1 solution and incubate at room temperature for 35 - 40 minutes. Centrifuge the mixed solution again to collect the precipitate. After washing with water, add a certain amount of water to suspend the precipitate to obtain the NH2-MIL-88(Fe) / Apt1-Apt2 solution. Finally, add a 1 mg / mL HgSO4 solution and incubate at room temperature for 35 minutes to finally obtain the NH2-MIL-88(Fe) / Apt1-Hg 2+ -Apt2 solution, store it at room temperature for standby; The Apt1 described above is the DAP aptamer, with the sequence: 5 '-CTTTCTGTCCCCGTCACATCCCACGCATTCTCCACAT-3 ' The Apt2 is Hg 2+ aptamer, with the sequence as follows: 5 '-TTGTGGTGTTTGCGTGGGTTGTGTCGGTTGGTCTGTTTG-3 ' (3) Preparation of MXene / GCE First, weigh a certain amount of Ti3AlC2 and add it to an appropriate amount of 50 mg / mL LiF solution. Magnetically stir and etch at 40 - 50 °C to obtain Ti3C2T x MXene; obtain multi-layered Ti3C2T by centrifugal separation x , and wash it thoroughly with distilled water. Then, after ultrasonic treatment of the multi-layered Ti3C2T x in an inert gas atmosphere, centrifuge the multi-layered Ti3C2T x solution, collect the supernatant and freeze-dry it; Next, polish the GCE electrode with α-aluminum suspension, wash it with water to remove the residual α-aluminum, and then use cyclic voltammetry to detect the performance of the GCE electrode until the voltammetric response is stable; Finally, dropwise deposit 1 mg / mL Ti3C2T x MXene dispersion onto the surface of the pre-prepared GCE electrode, and then dry it in an oven to obtain MXene / GCE; (4) Construction of NH2-MIL-88(Fe) / Apt1-Hg 2+ -Apt2 / MXene / GCE sensor To NH2-MIL-88(Fe) / Apt1-Hg 2+ -Apt2 solution, add phthalate esters (PAEs) solution, incubate at 35 - 40 °C for 40 - 60 minutes. The addition amount of PAEs solution and the volume ratio of NH2-MIL-88(Fe) / Apt1-Hg 2+ -Apt2 solution is 1:100; centrifuge to remove any excess substances, add an appropriate amount of 10 mM PBS to suspend the precipitate to obtain a solution containing the target PAEs; subsequently, take the above solution in a beaker and immerse the MXene / GCE into it to construct an NH2-MIL-88(Fe) / Apt1-Hg 2+ -Apt2 / MXene / GCE sensor.

3. The preparation method of an MOF-based electrochemical aptamer sensing according to claim 2, characterized in that: In step (2), the addition amount of glutaraldehyde to the volume ratio of the NH2-MIL-88(Fe) solution is 1:10, the addition amount of Apt1 to the volume ratio of the NH2-MIL-88(Fe) solution is 1:100; the addition volume of Apt2 is the same as the addition volume of Apt1; the addition amount of the HgSO4 solution to the volume ratio of the NH2-MIL-88(Fe) / Apt1-Apt2 solution is 1:

10.

4. An electrochemical aptamer sensor based on MOF according to claim 1 or an electrochemical aptamer sensor prepared by the preparation method according to any one of claims 2-3, for the detection of PAEs.

5. An electrochemical aptamer sensor based on MOF according to claim 1 or an electrochemical aptamer sensor prepared by the preparation method according to any one of claims 2-3, for the detection of DAP.

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