Nano-enzyme filtering membrane as well as preparation method and application thereof

Abstract

The invention discloses a nano-enzyme filtering membrane as well as a preparation method and application thereof. According to the preparation method disclosed by the invention, uniform dispersion on polyamide acid (PAA) is realized by utilizing the amphipathy of cobalt salicylic acid N-octyl imidazole ionic liquid, and the activity of the obtained polyamide acid cobalt (PAA-Co) composite membrane is endowed by reduction activation. The PAA-Co composite membrane has excellent catalytic performance and stability, so that the PAA-Co composite membrane becomes an ideal candidate for dimethoate colorimetric detection. In addition, the PAA-Co membrane is integrated into the filtering device, and catalytic oxidation occurs in the process of filtering the soil test solution. When the solution passes through the membrane, impurities are effectively filtered out, so that the detection precision is improved, and meanwhile, a catalytic oxidation reaction occurs. According to the method, continuous testing is achieved, the trouble of artificial enzyme recovery is eliminated, the detection efficiency is remarkably improved, and it is proved that the method has huge potential in actual pesticide residue detection application.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanoenzyme materials, and in particular relates to a composite membrane material with oxidase activity, a preparation method thereof, and an application thereof in rapid detection of pesticide residues. Background Art

[0002] Dimethoate, a highly effective, broad-spectrum insecticide and acaricide, has been widely used in agricultural production. However, its persistent environmental residues have raised serious concerns. Dimethoate is highly water-soluble and environmentally persistent, easily accumulating in soil and migrating through environmental media. As the primary reservoir of dimethoate residues, contaminated soil not only directly harms soil biodiversity and ecosystem health but can also be transferred to aquatic systems through leaching / runoff or absorbed by crops and enter the food chain, posing a cascading threat to aquatic ecosystems and human health. Studies have shown that dimethoate inhibits acetylcholinesterase activity, leading to acetylcholine accumulation in the body, causing nerve damage, headaches, respiratory paralysis, convulsions, and even death. In recent years, the widespread use of dimethoate has led to excessive pesticide residues in the environment, posing a significant threat to ecosystems and human health. Even long-term, low-dose exposure can induce neurotoxicity risks to non-target organisms and humans. Therefore, strict monitoring of dimethoate residues and control of its use are key measures to reduce environmental and health risks and are urgent needs for environmental and food security governance.

[0003] Currently, chromatography is the mainstream analytical method for quantifying dimethoate residues. Representative methods include high-performance liquid chromatography (HPLC), gas chromatography (GC), and its coupled technique, GC-mass spectrometry (GC-MS) [7-9]. In addition, surface-enhanced Raman scattering, fluorescence spectroscopy, electrochemical analysis, and colorimetry are also used. Although chromatography offers high sensitivity and good selectivity, it requires complex sample pretreatment, expensive instruments, and specialized operators, limiting its application for rapid on-site detection and high-throughput screening. Surface-enhanced Raman scattering offers excellent sensitivity and selectivity, but lacks signal stability. Fluorescence methods are rapid and sensitive but are susceptible to background fluorescence interference from biomacromolecules, affecting the accuracy of low-concentration pesticide detection. Electrochemical methods are highly sensitive and rapid but are susceptible to interference from electroactive molecules. In contrast, colorimetry has attracted much attention due to its low cost, simple operation, and intuitive results. However, traditional colorimetric methods often rely on natural oxidases, which are easily inactivated and sensitive to temperature and pH, making them difficult to store for a long time, restricting their practical application. In contrast, artificial enzymes based on metal nanomaterials offer superior stability and lower cost.

[0004] Although various nanomaterials, such as gold nanocrystals, have been explored as artificial enzymes for organophosphorus pesticide detection, their high cost and limited catalytic activity have hindered widespread application. Furthermore, the separation and recovery of artificial enzymes remain significant challenges. For example, Wang et al. developed Au-Fe3O4 magnetic nanoparticles with peroxidase-like activity, which were 1.3 times and 5.2 times more active than pure gold nanocrystals and Fe3O4 nanoparticles, respectively. The magnetic properties of these nanoparticles enabled separation from sample solutions, reduced spectral interference, and facilitated recovery. Similarly, Li et al. synthesized cobalt-functionalized graphene quantum dot-graphene composite nanozymes for pesticide detection in fresh food, accelerating the detection and recovery process using magnetic gel sheets. However, the small amount of artificial enzyme used in a single test and its high dispersibility made the magnetic separation and recovery process cumbersome and inefficient. Therefore, the development of highly active artificial enzymes and their efficient preparation and recovery methods remain pressing challenges for practical applications.

[0005] In existing research, artificial enzymes are usually dispersed in test solutions for colorimetric detection. However, inorganic nanoparticles have poor dispersibility and compatibility in aqueous solutions, which seriously impairs their activity and utilization efficiency. In addition, nanoparticles interfere with the color and turbidity of the sample, resulting in spectral interference in colorimetric measurements. What's more, nanoparticles are difficult to recycle, resulting in waste of precious metals or the release of heavy metals into the environment, causing secondary pollution. Membrane materials can cleverly solve the above problems: composite membrane materials not only have high functional activity, but also overcome defects such as poor dispersibility and sample interference, and are more conducive to recycling and reuse, thereby avoiding environmental problems. However, the preparation methods and applications of such composite membranes still lack universality.

[0006] Current Status of Dimethoate Residue Detection: Dimethoate, a broad-spectrum insecticide, can pose a threat to ecosystems and human health if left in soil through the food chain. Existing detection methods (such as chromatography and fluorescence) rely on large instruments, are complex to operate, and are difficult to perform rapid on-site screening.

[0007] Limitations of artificial enzymes: Although nano-artificial enzymes are low-cost and stable, they have problems such as insufficient catalytic activity, poor dispersibility, and difficulty in recovery (such as low magnetic separation efficiency), and nanoparticles can easily interfere with the accuracy of colorimetric detection.

[0008] Potential of membrane materials: Composite membranes can improve the dispersion of nanozymes and simplify their recovery, but existing technologies lack universal preparation methods and efficient catalytic membrane design. Summary of the Invention

[0009] In view of the deficiencies of the prior art, the present invention provides a nanoenzyme filtration membrane and a preparation method and application thereof.

[0010] 1. Technical Problems Solved

[0011] A highly active and easily recyclable nanoenzyme filtration membrane is provided to solve the problems of low catalytic efficiency, difficult separation and large detection interference of traditional artificial enzymes.

[0012] 2. Core of the Technical Solution

[0013] Material Design:

[0014] Cobalt-based salicylic acid N-octylimidazolium ionic liquid was used as a directing agent, and its amphiphilicity and affinity with PAA were utilized to achieve uniform loading of cobalt nanoparticles (particle size ~10 nm);

[0015] The PAA fiber skeleton restricts the growth of cobalt particles, exposes more active sites, and synergistically enhances the oxidase activity.

[0016] Preparation key:

[0017] The cobalt ions in the ionic liquid form a coordination structure with salicylic acid to prevent aggregation during the reduction process;

[0018] Sodium borohydride reduction of Co 2+ Converted to zero-valent cobalt (confirmed by XPS), activating the catalytic activity.

[0019] Application innovation:

[0020] The composite membrane is embedded in the filtration device, and the impurity filtration and TMB catalytic oxidation are completed simultaneously when the test solution flows through it;

[0021] By inhibiting the inhibitory effect of dimethoate on enzyme activity ( Figure 3 D) to achieve colorimetric quantitative detection.

[0022] 3. Beneficial Effects

[0023] Catalytic activity improvement: Michaelis constant (K M ) as low as 0.47 mM, with an affinity superior to similar materials (Table 1);

[0024] Excellent detection performance: the linear range of dimethoate detection is 10~5000ng / mL (R 2 =0.9996), detection limit 5.9 ng / mL;

[0025] Convenient operation: The membrane can be reused more than 10 times ( Figure 4 D), no separation and recovery steps are required;

[0026] Environmentally friendly: Cobalt dissolution is negligible (verified by ICP-MS).

[0027] The technical solutions of the present invention are as follows:

[0028] A nanoenzyme filtration membrane is based on polyamic acid (PAA) and has cobalt nanoparticles uniformly loaded on the surface.

[0029] Furthermore, the cobalt content is 20-30 wt.%, and the average particle size of the cobalt nanoparticles is 5-15 nm.

[0030] Furthermore, the cobalt nanoparticles are prepared by dispersing cobalt-based salicylic acid N-octylimidazolium ionic liquid on the surface of the PAA membrane and then reducing it.

[0031] A method for preparing a nanoenzyme filtration membrane comprises the following steps:

[0032] (a) ODA (4,4-diazodiphenyl ether) was dissolved in N,N-dimethylformamide, and then pyromellitic dianhydride (PMDA) was added. After stirring for a period of time, N,N-dimethylformamide (DMF) was added for dilution to prepare a PAA spinning solution, which was then electrospun to prepare a PAA film.

[0033] (b) Salicylic acid, N-octylimidazole and cobalt salt are mixed in an aqueous solution, stirred to obtain a pink mixture, and then extracted and the solvent is removed to obtain a cobalt-based salicylic acid N-octylimidazole ionic liquid.

[0034] (c) The PAA membrane is dispersed in the prepared cobalt-based salicylic acid N-octylimidazolium ionic liquid, an appropriate amount of solvent is added, the membrane is dispersed for a period of time and dried, and then the PAA-Co composite membrane nanozyme is obtained after reduction.

[0035] Furthermore, in step (b), the molar ratio of salicylic acid, N-octylimidazole and cobalt chloride is 8:8:2.

[0036] Furthermore, in step (c), the PAA membrane is immersed in the ethanol solution of the ionic liquid obtained in step (b), ultrasonically dispersed, and then vacuum dried.

[0037] Furthermore, in step (c), the reduction process is as follows: the PAA membrane is dispersed in water, sodium borohydride is added, the reaction is carried out at 60-120° C. for 0.6-2.0 hours, and the PAA-Co composite membrane is obtained after drying.

[0038] Furthermore, in step (c), the mass ratio of reducing agent sodium borohydride to PAA film is 1:3-10; the reaction time is 2 hours and the temperature is 80° C.; the drying temperature is 60-100° C. and the time is 1-5 hours.

[0039] A nanoenzyme filter membrane is used in detecting organophosphorus pesticide residues. The composite membrane is integrated into a filtration device. When the test liquid flows through the membrane, a catalytic oxidation reaction occurs, and the oxidation product is detected by colorimetry.

[0040] The use according to claim 9, characterized in that the organophosphorus pesticide is dimethoate, and the detection limit is 5.9 ng / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1:SEM images of PAA film (A) and PAA-Co film (BD); (E) general element distribution map; (FI) C, N, O, Co element mapping;

[0042] Figure 2 : XPS full spectrum (A), C 1s (B), N 1s (C), Co 2p (D) spectra, XRD (E) and Raman spectrum (F) of PAA-Co;

[0043] Figure 3 :PAA-Co catalyzed TMB oxidation kinetics curve (A), Lineweaver-Burk fitting (B), free radical quenching experiment (C), dimethoate inhibition effect (D);

[0044] Figure 4 :Dimethoate concentration gradient absorption spectrum (A), standard curve (B), ion interference test (C), membrane reusability (D);

[0045] Figure 5 PAA-Co composite membrane prepared using cobalt chloride solution;

[0046] Figure 6 Scanning electron microscopy (SEM) images of materials prepared with different ratios of co-ionic liquids at 0.5× (A), 2× (B), and 4× (C);

[0047] Figure 7 Contact angle images of PAA (A) and PAA-Co (B) composite films, visible light absorption spectra of PAA-Co, PAA, and Co catalytic systems (C), and absorbance of PAA-Co-catalyzed N2-, air-, and O2-saturated TMB solutions at 658 nm.

[0048] Figure 8 Schematic diagram of PAA-Co used for continuous detection of dimethoate. DETAILED DESCRIPTION

[0049] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention. It should be noted that the embodiments of the present invention and the features in the embodiments can be combined with each other in the absence of conflict.

[0050] Example 1: Preparation of PAA-Co composite membrane

[0051] PAA membrane synthesis:

[0052] Dissolve 8.0 g of ODA in 86.6 g of DMF and stir in an ice bath until dissolved;

[0053] Slowly add 9.0 g of PMDA, stir for 1 hour, and then add 16.3 g of DMF to dilute to 13 wt%;

[0054] Electrospinning: voltage +15 kV / -2 kV, flow rate 0.06 mm / min, receiving distance 10 cm.

[0055] Preparation of ionic liquids:

[0056] Mix 80 mL of 0.1 M salicylic acid, 80 mL of 0.1 M N-octylimidazole, and 20 mL of 0.1 M CoCl2 aqueous solution and stir at 70 °C for 90 minutes;

[0057] The oil phase was extracted with dichloromethane, the solvent was removed by rotary evaporation and then dried in vacuum at 85°C.

[0058] Composite membrane reduction activation:

[0059] 3 g of PAA membrane was immersed in 3 g of ionic liquid (containing 50 mL of ethanol), ultrasonicated for 10 min, and then vacuum dried;

[0060] Disperse in 100 mL of water, add 2 g of NaBH4, react at 80 ° C for 2 hours, and dry to obtain the finished product.

[0061] Example 2: Dimethoate residue detection

[0062] Standard solution test:

[0063] 100 μL of PAA-Co dispersion (5 mg / mL) was mixed with 200 μL of dimethoate standard solution and incubated at 50 °C for 15 min;

[0064] Add 300 μL of TMB solution (10 mM) and 400 μL of acetate buffer (pH 4.0) and vortex to mix;

[0065] After standing for 3 minutes, the absorbance at 658 nm was measured and a standard curve was drawn ( Figure 4 B).

[0066] Soil sample testing:

[0067] 5 g of soil was added with 5 mL of PBS (pH 7.4) and 1 mL of acetone, extracted by ultrasonication for 15 min, and the supernatant was collected by centrifugation;

[0068] The supernatant was mixed with TMB / buffer, passed through a PAA-Co membrane filtration device, and the filtrate was collected and the absorbance was measured.

[0069] Comparative Example

[0070] Comparative Example 1 (without ionic liquid mediation):

[0071] When CoCl2 solution was directly loaded onto the PAA membrane, SEM showed that the cobalt particles were severely agglomerated (>100nm) and the catalytic activity was only 32% of that of PAA-Co.

[0072] Comparative Example 2 (no reduction treatment):

[0073] Omitting the NaBH4 reduction step, XPS shows that cobalt is Co 2+ It exists in the form of β-actin and has no catalytic activity for TMB oxidation (absorbance ≤ 0.05).

[0074] Experimental results

[0075] index The present invention (PAA-Co) <![CDATA[Traditional Au-Fe3O4 nanozyme]]> Dimethoate detection limit 5.9 ng / mL 97ng / mL3333 Recovery rate 99.16～101.84％ 92～105％ Number of reuses >10 times ≤5 times (magnetic loss)

[0076] Example 3

[0077] A nanoenzyme filtration membrane, the preparation method of which comprises the following steps:

[0078] 10g of ODA (4,4'-diaminodiphenyl ether) and 10g of PMDA (pyromellitic anhydride) were placed in a test tube and dried at 100°C for 30 minutes. 8.0096g of ODA was weighed and placed in a three-necked flask. 86.5695g of N,N-dimethylformamide (DMF) was added and stirred in an ice-water bath until completely dissolved. 9g of PMDA was weighed and slowly added to the stirring flask. 10g of DMF was weighed and added to the flask, and stirring was continued for 1 hour. Then, 16.3g of DMF was added to dilute the solution to obtain a 13wt% PAA solution. Finally, PAA membranes were prepared by electrospinning. The electrospinning conditions were: positive voltage of 15kV, negative voltage of 2kV, propulsion speed of 0.06mm / min, temperature of 30°C, humidity of 30%, and a distance of 10cm between the spinneret and the roller receiver.

[0079] 80 mL of salicylic acid (0.1 mol / L), 80 mL of N-octylimidazole (0.1 mol / L), and 20 mL of cobalt chloride (0.1 mol / L) were mixed in an aqueous solution and stirred at 70°C for 90 minutes to produce a pink mixture. The mixture was then extracted with dichloromethane, and the oily solution was rotary evaporated to remove the solvent. After vacuum drying at 85°C overnight, a blue oily liquid, the cobalt-based salicylic acid N-octylimidazole ionic liquid, was obtained.

[0080] 3g of dry PAA membrane was dispersed in 3g of the prepared cobalt-based salicylic acid ionic liquid, 50mL of ethanol was added, and the mixture was sonicated for 10 minutes before vacuum drying. Subsequently, the membrane was dispersed in 100mL of water, 2g of sodium borohydride was added, and the mixture was reacted at 80°C for 2 hours. After drying, the PAA-Co composite membrane artificial enzyme was obtained.

[0081] Oxidase activity assay

[0082] Prepare a standard assay mixture by mixing acetic acid buffer (700 μL, pH 4.0) and 3,3',5,5'-tetramethylbenzidine (TMB) aqueous solution (100 μL, 1 mM). Add PAA-Co or other catalyst suspension (200 μL, 1 mg mL -1 ) and vortexed for 60 seconds. Quantification of oxidase mimetic activity involved separation of PAA-Co immediately prior to colorimetric measurement of absorbance at 658 nm.

[0083] Dimethoate testing

[0084] First, 100 μL of PAA-Co dispersion (5 mg / mL) was mixed with 200 μL of dimethoate standard or sample solutions at varying concentrations. After incubation at 50°C for 15 minutes, 300 μL of TMB solution (10 mmol / L) and 400 μL of acetate buffer (pH 4.0) were added to the mixture. After vortex mixing, the reaction solution was allowed to stand for 3 minutes before spectrophotometric scanning and absorbance quantification at 658 nm. Repeatability testing involved dividing a single dimethoate sample into 30 aliquots, each tested using a separate artificial enzyme unit to assess the reproducibility of the method.

[0085] Actual sample testing

[0086] Agricultural soil samples were collected from the field. A 5-gram soil sample was mixed with 5 mL of PBS buffer (0.1 mol / L, pH 7.4) and 1 mL of acetone. The mixture was ultrasonically extracted for 15 minutes and then centrifuged (10,000 rpm for 15 minutes). The supernatant was aspirated for subsequent quantification of dimethoate. To the supernatant, 300 μL of TMB solution (10 mmol / L) and 400 μL of acetic acid buffer (pH 4.0) were added. The resulting solution was passed through a filtration device equipped with a PAA-Co membrane for colorimetric analysis.

[0087] Effect of the embodiment:

[0088] The immobilization and dispersion of metal nanoparticles on polyamic acid (PAA) membranes are crucial for the preparation of highly active PAA-Co composite membrane artificial enzymes. To achieve high dispersibility and small cobalt nanoparticles, a novel ionic liquid-mediated synthesis route was developed. The synthesis begins with the preparation of a cobalt-based ionic liquid (IL), in which N-octylimidazole is ion-exchanged with salicylic acid to form an N-octylimidazolium salicylate ionic liquid (N-octylimidazolium salicylate IL). Subsequently, cobalt chloride is coordinated with this IL to yield the N-octylimidazolium salicylate IL. In this reaction, each cobalt ion forms coordination bonds with two salicylate ions, forming a stable cobalt salicylate anion. This coordination structure prevents cobalt ion aggregation, ensuring the formation of small cobalt nanoparticles during the reduction process. After the IL preparation, the N-octylimidazolium salicylate IL is uniformly dispersed onto the PAA membrane by leveraging the affinity between the IL and PAA. This swelling and dispersion effect promotes the three-dimensional distribution of the cobalt-based IL within the PAA matrix, significantly enhancing the dispersion of the metal nanoparticles and their interaction with the support. The uniform distribution of cobalt nanoparticles within the PAA membrane not only improves the activity of the composite membrane, but also enhances its stability. Finally, the oxidase activity of the composite membrane is activated by reducing cobalt ions. Under the reducing action of sodium borohydride, the cobalt in the N-octylimidazolium ionic liquid is reduced to zero-valent cobalt atoms and stably anchored on the PAA membrane. The reduction of cobalt ions to elemental cobalt significantly enhances the catalytic activity of the composite membrane. The cobalt content in the PAA-Co membrane was quantitatively analyzed using inductively coupled plasma mass spectrometry (ICP-MS), and the results showed that the cobalt content in the PAA-Co composite membrane artificial enzyme was 25.64wt.%.

[0089] Membrane structure characteristics

[0090] The synthesized PAA-Co composite membrane was characterized by scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD) and Raman spectroscopy. Figure 1 As shown in A, the PAA membrane has a stable structure and a clear three-dimensional porous fiber skeleton. In the PAA-Co composite membrane ( Figure 1 BD), cobalt nanoparticles with an average particle size of about 10nm are evenly distributed on the PAA fiber skeleton. Each fiber is densely decorated with evenly dispersed cobalt nanoparticles, showing excellent dispersion. This observation was supported by elemental distribution imaging analysis ( Figure 1 E) is further supported by the elemental distribution of carbon (C 1s), nitrogen (N 1s), oxygen (O 1s) and cobalt (Co 2p) ( Figure 1FI) revealed their spatial distribution within the composite. The results showed that carbon and nitrogen were evenly distributed on the carbon skeleton, confirming the excellent dispersion of the PAA-Co composite membrane. In contrast, this uniform dispersion effect was not achieved when cobalt chloride solution was used instead of cobalt-based salicylic acid N-octylimidazolium ionic liquid ( Figure 5 In addition, the density of cobalt nanoparticles can be precisely controlled by adjusting the ratio of metal ionic liquid added during the synthesis process ( Figure 6 These findings highlight the crucial role of the cobalt-based N-octylimidazolium salicylate ionic liquid in the material preparation process. The nitrogen-containing structure of PAA and the N-octylimidazolium ions in the ionic liquid ensure that the cobalt is completely derived from the metal ionic liquid. The results further demonstrate the excellent dispersion ability of this ionic liquid on PAA fibers, which is crucial for achieving uniform distribution of cobalt nanoparticles and optimizing the catalytic performance of the composite membrane.

[0091] The XPS analysis results of PAA-Co composite membrane are shown in Figure 2 As shown in AD. The composite membrane contains constituent elements such as carbon (C), nitrogen (N), oxygen (O) and cobalt (Co). The high-resolution C1s XPS spectrum was separated to identify the characteristic peaks of CC, CO and C=O bonds, while the N1s spectrum showed the characteristic peaks of pyridinic nitrogen and pyrrolic nitrogen. These observations confirm the partial retention of carbon / nitrogen groups in PAA and ionic liquid precursors, and that C, N and O are uniformly dispersed throughout the PAA fiber matrix. This elemental distribution is consistent with previous distribution imaging analysis. Co 2p spectrum ( Figure 2 D) shows two distinguishable peaks, attributed to the Co 2p3 / 2 and Co-O bond configurations. The Co-O peak is common in carbon materials containing cobalt and oxygen, consistent with the O1s spectrum. The Co 2p3 / 2 peak is characteristic of zero-valent metallic cobalt, confirming the successful reduction of cobalt ions to metallic cobalt.

[0092] XRD patterns of PAA-Co composite films ( Figure 2 E) shows two strong diffraction peaks at 16.9° and 43.8°. The peak at 16.9° is attributed to the characteristic diffraction of PAA, while the peak at 43.8° corresponds to the (111) crystal plane of cobalt. The small size of cobalt nanoparticles, combined with the fixation and barrier effect of the ionic liquid, limits the formation of more complete crystals, resulting in fewer exposed crystal planes. This amorphous and hybrid state enables the PAA-Co composite to expose more active sites, thereby enhancing its catalytic activity. Raman spectroscopy ( Figure 2 F) further supports these findings. Compared to pure PAA, the intensity of the characteristic Raman peak of PAA-Co is significantly reduced, indicating that the composite film is successfully reduced. The decrease in peak intensity is attributed to the reduction of functional groups with symmetrical vibration structures during the reduction process.

[0093] Oxidase activity

[0094] The oxidase activity of the synthesized materials was evaluated using the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) to generate a blue product as a model reaction. Figure 7 As shown. Contact angle measurements show that the contact angle of pure PAA is 46.5° ( Figure 7 A), indicating poor wettability and limited interaction with the test solution. In contrast, the PAA-Co composite membrane exhibited complete wetting due to the presence of the ionic liquid, with a contact angle of 0° ( Figure 7 B) This enhanced affinity enables the active sites of PAA-Co to interact more effectively with the test solution, resulting in higher catalytic efficiency and superior oxidase activity compared to other inorganic nanocrystals. Figure 7 As shown in Figure C, in the presence of PAA alone, the colorimetric system remained colorless, with no discernible absorption peak in the UV-visible spectrum and low absorbance, confirming that PAA had no catalytic effect on the reaction. When pure cobalt nanoparticles were introduced, the system turned light blue with a prominent absorption peak at 658 nm, indicating significant catalytic activity. However, when a PAA-Co composite membrane was used, the absorbance increased dramatically, demonstrating that the composite membrane exhibited superior catalytic activity compared to pure cobalt nanoparticles. The enhanced catalytic performance of the PAA-Co composite membrane can be attributed to a synergistic effect between the cobalt nanoparticles and the PAA fibers. On the one hand, the hydrophilicity of PAA-Co facilitated the penetration of TMB molecules into the composite membrane, enhancing effective contact and electron transfer between the membrane and TMB. On the other hand, the PAA fibers acted as a barrier, limiting the growth of the cobalt nanoparticles and resulting in a smaller particle size. These smaller cobalt nanoparticles exposed more active sites, allowing for greater interaction with TMB molecules and thus enhancing catalytic activity. These factors combined enhanced the TMB oxidation kinetics, leading to a significant increase in absorbance at 658 nm.

[0095] To elucidate the catalytic mechanism, the PAA-Co composite membranes were evaluated in nitrogen-saturated, air-saturated, and oxygen-saturated solutions. Figure 7 D indicates negligible absorbance in a nitrogen-saturated system, confirming the inability of the composite to oxidize TMB in the absence of dissolved oxygen. The absorbance in air-saturated solutions was significantly higher than that in nitrogen-saturated solutions, confirming the key role of oxygen in the catalytic process. The further enhancement of absorbance under oxygen-saturated conditions reflects superior catalytic activity due to the elevated oxygen concentration. These findings confirm that the PAA-Co composite mimics the behavior of oxidases in TMB oxidation, stemming from its ability to activate oxygen and generate reactive oxygen species (ROS).

[0096] In order to further study the kinetic behavior of the catalytic process, the apparent steady-state kinetic parameters ( Figure 3A and B). The Michaelis constant (K M ) and the maximum initial rate (Vmax) were 0.47 mM and 5.2 × 10 -7 Ms -1 . Lower K M Values ​​generally indicate a high affinity between the nanozyme and the substrate. This suggests that PAA-Co exhibits excellent affinity for TMB, which is consistent with the contact angle characterization results. The PAA-Co composite membrane not only promotes the rapid diffusion of dissolved oxygen to the catalytic active center, significantly improving the electron transfer efficiency of the reaction system, but also exhibits excellent adsorption and reduction capabilities for O2~, which contributes to its high oxidase activity.

[0097] In order to explore the catalytic mechanism, a series of experiments were performed to identify potential reaction intermediates. Benzoquinone, sodium azide (NaN3) and tert-butyl alcohol (TBA) were used as superoxide radicals (O2·-), singlet oxygen ( 1 O2) and hydroxyl radical (·OH) scavengers. Figure 3 As shown in C, the addition of benzoquinone and NaN3 significantly reduced the catalytic activity of PAA-Co, while TBA had a smaller inhibitory effect. These results indicate that PAA-Co catalyzes the activation of oxygen, converting the dissolved oxygen in the PAA-Co-TMB system into active oxygen (O2·- and 1 O2), and then the colorless TMB is oxidized into a blue product through a strong oxidation reaction.

[0098] To evaluate the effect of dimethoate on the oxidase activity of PAA-Co composite membranes, dimethoate was added to the test solution containing PAA-Co, and the absorbance at 658 nm was measured. Figure 3 As shown in Figure D, the absorbance decreased significantly in the presence of dimethoate, indicating that dimethoate effectively inhibited the oxidase activity of the PAA-Co composite film. To clarify whether dimethoate inhibits PAA-Co activity by scavenging reactive oxygen species (ROS) or by directly binding to the active site, a sequential addition experiment was designed. When dimethoate and TMB were added to the PAA-Co solution at different time points, TMB was still oxidized to a blue product. The resulting absorbance was consistent with that observed when the dimethoate-TMB mixture was added simultaneously. Combined with free radical quenching experiments, this suggests that pre-existing ROS in the solution oxidized TMB, while the pre-added dimethoate failed to quench these free radicals. Therefore, dimethoate inhibits PAA-Co activity primarily by directly binding to its active site, rather than by scavenging ROS.

[0099] Analyze performance

[0100] In order to evaluate the feasibility of the proposed method for the detection of dimethoate, the absorption spectra of the reaction system at different dimethoate concentrations were measured, and the absorbance at 658 nm was recorded. Figure 4 As shown in Figure A, the absorbance of the reaction system decreases with the increase of dimethoate concentration. This is attributed to the inhibitory effect of dimethoate on the catalytic activity of PAA-Co, which slows down the oxidation reaction rate and leads to a decrease in absorbance. In addition, Figure 4 B shows that in the range of 10–5000 ng / mL, the absorbance decreases linearly with the increase of dimethoate concentration. The linear regression equation is A = -0.0556logC 乐果 +0.3557, correlation coefficient (R 2 ) was 0.9996. Based on a signal-to-noise ratio (S / N) of 3, the detection limit of this method was calculated to be 5.9 ng / mL. To evaluate the reproducibility of the method, 30 repeated measurements were performed on a 10 ng / mL dimethoate solution. The relative standard deviation (RSD) of the results was 2.87%, indicating excellent reproducibility. For ease of comparison, the analytical parameters used for the determination of organophosphorus pesticides are summarized in Table 1. The results show that the detection limit of this method is significantly lower than that of existing methods. In addition, this method does not require the use of biological enzymes, and the PAA-Co composite membrane is simple to prepare and easy to store. These advantages highlight the potential of PAA-Co composite membrane for the detection of dimethoate pesticides in environmental samples.

[0101] Selectivity is crucial for obtaining reliable analytical results. + Mg 2+ , Ca 2+ 、Zn 2+ 、Fe 3+ 、C2O4 2- PO4 3- 、SO4 2- and CO3 2- The effect on the absorbance of the system is less than 5% ( Figure 4 C). After repeated use for 10 times, the catalytic activity did not decrease significantly ( Figure 4 D) To assess the potential environmental impact of cobalt leaching during long-term use, we measured the cobalt content of the artificial enzyme after 10 cycles of use. The results showed no significant difference in cobalt content compared to the unused enzyme, indicating that cobalt leaching was negligible over the 10 cycles of use. This demonstrates the environmental friendliness of the prepared artificial enzyme. This method demonstrates good selectivity and can be directly applied to the determination of dimethoate in soil.

[0102] Comparison of colorimetric detection of organophosphorus pesticides using different nanoenzyme materials

[0103]

[0104] Practical Application

[0105] To demonstrate the practicality of this sensing strategy, a rapid, high-throughput colorimetric detection method for the pesticide dimethoate was developed using a PAA-Co composite membrane. The PAA-Co membrane was integrated into a filtration device, allowing a catalytic oxidation reaction to occur while filtering the soil test solution. Figure 8 As shown, when the test solution flows through the PAA-Co membrane, impurities are effectively filtered out, improving the detection accuracy, while the catalytic oxidation reaction occurs. This design allows continuous testing without the need for artificial enzyme recovery, significantly improving the detection efficiency. The method has been successfully applied to the determination of dimethoate residues in soil samples. To verify its accuracy, a standard addition recovery experiment was performed. As shown in the table below, the results obtained using this method are highly consistent with the results of gas chromatography-mass spectrometry (GC-MS), with recoveries ranging from 99.16% to 101.84%. These findings confirm the high accuracy and reliability of the proposed method for the detection of dimethoate in environmental samples.

[0106]

[0107] A variety of pesticides were selected as test substrates to evaluate the potential application of this assay. The results demonstrated broad-spectrum responsiveness to organophosphorus pesticides, with detection accuracy exceeding 30%. However, the assay was ineffective against other pesticide classes, as they did not inhibit the oxidase-mimicking activity. Despite this, further optimization is needed to enhance its broad-spectrum screening performance against organophosphorus pesticides.

[0108]

[0109] This example demonstrates that the prepared PAA-Co composite membrane exhibits excellent oxidase-like catalytic activity and stability. The use of the PAA-Co composite membrane as an artificial enzyme for colorimetric detection of dimethoate has significant advantages, including high sensitivity, rapid response, and excellent stability. This method has been successfully applied to the colorimetric detection of dimethoate residues in soil samples. The PAA-Co composite membrane exhibits significant reusability and can be operated continuously without a complex recovery process. This not only simplifies the detection procedure, but also significantly improves the efficiency of dimethoate pesticide detection. The proposed method provides a valuable strategy for the sustainable reuse of artificial enzymes and the continuous monitoring of environmental pesticide residues, providing a promising solution for practical environmental applications.

[0110] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or make equivalent substitutions for some of the technical features therein. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A nanozyme filtration membrane, characterized in that: Polyamic acid (PAA) is used as the substrate, and cobalt nanoparticles are uniformly loaded on the surface.

2. The nanoenzyme filtration membrane according to claim 1, characterized in that: The cobalt content is 20-30 wt.%, and the average particle size of the cobalt nanoparticles is 5-15 nm.

3. The nanoenzyme filtration membrane according to claim 2, characterized in that: The cobalt nanoparticles are prepared by dispersing cobalt-based salicylic acid N-octylimidazolium ionic liquid on the surface of the PAA membrane and then reducing it.

4. A method for preparing the nanoenzyme filtration membrane according to claim 1-3, characterized in that The following steps are involved: (a) ODA (4,4-diazodiphenyl ether) was dissolved in N,N-dimethylformamide, and then pyromellitic dianhydride (PMDA) was added. After stirring for a period of time, N,N-dimethylformamide (DMF) was added for dilution to prepare a PAA spinning solution, which was then electrospun to prepare a PAA film. (b) Salicylic acid, N-octylimidazole and cobalt salt are mixed in an aqueous solution, stirred to obtain a pink mixture, and then extracted and the solvent is removed to obtain a cobalt-based salicylic acid N-octylimidazole ionic liquid. (c) The PAA membrane is dispersed in the prepared cobalt-based salicylic acid N-octylimidazolium ionic liquid, an appropriate amount of solvent is added, the membrane is dispersed for a period of time and dried, and then the PAA-Co composite membrane nanozyme is obtained after reduction.

5. The preparation method according to claim 4, characterized in that: In step (b), the molar ratio of salicylic acid, N-octylimidazole and cobalt chloride is 8:8:

2.

6. The preparation method according to claim 4, characterized in that: In step (c), the PAA membrane is immersed in the ethanol solution of the ionic liquid obtained in step (b), ultrasonically dispersed, and then vacuum dried.

7. The preparation method according to claim 4, characterized in that: In step (c), the reduction process is as follows: the PAA film is dispersed in water, sodium borohydride is added, the reaction is carried out at 60-120° C. for 0.6-2.0 hours, and the PAA-Co composite film is obtained after drying.

8. The preparation method according to claim 4, characterized in that: In step (c), the mass ratio of reducing agent sodium borohydride to PAA film is 1:3-10; the reaction time is 2 hours and the temperature is 80° C.; the drying temperature is 60-100° C. and the time is 1-5 hours.

9. Use of the nanozyme filtration membrane according to claims 1-3 in detecting organophosphorus pesticide residues, characterized in that: The composite membrane is integrated into a filtering device, and a catalytic oxidation reaction occurs when the test liquid flows through the membrane, and the oxidation product is detected by colorimetry.

10. The use according to claim 9, characterized in that: The organophosphorus pesticide is dimethoate, and the detection limit is 5.9 ng / mL.

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