Preparation method of copper-based-DNA engineered hybrid nano enzyme and application of copper-based-DNA engineered hybrid nano enzyme to pesticide exposure monitoring

Abstract

The invention discloses a preparation method of a copper-based-DNA engineering hybrid nano enzyme and application of the copper-based-DNA engineering hybrid nano enzyme to pesticide exposure monitoring, and belongs to the technical field of biosensors. According to the invention, a copper-based-DNA engineered hybrid nano enzyme (Cu-BDEH) is prepared on the basis of a DNA coordination driven self-assembly technology, and the form and catalytic activity of the Cu-BDEH are regulated and controlled through DNA. According to the method, Cu-BDEH is used as a target recognition and signal amplification unit, and an individual AChE baseline is established by combining the specific inhibition effect of the organophosphorus pesticide on acetylcholin esterase (AChE) and the reactivation of the AChE triggered by phosphatidine iodide, so that an efficient colorimetric platform for monitoring the exposure of the organophosphorus pesticide is constructed. Compared with a single biomarker method (such as inhibition or adduct detection), parallel measurement of AChE activity after exposure before and after reactivation provides higher accuracy and reliability, and provides a reliable method for environmental monitoring, occupational health screening and human health management.

Description

Technical Field

[0001] The present invention belongs to the technical field of biosensors, and specifically relates to a preparation method of a copper-based-DNA engineered hybrid nanozyme (Cu-BDEH) and its application in pesticide exposure monitoring. Background Art

[0002] The continued development of agriculture has led to an increase in the use of agrochemicals worldwide to reduce crop losses and improve product quality. However, overreliance on pesticides has harmed human health, particularly due to poisoning caused by pesticide accumulation in the food chain. Therefore, the development of effective diagnostic technologies to rapidly detect pesticide exposure and assess human health is crucial. Enzyme-based biosensors have shown great potential in a variety of applications, ranging from clinical diagnostics to environmental monitoring. However, their current limitations, particularly poor enzyme stability, make them unsuitable for field use in complex environments. Nanozymes, a class of enzyme mimics capable of catalyzing substrate transformations, offer significant advantages in sensing applications due to their excellent stability under extreme conditions, low production costs, and high catalytic performance. Despite these advantages, regulating nanozyme activity remains a challenge. The catalytic performance of nanozymes is closely related to their morphological characteristics. Therefore, matching the morphology of nanozymes with the specific requirements of the enzyme-catalyzed reaction is crucial for enhancing enzyme activity, improving utilization efficiency, and ultimately improving the performance of enzyme-based biosensors.

[0003] Metal coordination-directed biomolecular cross-linking has been used to synthesize a variety of biopolymers, including DNA, peptides, proteins, and polysaccharides. As a precisely engineered biopolymer, DNA exhibits unique programmability through systematic variations in its charge distribution, molecular size, hydrophobicity, and binding mode. DNA-based nanozymes exploit the specific affinity of nucleobase functional groups for metal surfaces to sequence-dependently control metal deposition and morphology. This unique property facilitates the rational design of nanozyme structures with precisely tailored shapes and tunable catalytic properties, effectively addressing the challenge of regulating nanozyme activity. By systematically programming the nucleobase sequence, DNA-based nanozymes facilitate precise optimization of key morphological features, particularly size parameters and spatial configuration. This programmable control enables optimal alignment with specific enzymatic reaction requirements, thereby enhancing catalytic performance and broadening its applicability in advanced biosensing platforms.

[0004] This study, based on DNA-mediated ligand-driven self-assembly, uses DNA to manipulate the morphology and catalytic activity of a copper-based nanozyme (Cu-BDEH). This method combines Cu-BDEH-mediated catalysis with pralidoxime (PAM)-triggered enzyme reactivation to establish an individual acetylcholinesterase (AChE) baseline. This approach improves the accuracy of organophosphorus pesticide exposure assessments, providing a reliable method for environmental monitoring, occupational health screening, and human health management. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for preparing a copper-based-DNA engineered hybrid nanozyme and its application in pesticide exposure monitoring.

[0006] This invention prepares a copper-based DNA-engineered hybrid nanozyme (Cu-BDEH) with tunable laccase-mimicking activity and excellent stability. Using Cu-BDEH as a target recognition and signal amplification unit, combined with the specific inhibition of AChE by OPs and PAM-triggered AChE reactivation, a highly efficient colorimetric platform for organophosphorus pesticide exposure monitoring has been established. This platform can simultaneously detect both AChE activity and total enzyme content. Compared to single biomarker methods (such as inhibition or adduct detection), the parallel measurement of AChE activity before and after post-exposure reactivation offers greater accuracy and reliability, providing technical support for the medical diagnosis of pesticide exposure.

[0007] The preparation method and application of the copper-based-DNA engineered hybrid nanozyme of the present invention include the following steps: A. Preparation of Cu-BDEH: Dissolve the DNA powder in ultrapure water to 5-75 μM, then mix the DNA aqueous solution with copper sulfate pentahydrate aqueous solution ( , 5~75 mM) in a volume ratio of 19:1, and then incubated in a metal bath at 75~100°C for 180~240 min; the resulting precipitate was then centrifuged at 8000~12000 rpm for 5~10 min, and the resulting precipitate was washed 3~5 times with ultrapure water, and then dissolved in ultrapure water to prepare a Cu-BDEH aqueous solution; B. Study on laccase activity of Cu-BDEH: Epinephrine hydrochloride (EP) aqueous solution ( ) was mixed with morpholineethanesulfonic acid (MES) buffer (20-50 mM, pH = 5.0-7.0) at a volume ratio of 1:8, and then a Cu-BDEH aqueous solution was added to the mixture. After reacting at 60-80°C for 45-75 minutes, the absorbance of the supernatant was monitored at a wavelength of 470 nm. C. Construction of AChE colorimetric sensing platform: Aqueous solutions of AChE with different concentrations were mixed with aqueous thioacetylcholine solution (ATCh, 2-5 mM) and Tris-HCl buffer (5-15 mM, pH = 7.0-8.5) at a volume ratio of 2:1:1 and incubated in a metal bath at 35-45°C for 30-50 min. The mixed solutions were then mixed with aqueous EP solution ( ), MES buffer (20-50 mM, pH = 5.0-7.0) and Cu-BDEH aqueous solution were mixed at a volume ratio of 1:1:7:1, reacted at 60-80°C for 45-75 min, and the absorbance of the supernatant was monitored at a wavelength of 470 nm; D. Determination of reactivated AChE: Chlorpyrifos samples of varying concentrations were added to the AChE solution or RBC solution at a 1:1 volume ratio and incubated for 30–50 min to form chlorpyrifos-AChE or chlorpyrifos-RBC adducts. To reactivate AChE, the solutions were reacted with an equal volume of 0.8–1.5 mM pralidoxime iodide (PAM) for 15–35 min, and then assayed according to the method established in step C.

[0008] E. Calculation of organophosphorus pesticide exposure: The pesticide inhibition rate (AP%) calculated from the intact AChE control and the pesticide inhibition rate (IR%) calculated from the reactivated AChE were calculated as follows: in For the controlled activity of the complete enzyme, The activity of the enzyme is inhibited, and is the enzyme activity after reactivation.

[0009] The mechanism of the present invention is as follows: A DNA-based nanostructure (Cu-BDEH) with tunable laccase-mimicking activity and good stability was synthesized using coordination-driven self-assembly of copper(II) ions and DNA. Using Cu-BDEH as a target recognition and signal amplification unit, combined with the specific inhibition of AChE by OPs and PAM-triggered AChE reactivation, an efficient colorimetric platform for organophosphorus pesticide exposure monitoring was established.

[0010] The Cu-BDEH-based colorimetric sensing platform prepared in the present invention can reliably obtain pesticide exposure information. It can simultaneously detect the activity and total enzyme content of AChE. Compared with single biomarker methods (such as inhibition or adduct detection), the parallel measurement of AChE activity before and after reactivation after exposure provides higher accuracy and reliability.

[0011] Compared with the existing technology, the present invention has the following characteristics: (1) The present invention synthesizes DNA nanostructures (Cu-BDEH) based on the coordination-driven self-assembly technology of copper (II) ions and DNA, which have controllable laccase mimetic activity and good stability, providing a good material for constructing enzyme-based sensors.

[0012] (2) By utilizing the tunable catalytic activity and excellent stability of Cu-BDEH and PAM-triggered AChE reactivation, a personalized and reliable monitoring platform for organophosphorus pesticide exposure was constructed. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 (a) is a scanning electron microscope image of the Cu-BDEH described in Example 1; (b) is a transmission electron microscope image and energy dispersion spectrum image of the Cu-BDEH described in Example 1.

[0014] Figure 2 (a) is the ultraviolet absorption spectrum of the Cu-BDEH catalytic substrate EP described in Example 2; (b) is a comparison of the catalytic activities of Cu-BDEH (A), Cu-BDEH (G) and Cu-BDEH (C) described in Example 2; (c) is a scanning electron microscope image of Cu-BDEH (A) described in Example 2; (d) is a scanning electron microscope image of Cu-BDEH (G) described in Example 2; (e) is a scanning electron microscope image of Cu-BDEH (C) described in Example 2.

[0015] Figure 3 (a) The stability of Cu-BDEH described in Example 2 after treatment at pH (3 and 9), high temperature (100°C) and ethanol; (b) The temporal stability of Cu-BDEH described in Example 2.

[0016] Figure 4 Schematic diagram of the detection platform mechanism described in Example 3.

[0017] Figure 5 (a) is a scanning electron microscope image of Cu-BDEH after incubation with ATCh and AChE described in Example 3; (b) is a feasibility analysis of the AChE detection sensing platform described in Example 3; (c) is a curve diagram of the relationship between AChE concentration and normalized intensity described in Example 3.

[0018] Figure 6 This is the correlation between AP% and IR% described in Example 4. DETAILED DESCRIPTION

[0019] Example 1: Synthesis and Characterization of Cu-BDEH DNA-based nanostructures (Cu-BDEH) were synthesized using a coordination-driven self-assembly technique of copper (II) ions and DNA. DNA powder was dissolved in ultrapure water to 25 μM, and then the DNA aqueous solution was mixed with an aqueous solution of copper sulfate pentahydrate ( , 25 mM) were mixed in a volume ratio of 19:1 and then incubated in a metal bath at 95°C for 180 min; the resulting precipitate was then centrifuged at 10,000 rpm for 10 min, and the resulting precipitate was washed with ultrapure water, and then dissolved in ultrapure water to prepare a Cu-BDEH aqueous solution.

[0020] Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) images show that Cu-BDEH exhibits a coral-like structure consisting of many nanofibers of relatively uniform size, with an average diameter of approximately 120 nm ( Figure 1 Energy dispersion spectroscopy shows that the elements Cu, N, P, and O are evenly distributed in Cu-BDEH ( Figure 1 b).

[0021] Example 2: Study on the laccase activity of Cu-BDEH Epinephrine hydrochloride (EP) aqueous solution ( ) was mixed with morpholineethanesulfonic acid (MES) buffer (30 mM, pH = 6.0) at a volume ratio of 1:8, and then a Cu-BDEH aqueous solution was added to the mixture. After reacting at 75°C for 60 min, the absorbance of the supernatant was monitored at a wavelength of 470 nm.

[0022] The laccase-mimicking catalytic activity of Cu-BDEH was quantitatively evaluated using a chromogenic biochemical reaction with EP as the substrate. EP exhibits no absorption in the visible region; upon oxidation by Cu-BDEH, an orange adduct ( Figure 2 a). By varying the structural units (DNA bases), the morphology and laccase-mimicking catalytic activity of Cu-BDEH were regulated. Significant changes in the morphology of Cu-BDEH were observed by varying the DNA bases (A, G, C, T), demonstrating a clear correlation between DNA base composition and material morphology. Specifically, Cu-BDEH (A) exhibited a spherical morphology with an average diameter of approximately 320 nm ( Figure 2 c), Cu-BDEH (G) forms a coral-like structure ( Figure 2 d), Cu-BDEH (C) also shows a spherical morphology ( Figure 2 e), but with an average diameter of approximately 540 nm, while the T base failed to produce any definite structure. When compared with the CuSO4 high-temperature oxidation product, the Cu-based DNA nanozymes showed higher activity, among which Cu-BDEH (G) showed the best catalytic performance ( Figure 2 b). In addition, Cu-BDEH exhibits good stability to organic solvents, heat, and extreme pH conditions ( Figure 3a). During the 15-day storage period, the catalytic activity of Cu-BDEH can still be maintained above 90.08% ( Figure 3 b).

[0023] Example 3: Establishment and performance evaluation of AChE sensing platform Aqueous solutions of AChE at different concentrations were mixed with aqueous thioacetylcholine solution (ATCh, 4 mM) and Tris-HCl buffer (10 mM, pH = 8.0) at a volume ratio of 2:1:1 and incubated in a metal bath at 37 °C for 30 min. The mixed solutions were then mixed with aqueous EP solution (1 mg mL -1 ), MES buffer (30 mM, pH = 6.0) and Cu-BDEH aqueous solution were mixed at a volume ratio of 1:1:7:1. After reacting at 75°C for 45 min, the absorbance of the supernatant was monitored at a wavelength of 470 nm.

[0024] Thiocholine (TCh) is the hydrolysis product of thioacetylcholine (ATCh) catalyzed by AChE. It competes with Cu-BDEH for binding to the Cu site, thereby inhibiting the catalytic function of Cu-BDEH ( Figure 4 ). SEM images further show that in the presence of TCh, the network structure of Cu-BDEH collapses, accompanied by obvious changes in absorbance and color ( Figure 5 ab). With the increase of AChE concentration ( ), Cu-BDEH activity gradually decreased, the absorbance value gradually decreased, and the fitting curve equation between the normalized intensity response value and AChE concentration was: y = 0.80-0.45x, where x is the logarithmic value of AChE concentration and y is the normalized intensity response value ( Figure 5 c).

[0025] Example 4: Establishment and performance evaluation of an organophosphorus pesticide exposure monitoring sensor platform Chlorpyrifos samples of varying concentrations were added to AChE solutions or erythrocyte solutions at a 1:1 volume ratio and incubated for 35 minutes to generate chlorpyrifos-AChE or chlorpyrifos-erythrocyte adducts. To reactivate AChE, the solutions were reacted with an equal volume of 1.1 mM pralidoxime iodide (PAM) for 25 minutes. The reactivated AChE concentration was then measured and the organophosphorus pesticide exposure level was calculated according to the formula.

[0026] To verify the accuracy of the reactivation-based quantitative analysis of organophosphate exposure, samples were prepared after exposure to different concentrations of chlorpyrifos. The absorbance values ​​of the system were detected under PAM treatment and without treatment, and then the absorbance values ​​of the system were converted into enzyme activity using a standard curve. The results are shown in Table 1. Different concentrations of chlorpyrifos (12.5-375 ng ) reduced AChE activity and produced a wide range of inhibition (35.87% - 97.73%). After PAM treatment, the AChE concentration measured after reactivation was compared with the known (control) value (15 mU ) were almost consistent, and the pesticide inhibition rate (AP%) calculated from the intact control AChE and the pesticide inhibition rate (IR%) calculated from the reactivated AChE also showed strong consistency ( =0.998)( Figure 6 ), indicating that this method is applicable and reliable for monitoring exposure to organophosphorus pesticide poisoning.

[0027] Table 1: Application of the present invention to prepare a copper-based-DNA engineered hybrid nanozyme sensing platform for detecting organophosphorus pesticide exposure in actual samples

Claims

1. A method for preparing a copper-based DNA engineered hybrid nanozyme, comprising the following steps: A. Preparation of Cu-BDEH: Dissolve the DNA powder in ultrapure water to 5-75 μM, then mix the DNA aqueous solution with copper sulfate pentahydrate aqueous solution. The mixture is mixed in a volume ratio of 19:1, and then incubated in a metal bath at 75-100°C for 180-240 minutes. The resulting precipitate is then centrifuged at 8000-12000 rpm for 5-10 minutes. The resulting precipitate is washed 3-5 times with ultrapure water, and then dissolved in ultrapure water to prepare a Cu-BDEH aqueous solution. B. Study on laccase activity of Cu-BDEH: An aqueous solution of epinephrine hydrochloride (EP) (0.5-1.5 mg mL-1) and a morpholineethanesulfonic acid (MES) buffer (20-50 mM, pH = 5.0-7.0) were mixed in a volume ratio of 1:

8. Then, a Cu-BDEH aqueous solution was added to the mixture. After reacting at 60-80°C for 45-75 min, the absorbance of the supernatant was monitored at a wavelength of 470 nm.

2. The method for preparing a copper-based DNA engineered hybrid nanozyme according to claim 1, wherein: In step A The concentration is 5~75 mM.

3. A copper-based DNA engineered hybrid nanozyme, characterized by: The method is prepared by the method described in claims 1 and 2.

4. A method for preparing a copper-based-DNA engineered hybrid nanozyme as described in claim 3 and its application in pesticide exposure monitoring.

5. The preparation method and application of a copper-DNA engineered hybrid nanozyme for pesticide exposure monitoring according to claim 4, characterized in that: Aqueous solutions of AChE at different concentrations were mixed with aqueous thioacetylcholine solution (ATCh, 2-5 mM) and Tris-HCl buffer (5-15 mM, pH = 7.0-8.5) at a volume ratio of 2:1:1 and incubated in a metal bath at 35-45 °C for 30-50 min. The mixed solutions were then mixed with aqueous EP solution (0.5-1.5 mg mL -1 ), MES buffer (20-50 mM, pH = 5.0-7.0), and Cu-BDEH aqueous solution were mixed in a volume ratio of 1:1:7:

1. The mixture was reacted at 60-80°C for 45-75 min, and the absorbance of the supernatant was monitored at a wavelength of 470 nm.

6. The method for preparing a copper-DNA engineered hybrid nanozyme and its application in pesticide exposure monitoring according to claim 4, characterized in that: To determine the concentration of reactivated AChE, different concentrations of chlorpyrifos samples were added to the AChE solution or erythrocyte solution at a volume ratio of 1:1 and incubated for 30-50 min to form chlorpyrifos-AChE or chlorpyrifos-erythrocyte adducts. To reactivate AChE, the above solutions were reacted with an equal volume of 0.8-1.5 mM pralidoxime iodide (PAM) for 15-35 min.

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