Method for detecting patulin in fruits by ratio-type molecularly imprinted electrochemical sensor and modified electrode

Abstract

The present application relates to the technical field of food safety detection, in particular to a ratio type molecular imprinting electrochemical sensing detection method for patulin in fruits and a modified electrode thereof, and the AgNPs@Ti2NT x IL nanocomposite is synthesized by etching and ultrasonic assisted method, the nanocomposite is used as a base material, thionine (Thi) is used as a functional monomer, and patulin (PAT) is used as a template molecule, and a ratio type molecular imprinting electrochemical sensor (MIPs-RECS) for high-sensitivity detection of PAT is prepared by an electro-polymerization method. In the sensor, signals generated by silver nanoparticles (AgNPs) and Thi are used as working signals and reference signals respectively, and the MIPs-RECS is successfully constructed. The modified electrode has good reproducibility, repeatability and anti-interference ability, and shows high specificity for PAT detection.

Description

Technical Field

[0001] This invention relates to the field of food safety testing technology, and in particular to a ratio-type molecularly imprinted electrochemical sensing method for detecting patulin in fruits and its modified electrode. Background Technology

[0002] Patulin (PAT) is a fungal toxin produced by fungi of the genera *Penicillium* or *Aspergillus*, which commonly grow in fruits, vegetables, and their products. Initially used as an antibiotic, it was quickly discovered that this substance causes a range of health risks in animals, including tissue edema, ulcers, intestinal inflammation, and epithelial cell degeneration. Therefore, in addition to ceasing the use of PAT as an antibiotic, countries have strictly limited the maximum residue limits (MRLs) for PAT in agricultural products. For example, the European Union stipulates that the PAT content in food must not exceed 10 μg / kg, and my country has set the MRL for PAT in fruits and their products at 50 μg / kg. Therefore, monitoring the PAT content in agricultural products is crucial.

[0003] Due to their large surface area and excellent electrochemical activity, MXenes are widely used in the construction of electrochemical sensors (ECS). Although MXenes are defined as a class of transition metal carbides or nitrides, Ti3C2 is the most frequently reported, followed by other carbon-based MXenes, while nitrogen-based MXenes are rarely reported. This is because conventional carbon-based MXenes can generally be obtained by etching the corresponding MAX phase using aqueous HF, while nitrogen-based MXenes are relatively unstable in aqueous HF, making it difficult to successfully prepare sheet-like nitrogen-based MXenes. However, compared to carbon-based MXenes, nitrogen-based MXenes have better conductivity, thus being more suitable for constructing high-performance ECSs.

[0004] Due to their high specificity, low cost, simple operation, and high sensitivity, molecularly imprinted electrochemical sensors (MIPs-ECS) have been extensively studied for detecting mycotoxins. Currently, most MIPs-ECS employ a single-signal output mode, utilizing a single redox signal generated by the electrocatalysis of the mycotoxin itself. However, patulin has low electroactivity, making it difficult to achieve a low detection limit. Therefore, researchers have introduced probe molecules into MIPs-ECS. Among them, [Fe(CN)6] is a suitable probe. 4- / 3-Most probe molecules are used, but small probe molecules cannot be effectively eluted after entering the imprinted pores due to capillary adsorption, ultimately leading to poor detection repeatability. However, whether using the sensor's own redox signal or probe molecule signal to construct it, a single-signal output mode is generally employed. This single-signal output mode is susceptible to intrinsic factors such as differences in electrode preparation and human operation, as well as external factors such as matrix effects and changes in the external environment, inevitably resulting in poor repeatability and low reproducibility. Another approach involves incorporating a built-in calibration mechanism into the sensor, creating a ratiometric sensor with a dual-signal output mode. This significantly eliminates the influence of systematic errors such as background current and external factors such as changes in the external environment, thereby improving the sensor's stability and repeatability. However, most current ratiometric molecularly imprinted electrochemical sensors (MIPs-RECS) use both probe molecule and analyte electrochemical redox signals as dual signals, but this method still cannot eliminate the problem of decreased stability of the molecularly imprinted film after repeated use, leading to poor data reliability. Therefore, researchers have explored functional monomers whose redox signals can represent indicators of molecularly imprinted membrane stability, thereby determining the reliability of current data from molecularly imprinted sensors. Currently, there is almost no research on using these functional monomers and probe molecules to construct MIPs-RECS for monitoring patulin. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a ratiometric molecularly imprinted electrochemical sensing detection method for patulin in fruits and its modified electrode.

[0006] Compared to the traditional [Fe(CN)6] 4- / 3- Compared to probe molecules, this invention reduces silver ions to silver nanoparticles (AgNPs) on MXene nanosheets through an in-situ process. This allows AgNPs to not only enhance the conductivity of the molecularly imprinted sensor but also provide better performance than [Fe(CN)6]. 4- / 3- A more stable electrochemical signal. Furthermore, to address the poor repeatability and low reproducibility issues of single-signal output modes, this invention introduces a functional monomer that can both form an imprinted molecular film with the analyte and provide an internal reference signal for internal calibration, building upon existing molecular imprinting sensing. This constitutes a dual-signal output mode (the other signal is an AgNPs signal correlated with the concentration of the target analyte). The patulin detection method constructed in this invention exhibits good stability, high repeatability, and strong reliability.

[0007] The modified electrode of this invention utilizes AgNPs@Ti2NT x A novel MIPs-RECS was developed using @IL nanocomposites. This invention synthesizes AgNPs@Ti2NT using an etching and ultrasound-assisted method.x Using @IL nanocomposite materials as the substrate, Thi as the functional monomer, and PAT as the template molecule, a highly sensitive MIPs-RECS for PAT detection was prepared by electropolymerization. In this sensor, the signals generated by AgNPs and Thi were used as the working and reference signals, respectively, successfully constructing a ratiometric MIPs-RECS. The results show that the sensor of this invention possesses excellent specificity, stability, and anti-interference ability, and demonstrates promising prospects for practical applications.

[0008] Specifically, this application provides the following technical solution:

[0009] A method for preparing a modified electrode for detecting patulin in fruits includes the following steps:

[0010] (1) Polishing treatment of substrate working electrode

[0011] 1.1 Polishing the working electrode: First, use alumina polishing powder to polish the working electrode into a mirror surface. Then, ultrasonically clean the polished working electrode in distilled water, anhydrous ethanol, and distilled water in sequence to thoroughly remove alumina powder and other contaminants adsorbed on the surface of the working electrode. Finally, dry it in cold air.

[0012] 1.2 Determining if electrode polishing is qualified: Place the working electrode, reference electrode, and counter electrode system dried in step 1.1 into an electrochemical probe solution, scan using cyclic voltammetry, and compare with the theoretical standard spectrum. If the potential difference between the two peaks is within the specified range, the electrode polishing is qualified.

[0013] (2)AgNPs@Ti2NT x Synthesis of @IL Nanocomposites

[0014] 2.1Ti2NT x Synthesis

[0015] Ti2NT was prepared using a KF-HCl mixture as an etchant. x Nanosheets. First, 10 mL of the prepared KF-HCl mixture was added to a centrifuge tube containing 1 g of Ti₂AlN powder, and the mixture was reacted at room temperature for 3 h, followed by sonication for 1 h. The resulting suspension was washed to neutral by centrifugation (6000 rpm, 5 min each time). After washing, the suspension was resuspended in isopropanol and centrifuged again at 6000 rpm for 30 min. The centrifuged product was then collected and dried in a vacuum drying oven to obtain multilayer Ti₂NT. x Weigh out 100 mg of multilayer Ti2NT. xDispersed in 10 mL of DMSO solution, and ultrasonically treated for 1 hour in an ultrasonic machine to prepare multilayer Ti2NT. x Further separation was performed. After separation, the DMSO solution was removed by centrifugation at 6000 rpm for 30 min. The obtained product was then resuspended in deionized water, followed by centrifugation at 3500 rpm for 30 min to collect the supernatant. Finally, the supernatant was placed in a vacuum freeze-drying oven and freeze-dried for 48 h to obtain Ti2NT. x Nanoparticle powder.

[0016] 2.2 Synthesis of Diallyl Hexafluorophosphate (IL)

[0017] First, 20 mmol of N-allyl imidazole was dissolved in 25 mL of toluene, and then 10 mmol of 1,4-dibromobutane was added dropwise while stirring at 75 °C. The mixture was stirred continuously for 24 h, and then cooled to room temperature. After the reaction was complete, the supernatant was poured off, and the product was washed three times with ethyl acetate and diethyl ether, respectively. The resulting product was then dried under vacuum at 60 °C for 1 h to obtain diallyl imidazole bromide. Next, 3 mmol of diallyl imidazole bromide was dissolved in 30 mL of deionized water, and 6 mmol of potassium hexafluorophosphate was added. The mixture was stirred at 85 °C for 6 h to obtain 1 L of diallyl hexafluorophosphate.

[0018] 2.3 Ultrasound-assisted synthesis of AgNPs@Ti2NT x @IL Nanocomposite Materials

[0019] First, weigh 10 mg Ti2NT x The powder was dispersed in 2.5 mL of 10 mM AgNO3 solution and reacted under ultrasonication for 1 h to obtain AgNPs@Ti2NT. x Composite material. Meanwhile, 30 mg IL was weighed and dissolved in 2.5 mL DMSO. Then, AgNPs@Ti2NT was added. x The prepared IL solution was added to the suspension, and the mixture was sonicated for another 1 hour to obtain AgNPs@Ti2NT. x @IL suspension. The obtained suspension was centrifuged and washed several times. After washing, the precipitate obtained by centrifugation was placed in a vacuum freeze-drying oven and freeze-dried for 48 hours to obtain AgNPs@Ti2NT. x @IL nanocomposite powder. Weigh 2mg AgNPs@Ti2NT x AgNPs@Ti2NT can be obtained by dispersing the IL nanocomposite powder in a 1:1 mixture of 1.0 mL DMF and water. x @IL suspension.

[0020] (3)MIP / AgNPs@Ti2NTx @IL / GCE build

[0021] GCE was carefully polished and ultrasonically cleaned with deionized water before modification. Then, 5.0 μL of AgNPs@Ti2NT was added. x The @IL suspension was dropped onto a clean surface of the GCE and then dried in an infrared oven. After the electrode cooled to room temperature, the prepared AgNPs@Ti2NT were... x @IL / GCE was immersed in an electrolyte solution containing 1.0 mM PAT and 6.0 mM functional monomers. A three-electrode system was constructed, and electropolymerization was performed using CV at a scan rate of 100 mV / s and a potential range of -0.85 to 0.85 V for 20 cycles. After electropolymerization, the modified GCE was immersed in 2.0 M sulfuric acid for 6 min to remove PAT molecules. Finally, the modified GCE was carefully washed with deionized water and dried under a tungsten filament lamp. The resulting electrode was labeled MIP / AgNPs@Ti2NT. x @IL / GCE.

[0022] Furthermore, in step 1.1, the particle size of the alumina polishing powder is 0.04–0.06 μm.

[0023] Furthermore, in step 1.2, the working electrode is any one of a glassy carbon electrode, a graphite electrode, a gold electrode, or a platinum electrode.

[0024] Furthermore, in step 1.2, the reference electrode is a saturated calomel electrode or a silver / silver chloride electrode.

[0025] Furthermore, in step 1.2, the counter electrode is any one of the inert metals that does not undergo a redox reaction in the detection circuit. Preferably, the counter electrode is platinum, gold, or tungsten.

[0026] Furthermore, in step 1.2, the electrochemical probe solution is a 5 mmol / L [Fe(CN)6] solution containing 0.1 mol / L KCl. 3- / 4- The solution may contain 5 mmol / L [Ru(NH3)6] with 0.1 mol / L KCl. 2+ / 3+ Solution.

[0027] Furthermore, in step 1.2, the specified range refers to below 100mV.

[0028] Further, in step (3), the electrolyte solution is phosphate buffer, acetate buffer or BR buffer.

[0029] Furthermore, in step (3), the functional monomer is any one of thionine, methylene blue, p-aminothiophenol, and pyrrole.

[0030] The method for ratiometric molecularly imprinted electrochemical sensing detection of patulin in fruits using the modified electrode prepared by the above method includes the following steps:

[0031] (1) Establish the standard working curve for PAT testing:

[0032] ① Prepare an electrolyte solution in an electrolytic cell that does not participate in chemical reactions and is conductive, and introduce an inert gas into the electrolyte solution;

[0033] ② Dissolve PAT in a solvent that can dissolve PAT and does not react with PAT to prepare a PAT standard stock solution;

[0034] ③MIP / AgNPs@Ti2NT x The IL / GCE modified electrode, reference electrode, and counter electrode were placed in the electrolyte solution treated with inert gas in step ①, and the Ag oxidation peak current (Ig) before PAT incubation was detected using square wave voltammetry (SWV). Ag ) and Thi oxidation peak current (I Thi MIP / AgNPs@Ti2NT x The IL / GCE modified electrode was placed in the electrolyte solution treated with inert gas in step ①. Then, different volumes and concentrations of the PAT standard stock solution prepared in step ② were added to the electrolyte solution. After stirring and incubation, the electrode was rinsed with deionized water to remove non-specifically adsorbed PAT. After standing and drying, the modified electrode, reference electrode, and counter electrode were placed in the electrolyte solution with optimized pH. The Ag oxidation peak current (Ig) after PAT incubation was detected using square wave voltammetry. Ag ) and Thi oxidation peak current (I Thi ); using the Ag oxidation peak current (I) before and after PAT incubation Ag ) and Thi oxidation peak current (I Thi The difference in the ratio [Δ(I)] Ag / I Thi A linear relationship was established between the concentration of PAT incubated and the concentration of PAT, with the PAT concentration as the x-axis and the Ag oxidation peak current (I) before and after PAT incubation as the y-axis. Ag ) and Thi oxidation peak current (I Thi The difference in the ratio [Δ(I)] Ag / I Thi Using [] as the ordinate, establish the PAT testing standard working curve.

[0035] (2) Rapid quantitative analysis of PAT in samples:

[0036] An electrolyte solution treated with an inert gas in step ① was added to a certain volume of solution containing an unknown PAT concentration, and the experimental parameters were adjusted. Then, the MIP / AgNPs@Ti2NT prepared according to this invention was added. x The IL / GCE modified electrode was immersed in solution for incubation. After incubation, the non-specifically adsorbed PAT was rinsed with deionized water. After standing and drying, the modified electrode, reference electrode, and counter electrode were placed in an electrolyte solution with optimized pH. The Ag oxidation peak current (Ig) before and after PAT incubation was measured using square wave voltammetry. Ag ) and Thi oxidation peak current (I Thi Finally, based on the PAT detection standard working curve established in step (1), the unknown PAT concentration in the actual sample is obtained.

[0037] Further, the electrolyte solution is a phosphate buffer solution; preferably, in some embodiments of the present invention, the phosphate buffer solution is a 0.1 mol / L mixed solution of disodium hydrogen phosphate, sodium dihydrogen phosphate and sodium chloride.

[0038] Furthermore, the inert gas is nitrogen, helium, or neon.

[0039] Furthermore, the preparation method of the PAT standard stock solution is as follows: PAT is dissolved in ethanol to prepare a PAT standard stock solution with a concentration of 10.00 mg / mL.

[0040] Furthermore, the reference electrode is a saturated calomel electrode or a silver / silver chloride electrode, and the counter electrode is any one of the inert metals that does not undergo redox reactions in the detection circuit.

[0041] Compared with the prior art, the ratiometric molecularly imprinted electrochemical sensing detection method for patulin in fruits and its modified electrode of the present invention have at least the following beneficial effects:

[0042] (1) This invention uses electropolymerization to directly polymerize MIP on the working electrode. The Ag oxidation peak current (Ig) before and after PAT incubation is used to measure this. Ag To determine the PAT concentration, as the added PAT concentration increases, the Ag oxidation peak current (Ig) increases. Ag The smaller the value of the internal reference signal peak Thi oxidation peak current (I) will be, the lower the value of the internal reference signal peak. Thi Since the current remains constant, the ratio of the two detection signals can be used to reduce instrument or environmental errors, thereby providing more accurate detection results for trace analysis of PAT.

[0043] (2) This invention uses the Ag oxidation peak current (I) before and after PAT incubation. Ag ) and Thi oxidation peak current (I Thi The difference in the ratio [Δ(I)] Ag / I Thi A linear relationship was established between the concentration of PAT and the concentration of PAT during incubation, with the concentration of PAT as the x-axis and the Ag oxidation peak current (I) before and after PAT incubation as the y-axis. Ag ) and Thi oxidation peak current (I Thi The difference in the ratio [Δ(I)] Ag / I Thi Using [] as the ordinate, a standard working curve for PAT detection was established. The method for measuring unknown concentrations of PAT using the standard working curve is simple, convenient, fast, has good repeatability and reproducibility, and strong anti-interference ability. It can be used for rapid quantitative detection of PAT in actual samples such as apples and pears.

[0044] The following description, in conjunction with the accompanying drawings, further illustrates the ratio-type molecularly imprinted electrochemical sensing detection method for patulin in fruits according to the present invention and its modified electrode. Attached Figure Description

[0045] Figure 1 This is a schematic diagram of the preparation process of the modified electrode for detecting patulin in fruits according to the present invention;

[0046] Figure 2 MIP / AgNPs@Ti2NT in this embodiment of the invention x @IL / GCE modified electrode, Ti2NT x / GCE, AgNPs@Ti2NT x / GCE, AgNPs@Ti2NT x @IL / GCE, NIP / AgNPs@Ti2NT x Square wave voltammetry plots of the @IL / GCE and unmodified substrate working electrodes in a buffer solution containing 0.1 ng / mL PAT.

[0047] Figure 3 MIP / AgNPs@Ti2NT in this embodiment of the invention x Square wave voltammetry curves of the @IL / GCE modified electrode in buffer solutions containing different concentrations of PAT.

[0048] Figure 4 MIP / AgNPs@Ti2NT in this embodiment of the invention x Standard curves of the @IL / GCE modified electrode in buffer solutions containing different concentrations of PAT.

[0049] Figure 5 MIP / AgNPs@Ti2NT in this embodiment of the invention x Anti-interference capability diagram of @IL / GCE modified electrode.

[0050] Figure 6MIP / AgNPs@Ti2NT in this embodiment of the invention x Reproducibility plot of the @IL / GCE modified electrode.

[0051] Figure 7 MIP / AgNPs@Ti2NT in this embodiment of the invention x Repeatability plot of the @IL / GCE modified electrode.

[0052] Figure 8 MIP / AgNPs@Ti2NT in this embodiment of the invention x Material stability diagram of the @IL / GCE modified electrode.

[0053] Figure 9 MIP / AgNPs@Ti2NT in this embodiment of the invention x Long-term stability graph of the @IL / GCE modified electrode. Detailed Implementation

[0054] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. Additional aspects and advantages of this invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. It should be understood that the following description is merely illustrative and not intended to limit the invention.

[0055] The terms “comprising,” “including,” “having,” “containing,” or any other variations thereof, as used herein, are intended to cover a non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such composition, step, method, article, or apparatus.

[0056] When a quantity, concentration, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pair of any upper or preferred value with any lower or preferred value, regardless of whether the range is disclosed individually. For example, when the range “1 to 5” is disclosed, the described range should be interpreted as including the ranges “1 to 4”, “1 to 3”, “1 to 2”, “1 to 2 and 4 to 5”, “1 to 3 and 5”, etc. When numerical ranges are described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within that range.

[0057] The singular form includes the plural objects of discussion unless the context clearly indicates otherwise. "Optional" or "any one" means that the matter or event described thereafter may or may not occur, and the description includes both the possibility that the event occurs and the possibility that the event does not occur.

[0058] The indefinite articles “a” and “an” preceding an element or component of this invention do not impose any limitation on the quantity (i.e., number of times) of the element or component. Therefore, “an” or “a” should be interpreted as including one or at least one, and the singular form of an element or component also includes the plural form, unless the quantity clearly refers only to the singular form.

[0059] Furthermore, the terms "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., described below refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms are not necessarily directed at the same embodiment or example. Moreover, the technical features involved in the various embodiments of the present invention can be combined with each other as long as they do not conflict with each other.

[0060] The abbreviations involved in the embodiments of this invention include:

[0061] 1. Patulin (PAT)

[0062] 2. Thionium

[0063] 3. Ionic liquids (IL)

[0064] 4. Dimethyl sulfoxide (DMSO)

[0065] 5. N,N-Dimethylformamide (DMF)

[0066] 6. Calomel electrode (SCE)

[0067] 7. Glassy carbon electrode (GCE)

[0068] 8. Phosphate Buffered Solution (PBS)

[0069] 9. Silver nanoparticles (AgNPs)

[0070] Example 1

[0071] like Figure 1 As shown, a MIPs-RECS based on AgNPs@Ti2NTx@IL nanocomposite material is prepared by the following steps:

[0072] (1) Polishing treatment of glassy carbon electrode (GCE) substrate

[0073] 1.1) Electrode polishing: First, the glassy carbon electrode is polished into a mirror surface using 0.05μm alumina polishing powder. Then, the polished glassy carbon electrode is ultrasonically cleaned in distilled water, anhydrous ethanol, and distilled water for 5 minutes in sequence to thoroughly remove the alumina powder and other contaminants adsorbed on the surface of the glassy carbon electrode. After that, it is dried in cold air.

[0074] 1.2) To determine if the electrode polishing is satisfactory, place the dried glassy carbon electrode, saturated calomel reference electrode, and platinum counter electrode system from step ① into 5 mL of 5 mmol / L [Fe(CN)6] solution containing 0.1 mol / L KCl. 3- / 4- In solution, cyclic voltammetry (CV) was used to scan the potential difference (ΔE) between the two peaks and compared with the theoretical standard spectrum. p =56mV), which is within the specified range (below ~100mV) and the electrode grinding is considered qualified.

[0075] (2)AgNPs@Ti2NT x Synthesis of @IL Nanocomposites

[0076] 2.1)Ti2NT x Synthesis

[0077] Ti2NT was prepared using a KF-HCl mixture as an etchant. x Nanosheets. First, 10 mL of the prepared KF-HCl mixture was added to a centrifuge tube containing 1 g of Ti₂AlN powder, and the mixture was reacted at room temperature for 3 h, followed by sonication for 1 h. The resulting suspension was washed to neutral by centrifugation (6000 rpm, 5 min each time). After washing, the suspension was resuspended in isopropanol and centrifuged again at 6000 rpm for 30 min. The centrifuged product was then collected and dried in a vacuum drying oven to obtain multilayer Ti₂NT. x Weigh out 100 mg of multilayer Ti2NT. x Dispersed in 10 mL of DMSO solution, and ultrasonically treated for 1 hour in an ultrasonic machine to prepare multilayer Ti2NT. x Further separation was performed. After separation, the DMSO solution was removed by centrifugation at 6000 rpm for 30 min. The obtained product was then resuspended in deionized water, followed by centrifugation at 3500 rpm for 30 min to collect the supernatant. Finally, the supernatant was placed in a vacuum freeze-drying oven and freeze-dried for 48 h to obtain Ti2NT. x Nanoparticle powder.

[0078] 2.2) Synthesis of IL-1 Diallyl Hexafluorophosphate

[0079] First, 20 mmol of N-allyl imidazole was dissolved in 25 mL of toluene, and then 10 mmol of 1,4-dibromobutane was added dropwise while stirring at 75 °C. The mixture was stirred continuously for 24 h, and then cooled to room temperature. After the reaction was complete, the supernatant was poured off, and the product was washed three times with ethyl acetate and diethyl ether, respectively. The resulting product was then dried under vacuum at 60 °C for 1 h to obtain diallyl imidazole bromide. Next, 3 mmol of diallyl imidazole bromide was dissolved in 30 mL of deionized water, and 6 mmol of potassium hexafluorophosphate was added. The mixture was stirred at 85 °C for 6 h to obtain 1 L of diallyl hexafluorophosphate.

[0080] 2.3) Synthesis of AgNPs@Ti2NT using ultrasound-assisted method x @IL Nanocomposite Materials

[0081] First, weigh 10 mg Ti2NT x The powder was dispersed in 2.5 mL of 10 mM AgNO3 solution and reacted under ultrasonication for 1 h to obtain AgNPs@Ti2NT. x Composite material. Meanwhile, 30 mg IL was weighed and dissolved in 2.5 mL DMSO. Then, AgNPs@Ti2NT was added. x The prepared IL solution was added to the suspension, and the mixture was sonicated for another 1 hour to obtain AgNPs@Ti2NT. x @IL suspension. The obtained suspension was centrifuged and washed several times. After washing, the precipitate obtained by centrifugation was placed in a vacuum freeze-drying oven and freeze-dried for 48 hours to obtain AgNPs@Ti2NT. x @IL nanocomposite powder. Weigh 2mg AgNPs@Ti2NT x AgNPs@Ti2NT can be obtained by dispersing the IL nanocomposite powder in a 1:1 mixture of 1.0 mL DMF and water. x @IL suspension.

[0082] (3)MIP / AgNPs@Ti2NT x @IL / GCE build

[0083] GCE was carefully polished and ultrasonically cleaned with deionized water before modification. Then, 5.0 μL of AgNPs@Ti2NT was added. x The @IL suspension was dropped onto a clean surface of the GCE and then dried in an infrared oven. After the electrode cooled to room temperature, the prepared AgNPs@Ti2NT were... x@IL / GCE was immersed in PBS (pH 6.0) containing 1.0 mM PAT and 6.0 mM Thi. A three-electrode system was constructed, and electropolymerization was performed using CV at a scan rate of 100 mV / s and a potential range of -0.85 to 0.85 V for 20 cycles. After electropolymerization, the modified GCE was immersed in 2.0 M sulfuric acid for 6 min to remove PAT molecules. Finally, the modified GCE was carefully washed with deionized water and dried under a tungsten filament lamp. The resulting electrode was labeled MIP / AgNPs@Ti2NT. x @IL / GCE. NIP / AgNPs@Ti2NT were prepared using the same method. x @IL / GCE, except for the absence of PAT in the electropolymerization solution, is identical to the preparation of MIP / AgNPs@Ti2NT. x The conditions for @IL / GCE are the same.

[0084] Example 2

[0085] The MIPs-RECS prepared in Example 1 were used to detect PAT. The specific steps of the detection method are as follows:

[0086] (1) Establish a standard working curve for PAT testing

[0087] ① Prepare a mixed solution of disodium hydrogen phosphate, sodium dihydrogen phosphate, and sodium chloride with a conductivity of 0.1 mol / L in an electrolytic cell. Adjust the pH to different values ​​and optimize to obtain a PBS buffer solution. Select the optimal pH of 6.0 for later use. Purge nitrogen gas into the PBS buffer solution to prevent gas interference in the PBS buffer solution.

[0088] ② Add 0.500 mL of ethanol to a bottle containing 5.000 mg of PAT powder. After complete dissolution, a PAT stock solution with a concentration of 10.00 mg / mL is obtained. Other series of PAT standard solutions are obtained by diluting the stock solution with ethanol. The stock solution and other series of standard solutions should be stored in a refrigerator.

[0089] The MIP / AgNPs@Ti2NT obtained in Example 1 x The IL / GCE modified electrode, reference electrode, and counter electrode were placed in 5 mL of 0.1 mol / L PBS buffer solution at pH 6 after inert gas treatment. The Ag oxidation peak current (Ig) before PAT incubation was measured using square wave voltammetry (SWV). Ag ) and Thi oxidation peak current (I ThiDifferent volumes of PAT standard stock solution of a certain concentration were added to PBS buffer solution to obtain PAT standard working solutions with concentrations ranging from 0.01 ng / mL to 1000 ng / mL. The modified electrode was placed in the solution, stirred and incubated, and then rinsed with deionized water to remove non-specifically adsorbed PAT. After standing and drying, the modified electrode, saturated calomel reference electrode, and platinum counter electrode were placed in 5 mL of the above-mentioned 0.1 mol / L PBS buffer solution at pH 6 after inert gas treatment. SWV was used for detection and analysis to obtain the Ag oxidation peak current (Ig) after incubation with PAT at different concentrations. Ag ) and Thi oxidation peak current (I Thi The oxidation peak current of the internal reference signal peak Thi is denoted as I. Thi PAT concentration is plotted on the x-axis, and Ag oxidation peak current (I) before and after PAT incubation is plotted on the y-axis. Ag ) and Thi oxidation peak current (I Thi The difference in the ratio [Δ(I)] Ag / I Thi Using [] as the ordinate, establish the PAT testing standard working curve. MIP / AgNPs@Ti2NT x The @IL / GCE modified electrode exhibits a good linear relationship with PAT (R 2 =0.9974, as Figure 4 As shown in the figure, square wave voltammetry curves of 0.1 mol / L PBS (pH 6.0) standard solutions containing different concentrations of PAT are plotted. Figure 3 The detection limit can be calculated to be as low as 0.007 ng / mL, which meets international limit standards.

[0090] Electrochemical response of modified electrode:

[0091] In 0.1 mol / L PBS (pH 6.0) solution, due to the absence of probe molecules, the bare electrode and Ti2NT... x No obvious peak was observed in / GCE. x The SWV response of / GCE shows a distinct oxidation peak around 0.1V, which is the oxidation peak of Ag. After adding IL, due to the excellent conductivity of IL and its affinity for Ti₂NT… x Stronger interactions make AgNPs@Ti2NT x The signal of @IL / GCE is further increased, which is more conducive to improving the sensing performance of MIPs-RECS for PAT; using Thi and PAT as functional monomers and template molecules, respectively, in AgNPs@Ti2NT xAfter electropolymerization of MIP on @IL / GCE, a distinct oxidation peak generated by Thi was observed at -0.22V; simultaneously, the Ag oxidation peak was significantly reduced, likely due to the inhibition of electron transfer caused by Thi polymerization, leading to a decrease in the Ag oxidation signal. Following sulfuric acid elution, the removal of template molecules resulted in the formation of MIP / AgNPs@Ti2NT. x @IL / GCE regained a large current response at 0.1V. After further incubation with PAT, the specific cavity was significantly reduced, resulting in a further decrease in the Ag signal. The results are as follows... Figure 2 As shown.

[0092] Interference immunity assessment of electrochemical sensors:

[0093] The MIP / AgNPs@Ti2NT prepared in this invention x The @IL / GCE sensor exhibits strong anti-interference capabilities. The electrochemical signal showed no significant change after the addition of ochratoxin A, aflatoxin B1, citrinin, zearalenone, vitamin C, fructose, KCl, and KNO3, indicating no significant interference in detection. Figure 5 As shown.

[0094] Reproducibility evaluation of electrochemical sensors:

[0095] Use 5 MIP / AgNPs@Ti2NT x The @IL / GCE modified electrode was incubated with 10 ng / mL PAT solution under optimal conditions, and measurements were performed in parallel. The relative standard deviation (RSD) of the peak current was 1.2%, indicating that the MIP / AgNPs@Ti2NT modified electrode... x The @IL / GCE modified electrode exhibits good reproducibility, such as Figure 6 As shown.

[0096] Repeatability evaluation of PAT detection by electrochemical sensor:

[0097] Using a MIP / AgNPs@Ti2NT x The sensor was eluted with IL / GCE and incubated in 10 ng / mL PAT solution for five cycles. The RSD of the five cycles was 3.6%, indicating that the constructed sensor has good reusability. Figure 7 As shown.

[0098] Stability assessment of electrochemical sensors:

[0099] MIP / AgNPs@Ti2NT xThe @IL / GCE modified electrode was subjected to 10 consecutive SWV measurements in PBS without PAT. The RSD of the results was 1.5%, indicating that the oxidation signals of Ag and Thi have excellent stability and can be used as probe signals. Figure 8 As shown.

[0100] MIP / AgNPs@Ti2NT x The @IL / GCE modified electrode was placed at 4°C for 1, 3, 5, 7, 9, 11, 13, and 15 days, respectively, and then incubated with 10 ng / mL PAT under optimal conditions. Two weeks later, MIP / AgNPs@Ti2NT were added. x The @IL / GCE response remains at 93.02% of its initial value, indicating that the sensor has excellent long-term stability. Figure 9 As shown.

[0101] (2) Recovery rate and rapid quantitative detection of PAT in actual samples

[0102] ① This method is used to calculate the recovery rate of PAT in actual samples.

[0103] A known PAT concentration was added to a real sample that contained no PAT (the real sample had been tested by high performance liquid chromatography according to local standards DB32 / T3771-2020 and DB35 / T 898-2009 and no PAT was found). PBS buffer was then added, and the pH and other experimental parameters were adjusted to the conditions used for establishing the standard curve. The MIP / AgNPs@Ti2NT prepared in Example 1 was then added. x The @IL / GCE modified electrode was incubated in a buffer solution with pH 6 containing the actual sample. After incubation, the non-specifically adsorbed PAT was rinsed with deionized water and allowed to stand and dry. The modified electrode, reference electrode (calomel electrode), and counter electrode (platinum electrode) were then placed in 5 mL of PBS buffer solution with pH 6 treated with inert gas. The square wave voltammetry was used to measure the PAT in parallel three times and the average signal ratio difference was obtained. Finally, the PAT concentration in the actual sample was obtained according to the PAT detection standard working curve established in step (1), and the recovery rate and relative standard deviation were calculated.

[0104] ②This method enables rapid quantitative detection of PAT in real samples.

[0105] A certain volume of pretreated apple and pear solution containing an unknown concentration of PAT was added to the test solution, and the pH was adjusted to 6.0. MIP / AgNPs@Ti2NT was then added. x The @IL / GCE modified electrode was immersed in the solution and stirred for incubation, resulting in MIP / AgNPs@Ti2NT. xThe @IL / GCE modified electrode was used as the working electrode, the saturated calomel electrode as the reference electrode, and the platinum electrode as the counter electrode. The electrode was placed in a PBS buffer solution with pH 6 that had been treated with inert gas. The signal ratio difference was measured and calculated by square wave voltammetry. Finally, the concentration of PAT in the test solution of unknown concentration was obtained according to the PAT detection standard working curve established in step (1).

[0106] The conditions for the square wave voltammetry method are: voltage scan range -0.6 to 0.6V, potential increment 0.004V, amplitude 0.025V, and pulse frequency 15Hz.

[0107] Table 1

[0108]

[0109] As shown in Table 1, after multiple repeated experiments, the recovery rate of apples and pears using this method was 96.90-103.8%, and the RSD was 2.6-5.0%, indicating that the constructed MIPs-RECS has high potential for the detection of PAT in actual samples.

[0110] The results demonstrate that the MIP / AgNPs@Ti2NT prepared in this invention x The @IL / GCE electrode has high accuracy and precision, and can be used for the detection and analysis of actual samples. It is simple to prepare, convenient to operate, fast to detect, has good repeatability and reproducibility, strong anti-interference ability, and high sensitivity. It can be used for rapid quantitative detection of PAT in actual samples such as apples and pears.

[0111] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A method for preparing a modified electrode for detecting patulin in fruits, characterized in that: Includes the following steps: (1) Polishing treatment of substrate working electrode (2) AgNPs@Ti2NT x Synthesis of @IL nanocomposites; wherein IL is diallyl hexafluorophosphate. (3) Construction of MIPs-RECS AgNPs@Ti2NT synthesized by self-assembly x @IL nanocomposite material is used as the substrate material of the electrode, and MIPs are prepared on the modified electrode by electropolymerization. The substrate material AgNPs and functional monomers are used as working probes and reference probes, respectively, to form MIPs-RECS with dual signal output mode, wherein the functional monomers are any one of thionine, methylene blue, p-aminothiophenol and pyrrole.

2. The method for preparing the modified electrode for detecting patulin in fruits according to claim 1, characterized in that, The specific steps (1) are as follows: 1.1 Polishing the working electrode: First, use alumina polishing powder to polish the working electrode into a mirror surface. Then, clean the polished working electrode with a mirror surface by ultrasonic cleaning in distilled water, anhydrous ethanol, and distilled water in sequence. Finally, dry it in cold air. 1.2 Determining if electrode polishing is qualified: Place the working electrode, reference electrode, and counter electrode system (dried in step ①) in an electrochemical probe solution, scan using cyclic voltammetry, and compare with the theoretical standard spectrum. If the potential difference between the two peaks is within the specified range, the electrode polishing is qualified.

3. The method for preparing the modified electrode for detecting patulin in fruits according to claim 2, characterized in that: In step 1.1, the particle size of the alumina polishing powder is 0.04~0.06 µm; In step 1.2, the working electrode is any one of glassy carbon electrode, graphite electrode, gold electrode or platinum electrode; the reference electrode is a saturated calomel electrode or silver / silver chloride electrode. The counter electrode is any one of the inert metals that does not undergo redox reactions in the detection circuit; the electrochemical probe solution is 5 mmol / L [Fe(CN)6] containing 0.1 mol / L KCl. 3- / 4- The solution may contain 5 mmol / L [Ru(NH3)6] with 0.1 mol / L KCl. 2+ / 3+ Solution; the specified range refers to below 100mV.

4. The method for preparing the modified electrode for detecting patulin in fruits according to claim 1, characterized in that, Step (2) specifically involves: 2.1 Ti2NT x Synthesis Ti2NT was prepared using a KF-HCl mixture as an etchant. x Nanoparticle powder; 2.2 Synthesis of IL-1 Diallyl Hexafluorophosphate First, 20 mmol of N-allyl imidazole was dissolved in 25 mL of toluene, and then 10 mmol of 1,4-dibromobutane was added dropwise while stirring at 75 °C. The mixture was stirred continuously for 24 h, and then cooled to room temperature. After the reaction was complete, the supernatant was poured off, and the product was washed three times with ethyl acetate and diethyl ether, respectively. The product was then dried under vacuum at 60 °C for 1 h to obtain diallyl imidazole bromide. Then, 3 mmol of diallyl imidazole bromide was dissolved in 30 mL of deionized water, and 6 mmol of potassium hexafluorophosphate was added. The mixture was stirred at 85 °C for 6 h to obtain 1 L of diallyl hexafluorophosphate. 2.3 Ultrasound-assisted synthesis of AgNPs@Ti2NT x @IL Nanocomposite Materials First, weigh 10 mg Ti2NT x The powder was dispersed in 2.5 mL of 10 mM AgNO3 solution and reacted under ultrasound for 1 h to obtain AgNPs@Ti2NT. x Composite material; simultaneously, weigh 30 mg IL and dissolve it in 2.5 mL DMSO; then add AgNPs@Ti2NT x The prepared IL solution was added to the suspension, and the mixture was sonicated for another 1 h to obtain AgNPs@Ti2NT. x @IL suspension; the obtained suspension was centrifuged and washed several times. After washing, the precipitate obtained by centrifugation was placed in a vacuum freeze dryer and freeze-dried for 48 h to obtain AgNPs@Ti2NT. x @IL nanocomposite powder; weigh 2 mg AgNPs@Ti2NT x AgNPs@Ti2NT can be obtained by dispersing the IL nanocomposite powder in a 1:1 mixture of 1.0 mL DMF and water. x @IL suspension.

5. The method for preparing the modified electrode for detecting patulin in fruits according to claim 1, characterized in that, The specific steps (3) are as follows: before modification, GCE is carefully polished and ultrasonically cleaned with deionized water; then 5.0 μL AgNPs@Ti2NT are applied. x The @IL suspension was dropped onto a clean surface of the GCE and then dried in an infrared oven; after the electrode cooled to room temperature, the prepared AgNPs@Ti2NT were then placed on the surface of the GCE. x @IL / GCE was immersed in an electrolyte solution containing 1.0 mM PAT and 6.0 mM functional monomers; a three-electrode system was constructed, and electropolymerization was performed using CV at a scan rate of 100 mV / s and a potential range of -0.85 ~ 0.85 V for 20 cycles; after electropolymerization, the modified GCE was immersed in 2.0 M sulfuric acid for 6 min to remove PAT molecules; finally, the modified GCE was carefully washed with deionized water and dried under a tungsten filament lamp; the resulting electrode was labeled MIP / AgNPs@Ti2NT. x @IL / GCE; The electrolyte solution is a phosphate buffer, an acetate buffer, or a BR buffer; the functional monomer is any one of thionine, methylene blue, p-aminothiophenol, and pyrrole.

6. A modified electrode prepared by any one of the preparation methods described in claims 1-5.

7. A method for ratiometric molecularly imprinted electrochemical sensing detection of patulin in fruits using the modified electrode described in claim 6, characterized in that: Includes the following steps: (1) Establish the standard working curve for PAT testing: ① Prepare an electrolyte solution in an electrolytic cell that does not participate in chemical reactions and is conductive, and introduce an inert gas into the electrolyte solution; ② Dissolve PAT in a solvent that can dissolve PAT and does not react with PAT to prepare a PAT standard stock solution; ③MIP / AgNPs@Ti2NT x The IL / GCE modified electrode, reference electrode, and counter electrode were placed in the electrolyte solution treated with inert gas in step ①, and the Ag oxidation peak current before PAT incubation was detected using square wave voltammetry (SWV). I Ag With Thi oxidation peak current I Thi ;MIP / AgNPs@Ti2NT x The IL / GCE modified electrode was placed in the electrolyte solution treated with inert gas in step ①. Different volumes and concentrations of the PAT standard stock solution prepared in step ② were added to the electrolyte solution. After stirring and incubation, the electrode was rinsed with deionized water to remove non-specifically adsorbed PAT. After standing and drying, the modified electrode, reference electrode, and counter electrode were placed in the electrolyte solution with optimized pH. The Ag oxidation peak current after PAT incubation was detected by square wave voltammetry. I Ag With Thi oxidation peak current I Thi Using Ag oxidation peak currents before and after PAT incubation I Ag With Thi oxidation peak current I Thi The difference in ratios [ ( I Ag / I Thi A linear relationship was established between the concentration of PAT incubation and the concentration of PAT, with PAT concentration as the x-axis and the Ag oxidation peak current before and after PAT incubation as the y-axis. I Ag With Thi oxidation peak current I Thi The difference in ratios [ ( I Ag / I Thi Using [] as the ordinate, establish the standard working curve for PAT testing; (2) Rapid quantitative analysis of PAT in the sample: Add the electrolyte solution treated with inert gas in step ① to a certain volume of solution containing unknown PAT concentration, and adjust the experimental parameters. Then, immerse the modified electrode described in claim 6 in the solution for incubation. After incubation, rinse the non-specifically adsorbed PAT with deionized water. After standing and drying, place the modified electrode, reference electrode and counter electrode in the electrolyte solution with optimized pH, and analyze them using square wave voltammetry. Finally, according to the PAT detection standard working curve established in step (1), the unknown PAT concentration in the actual sample is obtained.

8. The method for ratiometric molecularly imprinted electrochemical sensing detection of patulin in fruits according to claim 7, characterized in that: The electrolyte solution is a phosphate buffer solution; the phosphate buffer solution is a 0.1 mol / L mixed solution of disodium hydrogen phosphate, sodium dihydrogen phosphate, and sodium chloride; the inert gas is nitrogen, helium, or neon.

9. The method for ratio-imprinted electrochemical sensing detection of patulin in fruits according to claim 8, characterized in that: The PAT standard stock solution is prepared by dissolving PAT in ethanol to prepare a PAT standard stock solution with a concentration of 10.00 mg / mL.

10. The method for ratio-imprinted electrochemical sensing detection of patulin in fruits according to claim 9, characterized in that: The reference electrode is a saturated calomel electrode or a silver / silver chloride electrode, and the counter electrode is any one of the inert metals that does not undergo redox reactions in the detection circuit.

Images