Electrochemical aptamer sensor for detecting dibutyl phthalate as well as preparation method and application of electrochemical aptamer sensor

Abstract

The invention relates to a preparation method and application of an electrochemical aptamer sensor, in particular to an electrochemical aptamer sensor for detecting dibutyl phthalate as well as a preparation method and application of the electrochemical aptamer sensor. According to the invention, the electrochemical aptamer sensor with high sensitivity and high selectivity is constructed through compounding of Fe3O4 magnetic nanoparticles and a Hemin-coated HKUST-1 metal organic framework, and is used for detecting dibutyl phthalate (DBP); by adopting the Hemin-coated HKUST-1 as a signal amplification unit, the electron transfer rate of an electrode interface is effectively enhanced, so that the current response in square wave voltammetry measurement is obviously enhanced, and the signal stability and repeatability are ensured. The sensor disclosed by the invention has relatively high selectivity on PAEs substances with similar structures and common food interferents, and a DBP signal is obviously higher than that of other interferents, so that reliable detection in a complex matrix is ensured, and a novel, reliable and efficient analysis platform is provided for food safety and environmental monitoring.

Description

Technical Field

[0001] This invention relates to a method for preparing an electrochemical aptamer sensor and its application, and particularly to an electrochemical aptamer sensor for detecting dibutyl phthalate, its preparation method, and its application. Background Technology

[0002] Phthalate esters (PAEs) are a class of plasticizers widely used in plastics, pesticides, coatings, cosmetics, and various industrial products to improve product flexibility and processability. Because PAEs are bonded to the polymer matrix only through weak non-covalent bonds, they easily leach from materials, enter the environment, and accumulate in ecosystems and the human body. Long-term exposure may lead to reproductive toxicity, hormonal imbalances, and other health problems.

[0003] Currently, common methods for detecting PAEs include high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), and capillary electrophoresis (CE). While these methods offer high sensitivity and accuracy, they typically require expensive instruments, complex sample pretreatment, and lengthy analysis times, limiting their application in rapid on-site monitoring. Dibutyl phthalate (DBP) is one type of PAE. Traditional methods suffer from interference in the detection of trace amounts of DBP in complex food or environmental matrices, and a convenient, low-cost, and rapid on-site detection solution is lacking.

[0004] In recent years, electrochemical sensors based on nanomaterials have gradually become an emerging technology for the detection of PAEs (polyester esters). Functional materials such as metal-organic frameworks (MOFs), magnetic nanoparticles, and aptamer molecules have been introduced into sensor systems to improve detection sensitivity and selectivity. However, most existing electrochemical sensors still have the following shortcomings in terms of material composite strategies, signal amplification mechanisms, and selective recognition: First, the sensitivity and linear range of the sensors are limited, making it difficult to meet the detection requirements of trace DBP in complex matrices; second, the lack of systematic optimization in material assembly and surface modification during sensor construction leads to insufficient repeatability and stability; third, existing sensors struggle to achieve high selective recognition among various PAEs and are easily affected by analogues or food contaminants. Therefore, this invention aims to develop an electrochemical aptamer sensor based on magnetic nanoparticles and metal-organic frameworks for the rapid, sensitive, and selective detection of DBP in food and environmental samples. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides an electrochemical aptamer sensor for the detection of dibutyl phthalate, its preparation method, and its application. By combining Fe3O4 magnetic nanoparticles, an aptamer, and a heme-encapsulated MOF material (Hemin@HKUST-1), and using a reduced graphene oxide (rGO)-modified electrode, a highly sensitive and selective electrochemical aptamer sensor is constructed.

[0006] This invention provides an electrochemical aptamer sensor for the detection of dibutyl phthalate, comprising:

[0007] Fe3O4 magnetic nanoparticles modified with dibutyl phthalate (DBP) aptamer (Apt) are used for specific recognition, target enrichment and magnetic separation.

[0008] Hemin-encapsulated metal-organic framework (Hemin@HKUST-1) is used as a signal amplification unit;

[0009] Complementary DNA (cDNA) hybridizes with DBP aptamers to form a double-stranded structure; used for signal regulation after target molecule binding.

[0010] The above components are fixed on the surface of a glassy carbon electrode (GCE) substrate modified with reduced graphene oxide (rGO);

[0011] The DBP aptamer sequence is as follows:

[0012] 5'-NH2-CTTTCTGTCCCCGTCACATCCCACGCATTCTCCACAT-3';

[0013] The complementary DNA sequence is as follows:

[0014] 5'-NH2-ATGTGGAGAATGCGTGGGATGTGACGGAAGGACAGAAAG-3'.

[0015] The present invention provides a method for preparing an electrochemical aptamer sensor for the detection of dibutyl phthalate, comprising the following steps:

[0016] (1) Preparation of carboxylated Fe3O4 magnetic nanoparticles (Fe3O4–COOH):

[0017] A certain amount of Fe3O4 magnetic nanoparticles were weighed and dispersed in a 0.5 M citric acid monohydrate solution and stirred thoroughly at room temperature. After the reaction was completed, the precipitate was collected by magnetic separation and washed with acetone until the pH value was 7.0-7.5. Finally, the product was dried to obtain carboxyl-functionalized Fe3O4–COOH powder.

[0018] Preferably, the Fe3O4 magnetic nanoparticles have an average particle size of 200 nm.

[0019] (2) Preparation of Fe3O4–aptamer (Fe3O4–Apt) complex:

[0020] The Fe3O4–COOH prepared above was dispersed in water to prepare a Fe3O4–COOH aqueous dispersion with a concentration of 2 mg / mL; a certain amount of Fe3O4–COOH aqueous dispersion was measured and mixed with an equal volume of EDC / NHS solution, and activated at room temperature for 30-50 minutes.

[0021] Preferably, the mass ratio of EDC to NHS in the EDC / NHS solution is 4:1.

[0022] Subsequently, a certain amount of 100 μM DBP aptamer (Apt) solution was added, mixed evenly, and incubated at room temperature for 3-5 hours to allow the aptamer to be covalently grafted onto the surface of the nanoparticles through amidation reaction. After the reaction was completed, the mixed solution was centrifuged to remove unreacted aptamer molecules. The precipitate was collected, washed with ultrapure water, and redispersed in ultrapure water to obtain a Fe3O4–Apt complex dispersion.

[0023] (3) Preparation of Hemin@HKUST-1:

[0024] 1,3,5-Benzotricarboxylic acid and hemin were dissolved together in a volume ratio of 5:12 in a certain amount of a mixed solvent composed of N,N-dimethylformamide (DMF) and ethanol, and stirred until completely dissolved to obtain an organic solution.

[0025] Preferably, in the mixed solvent composed of N,N-dimethylformamide (DMF) and ethanol, DMF and ethanol are mixed in a volume ratio of 1:1.

[0026] Alternatively, a certain amount of Cu(NO3)2·3H2O is dissolved in ultrapure water to obtain an aqueous solution of copper nitrate; under continuous stirring, the aqueous solution of copper nitrate is slowly added dropwise to the above organic solution. After stirring for a period of time, the mixture is transferred to an autoclave and heated at 120℃-160℃ for 20-25 hours.

[0027] After the reaction was completed, the mixture was allowed to cool naturally to room temperature. The precipitate was collected by centrifugation. The precipitate was washed successively with DMF and anhydrous ethanol and then dried in a vacuum drying oven to obtain dark blue Hemin@HKUST-1 powder.

[0028] Preferably, the concentration of the copper nitrate aqueous solution is 0.5-0.6 mol / L.

[0029] (4) Preparation of Hemin@HKUST-1-cDNA:

[0030] The preparation method is the same as that for the Fe3O4–aptamer complex, except that Hemin@HKUST-1 is used to replace Fe3O4–COOH and complementary strand DNA (cDNA) is used to replace the aptamer (Apt); thus, a Hemin@HKUST-1–cDNA complex dispersion is obtained.

[0031] (5) Assembly of the Fe3O4 / Hemin@HKUST-1 complex:

[0032] A certain amount of Fe3O4–Apt dispersion and a certain amount of Hemin@HKUST-1–cDNA dispersion were mixed evenly in a centrifuge tube and incubated at room temperature for 50-75 minutes to allow the aptamer (Apt) and complementary strand DNA (cDNA) to fully hybridize. Subsequently, the mixture was separated using a magnetic separation device, and the magnetically adsorbed complex was collected and resuspended in ultrapure water to obtain the Fe3O4 / Hemin@HKUST-1 complex dispersion.

[0033] Preferably, the volume ratio of Fe3O4–Apt dispersion to Hemin@HKUST-1–cDNA dispersion is 1:3.

[0034] (6) Electrode modification:

[0035] The glassy carbon electrode (GCE) after polishing and cleaning was tested for electrode performance by cyclic voltammetry (CV) until the voltammetric response stabilized. A 1 mg / mL reduced graphene oxide (rGO) dispersion was drop-coated onto the pretreated GCE surface, allowed to air dry naturally, and then dried in an oven to obtain the rGO / GCE modified electrode.

[0036] (7) Sensor construction:

[0037] Add the sample solution containing DBP to the Fe3O4 / Hemin@HKUST-1 composite dispersion and incubate at room temperature for 35-45 minutes; then perform magnetic separation, collect the enriched component and drop it onto the surface of the rGO / GCE modified electrode, and dry it at room temperature to obtain the Hemin@HKUST-1 / rGO / GCE electrochemical aptamer sensor.

[0038] Preferably, in step (7), the volume ratio of the test sample solution containing DBP to the Fe3O4 / Hemin@HKUST-1 composite dispersion is 3:20.

[0039] The resulting Hemin@HKUST-1 / rGO / GCE electrochemical aptamer sensor can then be used for electrochemical detection.

[0040] The Hemin@HKUST-1 / rGO / GCE electrochemical aptamer sensor prepared in this invention can be used to detect dibutyl phthalate (DBP).

[0041] The method for detecting dibutyl phthalate using the Hemin@HKUST-1 / rGO / GCE electrochemical aptamer sensor prepared in this invention is as follows:

[0042] The Hemin@HKUST-1 / rGO / GCE electrochemical aptamer sensor electrode was used as the working electrode, forming a three-electrode system with a platinum wire counter electrode and an Ag / AgCl reference electrode. The system was immersed in 0.1 M PBS buffer (pH 7-7.4) and detected using square wave voltammetry (SWV). The oxidation peak current value near -0.2 V was recorded. The current value of the blank sample without DBP was I0, and the current value of the sample containing DBP was I. The current change ΔI = I0 - I / (logarithm of DBP concentration) within 3 × 10⁻¹⁰. -6 mg / mL to 3×10 -3 The sample exhibits a good linear relationship within the mg / mL range, with a linear regression equation of ΔI = 3.39527 lgC +6.2937 (R² = 0.99259). Based on this, DBP in the sample solution can be quantitatively analyzed.

[0043] As a preferred option, the SWV parameters are set as follows: scan range -0.6 V to 0 V, pulse amplitude 0.025 V, and rest time 10 seconds.

[0044] Working principle of the invention:

[0045] The Fe3O4 magnetic nanoparticles of this invention, after aptamer modification, form a Fe3O4-Apt composite. This composite can rapidly capture the target molecule DBP in solution, while simultaneously utilizing magnetism to achieve enrichment, thereby improving detection efficiency and sensitivity. The Hemin@HKUST-1 composite material serves as a signal amplification unit; its channels and surface-loaded Hemin exhibit catalytic activity, enhancing the electron transfer rate at the electrode interface and thus amplifying the electrochemical response signal. The rGO-modified glassy carbon electrode (GCE) provides the sensor with excellent conductivity and a large specific surface area, ensuring efficient electron transfer to the working electrode.

[0046] During the detection process, DBP molecules specifically bind to the aptamer, causing the hybrid structure to unwind and exposing the electrocatalytic activity of Hemin@HKUST-1, resulting in a significant enhancement of the oxidation peak current in square wave voltammetry (SWV) measurements. The change in current (ΔI) is linearly related to the DBP concentration and can be used for quantitative analysis.

[0047] The beneficial effects of this invention are:

[0048] First, the electrochemical aptamer sensor of this invention constructs a highly sensitive and selective electrochemical aptamer sensor for detecting dibutyl phthalate (DBP) by combining Fe3O4 magnetic nanoparticles with the Hemin@HKUST-1 metal-organic framework. The sensor utilizes the magnetic separation properties of Fe3O4, combined with the specific recognition function of the aptamer, to achieve efficient enrichment and detection of the target molecule DBP, thereby significantly improving detection sensitivity and response speed. Under optimal experimental conditions, the sensor exhibits a linear detection range of 3 × 10⁻⁻⁻⁻⁶ for DBP. 6 Up to 3×10⁻ 3 The method concentration was mg / mL, with a linear correlation coefficient R² of 0.99259 and a detection limit as low as 2.90 × 10⁻⁻⁻⁶. 6 mg / mL, demonstrating excellent detection performance.

[0049] Secondly, this invention employs Hemin@HKUST-1 as the signal amplification unit, effectively enhancing the electron transfer rate at the electrode interface. This results in a significant increase in current response during square-wave voltammetry measurement, ensuring signal stability and repeatability. Experimental results show that the relative standard deviation (RSD) of five batches of sensors detecting the same concentration of DBP was only 1.94%, and after being stored at 4℃ for 7 consecutive days, the signal response remained at 76.31% of the initial value, indicating that the sensor has good fabrication reproducibility and storage stability.

[0050] Furthermore, the sensor of this invention exhibits high selectivity for structurally similar PAEs and common food interfering substances, with a significantly higher DBP signal than other interfering substances, ensuring reliable detection in complex matrices. Compared to existing technologies, this invention overcomes the technical barriers of low sensitivity, slow response, and poor anti-interference ability in traditional DBP detection methods through material composite and functional design, achieving rapid, accurate, and repeatable trace DBP detection, and providing a novel, reliable, and efficient analytical platform for food safety and environmental monitoring. Attached Figure Description

[0051] Figure 1 This is a schematic diagram of the preparation method of the electrochemical aptamer sensor of the present invention.

[0052] Figure 2 The following is a schematic diagram of the characterization of the composite material in an embodiment of the present invention: (A) TEM image of hemin@HKUST-1; (B) SEM image of Fe3O4; (C) FT-IR spectra of heme, HKUST-1 and hemin@HKUST-1; (D) XRD patterns of HKUST-1 and hemin@HKUST-1.

[0053] Figure 3The following are schematic diagrams of XPS spectral characterization of hemin@HKUST-1 in this embodiment of the invention: (A) Survey spectrum; (B) High-resolution C 1s spectrum; (C) High-resolution O 1s spectrum; (D) High-resolution Fe 2p spectrum; (E) High-resolution Cu 2p spectrum; (F) Zeta potential analysis of different functionalized materials.

[0054] Figure 4 As an embodiment of the present invention, 5 mM Fe[(CN)6] containing 0.1 M KCl was used. 3- / 4- Electrochemical characterization of different electrodes was obtained in solution at a scan rate of 0.05 V / s; (A) CV curve and (B) EIS curve; (C) rGO / GCE in 5 mM [Fe(CN)6] containing 0.1 MKCl. 3– / 4– (A) CV curves at different scan rates; (B) Linear relationship between peak current of anode and cathode and scan rate; (C) Linear curve of fitted lgV and potential; (D) Feasibility of the sensor.

[0055] Figure 5 The following is a schematic diagram comparing parameters in the embodiments of the present invention: (A) incubation time of aptamer with Fe3O4; (B) Fe3O4 particle size; (C) pH; (D) Fe3O4 addition amount; (E) incubation time of Fe3O4-Apt with hemin@HKUST-1-cDNA; (F) optimization of DBP incubation time.

[0056] Figure 6 For the embodiments of the present invention, electrochemical detection of DBP standard solutions of different concentrations was performed: (A) Current curves of DBP at different concentrations; (B) Linear relationship between the logarithm of DBP concentration and current. Detailed Implementation

[0057] Example 1

[0058] like Figure 1 As shown in this embodiment, a method for preparing an electrochemical aptamer sensor for the detection of dibutyl phthalate includes the following steps:

[0059] (1) Preparation of carboxylated Fe3O4 magnetic nanoparticles (Fe3O4–COOH):

[0060] 1.0 g of Fe3O4 magnetic nanoparticles (approximately 200 nm in diameter) were weighed and dispersed in 100 mL of 0.5 M citric acid monohydrate solution. The mixture was mechanically stirred at 500 rpm for 5 hours at room temperature. After the reaction was completed, magnetic separation was performed using an external magnetic field. The supernatant was discarded, the precipitate was collected, and the precipitate was repeatedly washed with acetone until the pH of the washing solution was close to 7.0. Finally, the product was dried in a vacuum drying oven at 40 °C for 12 hours to obtain carboxyl-functionalized Fe3O4–COOH powder.

[0061] (2) Preparation of Fe3O4–aptamer (Fe3O4–Apt) complex:

[0062] The Fe3O4–COOH prepared above was dispersed in water to prepare an aqueous dispersion with a concentration of 2 mg / mL; 1 mL of Fe3O4–COOH aqueous dispersion was measured and mixed with 1 mL of EDC / NHS solution, and activated at room temperature for 30 minutes; the mass ratio of EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) to NHS (N-hydroxysuccinimide) in the EDC / NHS solution was 4:1;

[0063] Subsequently, 2 μL of a 100 μM DBP aptamer (Apt) solution was added, mixed thoroughly, and incubated at room temperature for 5 hours to allow the aptamer to be covalently grafted onto the nanoparticle surface via amidation. After the reaction was complete, the mixture was centrifuged at 10,000 rpm for 10 minutes, and the supernatant was discarded to remove unreacted aptamer molecules. The precipitate was washed three times with ultrapure water and finally redispersed in 1 mL of ultrapure water to obtain a Fe3O4–Apt complex dispersion, which was stored at 4 °C for later use.

[0064] (3) Preparation of Hemin@HKUST-1:

[0065] 0.50 g of 1,3,5-benzenetricarboxylic acid and 0.12 g of heme were dissolved together in 15.0 mL of a mixed solvent consisting of N,N-dimethylformamide (DMF) and ethanol in a volume ratio of 1:1, and stirred until completely dissolved to obtain an organic solution.

[0066] Separately, 1.04 g of copper nitrate trihydrate (Cu(NO3)2·3H2O) was dissolved in 7.5 mL of ultrapure water to obtain an aqueous solution of copper nitrate. Under continuous stirring, the aqueous solution of copper nitrate was slowly added dropwise to the above organic solution. After stirring for 20 minutes, the mixture was transferred to a 100 mL polytetrafluoroethylene-lined stainless steel autoclave and heated in an oven at 120°C for 24 hours.

[0067] After the reaction was completed, the mixture was allowed to cool naturally to room temperature. The precipitate was collected by centrifugation at 10,000 rpm for 10 minutes. The supernatant was discarded. The precipitate was washed three times each with DMF and anhydrous ethanol. Finally, the product was dried in a vacuum drying oven at 70 °C for 24 hours to obtain dark blue Hemin@HKUST-1 powder, which was stored in a desiccator for later use.

[0068] (4) Preparation of Hemin@HKUST-1-cDNA:

[0069] The preparation method is the same as that for the Fe3O4–aptamer complex, except that Hemin@HKUST-1 is used to replace Fe3O4–COOH and complementary strand DNA (cDNA) is used to replace the aptamer (Apt); thus, a Hemin@HKUST-1–cDNA complex dispersion is obtained.

[0070] (5) Assembly of the Fe3O4 / Hemin@HKUST-1 complex:

[0071] Take 25 μL of the prepared Fe3O4–Apt dispersion and 75 μL of Hemin@HKUST-1–cDNA dispersion and mix them evenly in a centrifuge tube. Incubate at room temperature for 65 minutes to allow the DBP aptamer (Apt) to fully hybridize with the complementary strand DNA (cDNA). Then, separate the mixture using a magnetic separation device, discard the supernatant, collect the magnetically adsorbed complex, and gently resuspend it in 100 μL of ultrapure water to obtain the Fe3O4 / Hemin@HKUST-1 complex dispersion.

[0072] The DBP aptamer sequence is as follows:

[0073] 5'-NH2-CTTTCTGTCCCCGTCACATCCCACGCATTCTCCACAT-3';

[0074] The complementary DNA sequence is as follows:

[0075] 5'-NH2-ATGTGGAGAATGCGTGGGATGTGACGGAAGGACAGAAAG-3'.

[0076] (6) Electrode modification:

[0077] A 3 mm diameter glassy carbon electrode (GCE) was polished sequentially on chamois leather using 0.3 μm and 0.05 μm alumina polishing powders (slurry) until a mirror-like finish was achieved. The electrode surface was then thoroughly rinsed with ultrapure water to remove any residual polishing agent. The polished and cleaned GCE electrode was then placed in a solution containing 5 mM [Fe(CN)6]. 3- / 4-Cyclic voltammetry (CV) scans were performed in a solution of 0.1 M KCl at a scan rate of 100 mV / s for 10 cycles until a stable and repeatable redox peak was obtained. The electrode performance was then assessed to verify the completion of the electrode surface treatment. 7 μL of a 1 mg / mL reduced graphene oxide (rGO) dispersion was drop-coated onto the pretreated GCE surface, allowed to air dry naturally, and then dried in a 60 ℃ oven for 30 minutes to obtain the rGO / GCE modified electrode.

[0078] (7) Sensor construction:

[0079] Add 3 μL of the sample solution (containing DBP) to 20 μL of Fe3O4 / Hemin@HKUST-1 complex dispersion and incubate at room temperature for 35 minutes. DBP specifically binds to the aptamer and competitively dissociates the originally hybridized cDNA, resulting in the release of some Hemin@HKUST-1–cDNA into the supernatant. Then, perform magnetic separation, accurately pipette 7 μL of the enriched fraction, drop it onto the surface of the rGO / GCE modified electrode, and dry at room temperature for 1 hour to obtain the Hemin@HKUST-1 / rGO / GCE electrochemical aptamer sensor.

[0080] The Hemin@HKUST-1 / rGO / GCE electrochemical aptamer sensor prepared in this embodiment can be used to detect dibutyl phthalate (DBP).

[0081] Example 2

[0082] The method for detecting dibutyl phthalate using the Hemin@HKUST-1 / rGO / GCE electrochemical aptamer sensor prepared in Example 1 is as follows:

[0083] In step 7, 3 μL of DBP standard solutions of different concentrations were added to 20 μL of Fe3O4 / Hemin@HKUST-1 complex dispersion. The resulting Hemin@HKUST-1 / rGO / GCE electrochemical aptamer sensor electrode was used as the working electrode, forming a three-electrode system together with the platinum wire counter electrode and the Ag / AgCl reference electrode. The electrode was then immersed in 10 mL of 0.1 M, pH 7.4 PBS buffer and detected using square wave voltammetry (SWV). SWV parameters were set as follows: scan range -0.6 V to 0 V, pulse amplitude 0.025 V, and settling time 10 seconds. The oxidation peak current value near -0.2 V was recorded. The current value of the blank sample without DBP was I0, and the current value of the sample containing DBP was I. The current change ΔI = I0 - I / (logarithm of DBP concentration (lgC)) was within 3 × 10⁻¹⁰. -6 mg / mL to 3×10 -3The sample exhibits a good linear relationship within the mg / mL range, with a linear regression equation of ΔI = 3.39527 lgC +6.2937 (R² = 0.99259). Based on this, DBP in the sample solution can be quantitatively analyzed.

[0084] Example 3

[0085] Actual water sample testing:

[0086] Spiked recovery experiments were conducted using commercially available tap water as the actual sample. 10 mL of tap water was taken and spiked with three concentration levels (3 × 10⁻⁶) of water at low, medium, and high concentrations, respectively. -5 3×10 -4 3×10 -3 DBP standard (mg / mL) was vortexed and then analyzed using the Hemin@HKUST-1 / rGO / GCE electrochemical aptamer sensor preparation method of Example 1 and the detection method of Example 2. Each concentration level was measured in parallel five times. The concentration was calculated based on the standard curve, and the spiked recovery rate and relative standard deviation (RSD) were calculated. The results (see Table 1) showed that the spiked recovery rate of DBP ranged from 98.03% to 107.21%, and the RSD was less than 3.76%, indicating that the method has good accuracy and precision and can be used for rapid and reliable detection of DBP in real samples.

[0087] Table 1. Results of DBP Spiking Recovery in Tap Water

[0088]

[0089] The composite material prepared in Example 1 was characterized in this invention:

[0090] like Figure 2 , 3As shown, the Hemin@HKUST-1 sample synthesized in step (2) was dropped onto a copper mesh carbon film, dried, and observed under a transmission electron microscope. The results showed that the particles were mostly irregular polygons, proving that the introduction of heme affected the growth of MOF crystals. Fe3O4 nanoparticles were uniformly dispersed on conductive adhesive and characterized by scanning electron microscopy. The results showed that they were regular spheres, uniformly dispersed, and had an average particle size of about 200 nm. Samples were prepared by KBr pellet method and detected by Fourier transform infrared spectroscopy. The results showed that Hemin@HKUST-1 had characteristic peaks of both heme and HKUST-1. The C=O stretching vibration peak position was slightly shifted, indicating that there was a hydrogen bond or coordination effect between heme and MOF. Further powder X-ray diffraction test showed that its diffraction peak position was basically consistent with that of HKUST-1, only the peak intensity was reduced, proving that heme existed in the framework in a dispersed or amorphous form. X-ray photoelectron spectroscopy (XPS) analysis revealed that the material contains C, O, N, Fe, and Cu elements, with Fe primarily existing in the Fe(III) state and Cu remaining in the Cu(II) state, indicating a stable material structure. Zeta potential analysis showed that the potential gradually shifted from positive to negative during the stepwise modification process, with the final Hemin@HKUST-1-Apt composite exhibiting a significant negative charge, demonstrating successful aptamer immobilization on the surface.

[0091] This invention employs a three-electrode system for electrochemical characterization:

[0092] like Figure 4 As shown, a glassy carbon electrode (3 mm in diameter) was used as the working electrode, a platinum wire as the counter electrode, and a saturated calomel electrode as the reference electrode. The electrolyte was 0.1 mol / L PBS (pH=7.0). Initially, the bare glassy carbon electrode showed a weak current signal. Subsequently, modification with reduced graphene oxide significantly enhanced the current, indicating improved electron transport capability. Further modification with Hemin@HKUST-1 resulted in a decrease in current, suggesting the composite material covered the electrode surface. Finally, modification with the aptamer further reduced the current, confirming successful aptamer immobilization. Electrochemical impedance spectroscopy results showed that the charge transfer resistance gradually increased with the modification process, as expected. Cyclic voltammetry at different scan rates showed that the peak current was proportional to the square root of the scan rate, indicating that the reaction process was diffusion-controlled and quasi-reversible.

[0093] Comparison of effects with different parameters:

[0094] like Figure 5As shown, this invention compares different parameters of the detection conditions. When comparing the aptamer incubation time, the electrode modified with rGO / Hemin@HKUST-1 / Fe3O4 was immersed in a PBS solution containing 1 μM of aptamer and incubated for 2 h, 3 h, 4 h, 5 h, 6 h, 12 h, and 24 h, respectively, and its response signal was detected. The results showed that the signal was optimal at 5 h, and remained slightly unchanged after 5 h. When comparing the Fe3O4 particle size, sensors were constructed using Fe3O4 particles of 100 nm, 200 nm, and 300 nm, respectively, and the DBP response was detected. The results showed that the signal was strongest at a particle size of 200 nm. By adjusting the pH of the PBS buffer in the range of 6.0–8.0, the results showed that the detection performance was best at pH 7.0. When comparing the ratio of Fe3O4 to Hemin@HKUST-1, detection was performed at ratios of 1:1, 1:2, 1:3, and 1:4, respectively, and the results showed that the 1:3 ratio performed best. Comparing hybridization incubation times, the electrodes were incubated in the target DNA solution for 30–90 min, and the results showed that the signal was strongest at 75 min. Comparing DBP incubation times, tests were conducted within the range of 10–50 min, and the results showed that 35 min was the optimal condition.

[0095] like Figure 6 As shown, this invention applies different concentrations of DBP standard solutions (3×10⁻⁻⁴). 6 Electrochemical detection was performed using a concentration of -3 × 10⁻³ mg / mL. Changes in the redox peak current were recorded and a standard curve was plotted. The results showed a good linear relationship between the current and the DBP concentration, with a correlation coefficient greater than 0.99. The limit of detection was calculated to be 2.90 × 10⁻³ mg / mL. 6 mg / mL.

[0096] To verify the reliability of the method, DBP was compared with potential interfering substances such as dimethyl phthalate, dioctyl phthalate, and phenol. The operating conditions were the same as in Examples 1 and 2. The results showed that only DBP elicited a significant current response, indicating that the method has good selectivity. In the reproducibility test, five independent electrodes were prepared and the same concentration of DBP was detected under the same conditions. The relative standard deviation was 1.94%, demonstrating the excellent repeatability of the method. In the stability test, the modified electrode was stored at 4°C, and detection was performed every other day. The results showed that it still maintained more than 76% of the initial current response after 7 days, demonstrating the good stability of the electrochemical aptamer sensor and detection method of this invention.

[0097] In summary, this invention provides an electrochemical aptamer sensing method based on Fe3O4 and Hemin@HKUST-1 composite materials. This method specifically includes steps such as material characterization, electrode modification, electrochemical detection, condition optimization, performance evaluation, selectivity verification, and actual sample analysis. It can achieve highly sensitive and selective detection of dibutyl phthalate and has the advantages of simple operation, wide detection range, low detection limit, good repeatability, and excellent stability. It can be widely used in the fields of food safety and environmental monitoring.

[0098] In this embodiment of the invention, the aptamer sequence is:

[0099] 5'-NH2-CTTTCTGTCCCCGTCACATCCCACGCATTCTCCACAT-3', its complementary strand sequence is:

[0100] 5'-NH2-ATGTGGAGAATGCGTGGGATGTGACGGAAGGACAGAAAG-3' was provided by Shanghai Shenggong Bioengineering Technology Service Co., Ltd. Fe3O4 nanoparticles were purchased from Nanjing Xianfeng Nanomaterials Technology Co., Ltd., and the Hemin@HKUST-1 composite material was prepared according to the embodiments of this invention. All solutions were prepared using ultrapure water. The morphology and elemental composition of the 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 performed on a Shanghai Chenhua CHI 760E electrochemical workstation using a conventional three-electrode system, including a modified glassy carbon electrode (GCE, Φ=3 mm) as the working electrode, a platinum wire as the counter electrode, and Ag / AgCl as the reference electrode. Square wave voltammetry (SWV) was performed directly in 0.1 M PBS buffer (pH = 7.0) with a scan potential range of -0.2 to 0.6 V, a pulse amplitude of 0.025 V, and a frequency of 15 Hz.

Claims

1. An electrochemical aptamer sensor for the detection of dibutyl phthalate, characterized in that it comprises: Fe3O4 magnetic nanoparticles modified with dibutyl phthalate (DBP) aptamer; Hemin encapsulates the metal-organic framework Hemin@HKUST-1 as a signal amplification unit; Complementary DNA strands hybridize with DBP aptamers to form double-stranded structures. The above components are fixed on the surface of the glassy carbon electrode GCE substrate modified with reduced graphene oxide (rGO); The DBP aptamer sequence is as follows: 5'-NH2-CTTTCTGTCCCCGTCACATCCCACGCATTCTCCACAT-3'; The complementary DNA sequence is as follows: 5'-NH2-ATGTGGAGAATGCGTGGGATGTGACGGAAGGACAGAAAG-3'.

2. The method for preparing an electrochemical aptamer sensor for detecting dibutyl phthalate according to claim 1, characterized in that: Includes the following steps: (1) Preparation of carboxylated Fe3O4 magnetic nanoparticles: A certain amount of Fe3O4 magnetic nanoparticles were weighed and dispersed in a hydrated citric acid solution and stirred thoroughly at room temperature. After the reaction was completed, the precipitate was collected by magnetic separation and washed with acetone until the pH value was 7.0-7.

5. Finally, the product was dried to obtain carboxyl-functionalized Fe3O4–COOH powder. (2) Preparation of Fe3O4–aptamer complex: The Fe3O4–COOH prepared above was dispersed in water to prepare an Fe3O4–COOH aqueous dispersion; a certain amount of Fe3O4–COOH aqueous dispersion was measured and mixed with an equal volume of EDC / NHS solution, and activated at room temperature for 30-50 minutes. Then, a certain amount of DBP aptamer solution was added, mixed evenly, and incubated at room temperature for 3-5 hours. After the reaction was completed, the mixed solution was centrifuged to remove unreacted aptamer molecules. The precipitate was collected, washed with ultrapure water, and redispersed in ultrapure water to obtain Fe3O4–Apt complex dispersion. (3) Preparation of Hemin@HKUST-1: 1,3,5-Benzotricarboxylic acid and heme were dissolved together in a volume ratio of 5:12 in a certain amount of a mixed solvent composed of N,N-dimethylformamide and ethanol, and stirred until completely dissolved to obtain an organic solution. Alternatively, a certain amount of Cu(NO3)2·3H2O is dissolved in ultrapure water to obtain an aqueous solution of copper nitrate; under continuous stirring, the aqueous solution of copper nitrate is slowly added dropwise to the above organic solution. After stirring for a period of time, the mixture is transferred to an autoclave and heated at 120℃-160℃ for 20-25 hours. After the reaction was completed, the mixture was allowed to cool naturally to room temperature. The precipitate was collected by centrifugation. The precipitate was washed successively with N,N-dimethylformamide and anhydrous ethanol, and then dried in a vacuum drying oven to obtain dark blue Hemin@HKUST-1 powder. (4) Preparation of Hemin@HKUST-1-cDNA: The preparation method is the same as that for the Fe3O4–aptamer complex, except that Hemin@HKUST-1 is used to replace Fe3O4–COOH and complementary strand DNA is used to replace DBP aptamer; a Hemin@HKUST-1–cDNA complex dispersion is obtained. (5) Assembly of the Fe3O4 / Hemin@HKUST-1 complex: A certain amount of Fe3O4–Apt dispersion and a certain amount of Hemin@HKUST-1–cDNA dispersion were mixed evenly in a centrifuge tube and incubated at room temperature for 50-75 minutes. Then, the mixture was separated using a magnetic separation device, and the magnetically adsorbed complex was collected and resuspended in ultrapure water to obtain the Fe3O4 / Hemin@HKUST-1 complex dispersion. (6) Electrode modification: The glassy carbon electrode GCE after polishing and cleaning was tested for electrode performance by cyclic voltammetry until the voltammetric response stabilized; the reduced graphene oxide (rGO) dispersion was drop-coated onto the pretreated GCE surface, air-dried naturally, and then dried in an oven to obtain the rGO / GCE modified electrode. (7) Sensor construction: Add a certain amount of the test sample solution containing DBP to the Fe3O4 / Hemin@HKUST-1 complex dispersion and incubate at room temperature for 35-45 minutes; Magnetic separation was then performed, and the enriched components were collected and drop-coated onto the surface of the rGO / GCE modified electrode. After drying at room temperature, the Hemin@HKUST-1 / rGO / GCE electrochemical aptamer sensor was obtained.

3. The method for preparing an electrochemical aptamer sensor for detecting dibutyl phthalate according to claim 2, characterized in that: In step (1), the concentration of the citric acid monohydrate solution is 0.5 M; the average particle size of the Fe3O4 magnetic nanoparticles is 200 nm.

4. The method for preparing an electrochemical aptamer sensor for detecting dibutyl phthalate according to claim 2, characterized in that: In step (2), the concentration of the Fe3O4–COOH aqueous dispersion is 2 mg / mL; the mass ratio of EDC to NHS in the EDC / NHS solution is 4:1; and the concentration of the DBP aptamer solution is 100 μM.

5. The method for preparing an electrochemical aptamer sensor for detecting dibutyl phthalate according to claim 2, characterized in that: In step (3), the N,N-dimethylformamide and ethanol are mixed in a volume ratio of 1:1 in the mixed solvent.

6. The method for preparing an electrochemical aptamer sensor for detecting dibutyl phthalate according to claim 2, characterized in that: In step (5), the volume ratio of Fe3O4–Apt dispersion to Hemin@HKUST-1–cDNA dispersion is 1:

3.

7. The method for preparing an electrochemical aptamer sensor for detecting dibutyl phthalate according to claim 2, characterized in that: In step (6), the concentration of the reduced graphene oxide (rGO) dispersion is 1 mg / mL.

8. The application of the electrochemical aptamer sensor for the detection of dibutyl phthalate according to claim 1, or the electrochemical aptamer sensor for the detection of dibutyl phthalate according to any one of claims 2-7, characterized in that: The Hemin@HKUST-1 / rGO / GCE electrochemical aptamer sensor was used to detect dibutyl phthalate (DBP).

9. The application according to claim 8, characterized in that: The method for detecting dibutyl phthalate using the prepared Hemin@HKUST-1 / rGO / GCE electrochemical aptamer sensor is as follows: The Hemin@HKUST-1 / rGO / GCE electrochemical aptamer sensor electrode was used as the working electrode, forming a three-electrode system with a platinum wire counter electrode and an Ag / AgCl reference electrode. The system was immersed in 0.1 M PBS buffer (pH 7-7.4) and detected using square wave voltammetry. The oxidation peak current value near -0.2 V was recorded. The current value of the blank sample without DBP was I0, and the current value of the sample containing DBP was I. The current change ΔI = I0 - I, with the logarithm of the DBP concentration within 3 × 10⁻¹⁰, was calculated. -6 mg / mL to 3×10 -3 The sample showed a good linear relationship within the mg / mL range, with a linear regression equation of ΔI = 3.39527 lgC + 6.2937 and R² = 0.99259. Based on this, the DBP in the test sample solution was quantitatively analyzed.

10. The application according to claim 9, characterized in that: Set the square wave voltammetry parameters: scan range -0.6 V to 0 V, pulse amplitude 0.025 V, rest time 10 seconds.

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