Preparation of photo-activated manganese ion bridged deoxyribozyme probe and application thereof in inhibiting acetylcholinesterase activity of cistanche

Abstract

The application discloses a kind of photoactivation manganese ion bridging type deoxyribozyme probe preparation and its application in Cistanche inhibiting acetylcholinesterase activity.The probe is pSD@MnO2 Composite structure, which is composed of manganese dioxide nanosheet and functional nucleic acid assembly adsorbed on its surface, the assembly includes S chain labeled with fluorescence / quenching group and containing deoxyribozyme cleavage site, and pD chain partially hybridized with S chain and containing photocleavage site.Its preparation includes nucleic acid probe assembly, MnO2 Nanosheet complexing and purification steps.When applied, AChE catalytic product decomposes MnO2, releases Mn 2+ And nucleic acid chain; after centrifugation, pD chain is cut by ultraviolet light activation trigger, and released DNAzyme is cut under the assistance of Mn 2+ It produces amplified fluorescence signal; by comparing signal change before and after adding inhibitor, inhibition activity can be quantitatively evaluated.The application combines photocontrol activation with enzyme cascade amplification, realizes high sensitivity and operation synchronism of detection process, and is especially suitable for high-throughput rapid screening of AChE inhibitor in Cistanche and other natural products.

Description

Technical Field

[0001] This invention relates to the field of biosensing and drug screening technology, specifically to a method for constructing a photoactivated biosensing probe based on functional nucleic acids and nanomaterials. More specifically, this invention relates to a photoactivated manganese ion bridging probe (pSD@MnO2) composed of manganese dioxide nanosheets and a specific deoxyribonuclease probe, its preparation method, and its application in the quantitative detection of acetylcholinesterase (AChE) activity, and further in the high-throughput screening of acetylcholinesterase inhibitors in the traditional Chinese medicine Cistanche deserticola. Background Technology

[0002] Acetylcholinesterase (AChE) is a key enzyme that hydrolyzes the neurotransmitter acetylcholine, and its abnormally elevated activity is closely related to the pathological progression of neurodegenerative diseases such as Alzheimer's disease (AD). Currently, first-line drugs for treating AD in clinical practice are mostly AChE inhibitors, such as donepezil, galantamine, and huperzine A. However, these synthetic drugs are often accompanied by side effects such as gastrointestinal discomfort and hepatotoxicity. Therefore, discovering highly effective and low-toxicity AChE inhibitors from natural products has become one of the important directions in new drug development. Cistanche deserticola, a traditional Chinese medicine that is both food and medicine, is considered a potential source of AChE inhibitors because it is rich in various active ingredients such as phenylethanol glycosides and iridoids, exhibiting good neuroprotective potential.

[0003] Currently, the classic method for AChE activity detection and inhibitor screening is the Ellman colorimetric assay. This method is based on the hydrolysis of the substrate thioacetylcholine (ATCh) by AChE to generate thiocholine (TCh). TCh then reacts with the chromogenic agent 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) to produce a yellow product, and the enzyme activity is indirectly reflected by measuring its absorbance at 412 nm. Although this method is widely used, its sensitivity is limited, it is easily affected by the color of the sample matrix, and its repeatability and accuracy are easily affected when used for high-throughput screening of complex natural product extracts (such as Cistanche deserticola extract).

[0004] To improve screening efficiency and reliability, immobilized enzyme technology is often combined with capillary electrophoresis and microplate detection. Existing technologies include various methods that use manganese dioxide (MnO2) microspheres or hollow structures as carriers to immobilize AChE via adsorption or cross-linking, followed by detection using the Ellman reaction (e.g., Chinese patents CN115197929A and CN114250220B). These methods improve enzyme stability and reusability. However, they still fundamentally rely on the traditional enzyme-catalyzed colorimetric principle, failing to fundamentally solve the problems of sensitivity improvement and background interference. Furthermore, in high-throughput operations such as 96-well or 384-well plates, differences in the timing of multi-step sample addition and reaction initiation can introduce significant systematic errors, affecting data consistency and comparability.

[0005] In recent years, deoxyribonucleases (DNAzymes), as functional nucleic acids with highly efficient catalytic cleavage activity, have been combined with fluorescent labeling technology, providing a new strategy for constructing highly sensitive biosensors. Meanwhile, manganese dioxide nanosheets, due to their excellent fluorescence quenching ability, biocompatibility, and the ability to be decomposed by reducing agents such as thiocholine, are often used as "switch" elements in sensing platforms. However, how to decompose MnO2 and utilize metal cofactors (such as MnO2) remains a challenge. 2+ The efficient and controllable coupling of the release of [a substance] with the activation of DNAzyme, and the precise spatiotemporal synchronization of the entire detection process, especially the signal amplification step, remains a challenge for current technology. Existing methods mostly rely on the order of chemical reagent addition to trigger cascade reactions, making it difficult to ensure that the signal initiation and progress of each reaction unit in high-throughput screening are completely uniform, which seriously restricts the reliability and efficiency of screening data.

[0006] Therefore, developing a novel AChE activity sensing platform with high sensitivity, strong anti-interference ability, and controllable signal triggering synchronization is of great practical significance and application value for rapidly and accurately screening AChE inhibitors from complex natural products such as Cistanche deserticola. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of the prior art and provide a photoactivated manganese ion-bridged deoxyribonuclease probe with high sensitivity and controllable signal triggering synchronization, its preparation method, and its application in the detection of acetylcholinesterase (AChE) activity and the screening of inhibitors, especially in evaluating the AChE inhibition activity of Cistanche deserticola.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a photoactivated manganese ion-bridged deoxyribozyme probe. The probe is a pSD@MnO2 composite structure, composed of manganese dioxide nanosheets and a functional nucleic acid assembly adsorbed on their surface; the functional nucleic acid assembly comprises: (a) an S chain, the sequence of which contains a Mn... 2+ (a) An ion-dependent deoxyribonuclease cleavage site, labeled with a quenching group and a fluorescent group at its 5' and 3' ends, respectively; (b) a pSD probe, which is formed by partial complementary hybridization of a pD chain with the S chain; the sequence of the pD chain contains a photocleavage site that can be specifically cleaved by ultraviolet light, which, when cleaved, releases a fragment that hybridizes with the S chain.

[0009] In the probe, fluorescence is quenched due to the fluorescence filtering effect of MnO2 and the proximity effect of the quenching groups. Its working principle is as follows: when the catalytic product thiocholine of AChE is present in the system, the MnO2 nanosheets are decomposed, simultaneously releasing Mn... 2+ and the functional nucleic acid assembly; activated by ultraviolet light irradiation and in Mn 2+ With the assistance of deoxyribozymes, the S-chain is cyclically cleaved, thereby restoring and amplifying the fluorescence signal.

[0010] Preferably, the nucleotide sequence of the S chain is as shown in SEQ ID NO: 1.

[0011] Preferably, the nucleotide sequence of the pD chain is as shown in SEQ ID NO: 2.

[0012] Secondly, the present invention provides a method for preparing the above-mentioned photoactivated manganese ion-bridged deoxyribozyme probe, comprising the following steps: (1) Probe assembly: Mix the S chain with a concentration of 8.0-12.0 μM and the pD chain with a concentration of 1.5-2.5 μM in a buffer solution at a volume ratio of 1:1, heat at 90-95℃ for 5-15 minutes to denature it, then slowly cool to 4℃ and let stand for 0.5-1.5 hours to allow the two to hybridize through complementary base pairing to form a pSD probe; (2) Composite: The pSD probe mixture obtained in step (1) is mixed with a manganese dioxide nanosheet dispersion with a concentration of 150-250 μg / mL at a volume ratio of 1:1, and incubated at 20-25℃ in the dark for 10-14 hours, so that the pSD probe and free S chain are adsorbed on the surface of manganese dioxide nanosheets through π-π stacking and electrostatic interaction. (3) Purification: Centrifuge the mixture after incubation in step (2) at 8000-12000 rpm for 8-12 minutes, discard the supernatant containing unadsorbed nucleic acid, and resuspend the resulting precipitate in phosphate buffer at pH 7.2-7.6 to obtain the purified pSD@MnO2 composite probe.

[0013] Thirdly, the present invention provides a method for detecting acetylcholinesterase activity using the above-mentioned probe, comprising the following steps: (1) Enzymatic reaction: The acetylcholinesterase in the sample to be tested reacts with its substrate acetylthiocholine to generate thiocholine; (2) Probe dissociation and substance release: The reaction system of step (1) is incubated with the composite probe. The reducing properties of thiocholine decompose the manganese dioxide nanosheets, and Mn is released simultaneously. 2+ and the adsorbed S chain and pSD probe; (3) Separation and photoactivation: The system after incubation in step (2) is subjected to solid-liquid separation (preferably centrifugation), and the supernatant is taken and irradiated with ultraviolet light (preferably wavelength 365 nm, irradiation time 1-10 minutes) to break the pD chain at the photocutting site; (4) Signal generation and detection: After illumination in step (3), the system in Mn 2+ In its presence, activated deoxyribonuclease cleaves the S chain, resulting in enhanced fluorescence signal. The activity of acetylcholinesterase is quantified by detecting the fluorescence intensity.

[0014] Fourthly, the present invention provides a method for screening acetylcholinesterase inhibitors, based on the above detection method, specifically including: (A) Pre-incubate test samples (preferably natural plant extracts) of different concentrations with a solution of acetylcholinesterase with immobilized activity; (B) Add the substrate acetylthiocholine to the mixture in step (A) to start the reaction. The subsequent operations are carried out according to steps (2) to (4) of the detection method described above. The fluorescence intensity value F in the presence of each concentration of the sample to be tested is measured. (C) Set up a negative control without the test sample and measure its fluorescence intensity value F0; (D) Calculate the inhibition rate at each concentration according to the formula: Inhibition rate = [1 - (F / F0)] × 100%, and use this to evaluate the inhibitory activity of the sample to be tested.

[0015] Preferably, the natural plant extract includes Cistanche deserticola extract.

[0016] Fifthly, the present invention provides the application of the above-mentioned photoactivated manganese ion-bridged deoxyribonuclease probe in the preparation of kits for detecting acetylcholinesterase activity or screening acetylcholinesterase inhibitors.

[0017] In a sixth aspect, the present invention provides a kit for detecting acetylcholinesterase activity or screening its inhibitors, comprising the above-mentioned photoactivated manganese ion-bridged deoxyribonuclease probe, acetylcholinesterase substrate acetylthiocholine, and reaction buffer.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention creatively constructs a "manganese dioxide nanosheet-functional nucleic acid" composite probe (pSD@MnO2), which converts the enzymatic activity of AChE into the chemical decomposition of MnO2 and Mn 2+ The release of DNA, the dissociation of DNA probes, and the final photo-activated DNAzyme cascade reaction enable multi-level signal transduction and amplification, significantly improving detection sensitivity.

[0019] This invention introduces "photoactivation" as a unified trigger switch for the final signal amplification step. This design achieves precise spatiotemporal control of the detection process. After all incubation and separation operations are completed, signal generation in all reaction wells is initiated by synchronous ultraviolet light irradiation, fundamentally eliminating the systematic errors introduced by differences in operation timing in traditional stepwise sample addition methods in high-throughput screening, and greatly improving the repeatability, reliability, and data comparability of the detection.

[0020] The detection method of the present invention is simple to operate, and can effectively remove interference from complex sample matrices (such as traditional Chinese medicine extracts) through centrifugation, thus exhibiting good anti-interference ability.

[0021] The method provided by this invention is particularly suitable for the rapid screening of AChE inhibitors from natural products. Taking Cistanche deserticola extract as an example, this method can accurately and efficiently quantify its inhibitory activity, providing a powerful technical tool for discovering novel, low-toxicity lead compounds for AD treatment from traditional Chinese medicine. Attached Figure Description

[0022] Figure 1 This is the ultraviolet-visible absorption spectrum of the manganese dioxide (MnO2) nanosheets prepared in the embodiments of the present invention (the inset is a photograph of the actual product).

[0023] Figure 2 This is a schematic diagram illustrating the construction of the photoactivated manganese ion-bridged deoxyribonuclease probe (pSD@MnO2) of this invention and its working principle for detecting acetylcholinesterase (AChE) activity and screening inhibitors.

[0024] Figure 3 This is a fluorescence spectrum used to verify the feasibility of the probe in an embodiment of the present invention.

[0025] Figure 4The fluorescence response curves (A) and linear relationship graphs (B) of the probe to different concentrations of AChE in the embodiments of the present invention are shown.

[0026] Figure 5 This is a bar chart showing the results of evaluating the inhibitory activity of different concentrations of Cistanche deserticola extract against AChE using the probe in this embodiment of the invention, and comparing it with the positive control drug donepezil; A and C - no ultraviolet light exposure, B and D - with ultraviolet light exposure. Detailed Implementation

[0027] 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. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without inventive effort are within the scope of protection of this invention.

[0028] I. Preparation of photoactivated manganese ion-bridged deoxyribonuclease probe (pSD@MnO2) Example 1: Preparation of manganese dioxide (MnO2) nanosheets An oxidation precipitation method was used. The specific steps are as follows: 20 mL of a 0.6 M tetramethylammonium hydroxide (TMA•OH) solution was placed in a beaker, and an appropriate amount of hydrogen peroxide was added to bring the final concentration to 3 wt%, then mixed thoroughly. Subsequently, 10 mL of a 0.3 M manganese chloride tetrahydrate (MnCl2•4H2O) aqueous solution was rapidly poured into the above mixture, and the system quickly changed from colorless to brownish-red. The mixture was stirred continuously at room temperature for 12 hours. After the reaction was completed, the resulting suspension was centrifuged at 10,000 rpm for 10 minutes, and the precipitate was collected. The precipitate was washed three times alternately with ultrapure water and methanol to thoroughly remove residual reactants and byproducts. The washed solid was placed in a freeze dryer and dried overnight to obtain brownish-black MnO2 nanosheet powder. Figure 1 As shown, the UV-Vis absorption spectrum of the obtained MnO2 nanosheets exhibits a characteristic absorption peak near 350 nm, confirming its successful synthesis. 10 mg of dried MnO2 nanosheet powder was weighed and dispersed in 10 mL of deionized water. After ultrasonic treatment for 10 hours, a uniformly dispersed MnO2 nanosheet stock solution with a concentration of approximately 1 mg / mL was obtained and stored at 4°C in the dark for later use.

[0029] Example 2: Construction of pSD@MnO2 composite probe This embodiment details the preparation method of the probe described in claim 1.

[0030] DNA strand sequence: S-chain (SEQ ID NO: 1): 5'-BHQ3-TTGACTTrAGGAGCAG-Cy5-3' pD chain (SEQ ID NO: 2): 5'-CTGCTCAGCGATCCTTAAGGCACCCATGTAGTCAA(PC-linker)GACTTGCAAGTCTTGACT-3' (Note: In the sequence, "rA" represents RNA adenine nucleotide, and PC-linker is the photocleavage site.) Assembly of the pSD probe: Mix 150 μL of 10.0 μM S-chain solution and 150 μL of 2.0 μM pD-chain solution in a PCR tube. Place the mixture in a PCR instrument and heat at 95°C for 10 minutes to ensure complete thermal denaturation. Then cool to room temperature and incubate at 4°C for 1 hour. This process allows the pD and S chains to partially hybridize through complementary base pairing, forming a stable double-stranded pSD probe structure.

[0031] Composite of the probe with MnO2 nanosheets: 200 μL of the pSD probe mixture prepared in the above steps was added to 200 μL of a 200 μg / mL MnO2 nanosheet dispersion (obtained by dilution of the stock solution from Example 1), and gently vortexed to mix. The mixture was then placed in a shaker at 25°C and incubated with gentle shaking in the dark for 12 hours. During this process, the pSD probe and excess free S chains in the system were efficiently adsorbed onto the surface of the MnO2 nanosheets through π-π stacking and electrostatic interactions.

[0032] Probe purification: Transfer the incubated mixture to a 1.5 mL centrifuge tube and centrifuge at 12,000 rpm for 10 minutes at 4°C. Carefully discard the supernatant to remove any unadsorbed free DNA strands. Add 200 μL of pH 7.4 phosphate buffer to the precipitate and gently pipette or briefly sonicate to fully resuspend it. This yields the purified pSD@MnO2 composite probe working solution (concentration approximately 200 μg / mL based on MnO2). Store at 4°C protected from light for later use.

[0033] II. Structural Characterization and Performance Evaluation of the Probe Example 3: Feasibility verification of probe detection of AChE activity To verify the working mechanism of the constructed probe, four sets of experiments were set up: ① probe only, no AChE enzyme reaction, no UV light; ② probe + AChE enzyme reaction, no UV light; ③ probe + UV light, no AChE enzyme reaction; ④ probe + AChE enzyme reaction + UV light.

[0034] The specific procedures are as follows: First, perform the enzymatic reaction. Add 10 μL of AChE solution (1000 U / mL) and 10 μL of substrate acetylthiocholine (ATCh, 4 mM) to the centrifuge tubes of groups ② and ④, and then add 80 μL of PBS buffer to a final volume of 100 μL. Incubate at 37°C for 30 minutes to generate thiocholine (TCh). For groups ① and ③, add an equal volume of PBS buffer instead of the enzyme-substrate mixture. Subsequently, add 100 μL of pSD@MnO2 probe working solution (200 μg / mL) to all groups and incubate at 37°C in the dark for 60 minutes to allow TCh to decompose MnO2. After incubation, centrifuge all samples at 10000 rpm for 10 minutes, and transfer 200 μL of the supernatant to a centrifuge tube. Only the supernatants of groups ③ and ④ are vertically irradiated with a 365 nm UV lamp for 5 minutes (light intensity approximately 10 mW / cm²), while groups ① and ② are kept in the dark. Finally, the fluorescence intensity was immediately measured using a fluorometer (excitation wavelength 640 nm, emission wavelength 670 nm).

[0035] The results are as follows Figure 3 As shown, only in group ④, where AChE was present and UV light irradiation was applied, was a significant enhancement of the Cy5 fluorescence signal observed. Groups ① (no enzyme, no light) and ② (with enzyme, no light) showed no significant signal, indicating a low background and that signal generation was strictly dependent on photoactivation. Group ③ (no enzyme, light) showed an extremely weak signal, indicating that in the absence of AChE, MnO2 was not decomposed, the DNA probe was not released, and there was no signal amplification even under light irradiation. This result fully demonstrates the feasibility of the dual control mechanism of the probe in this invention: "AChE trigger substance release - photoactivated signal amplification".

[0036] Example 4: Sensitivity evaluation of the probe for AChE detection A series of AChE standard solutions with different activities were prepared (final concentrations: 0, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, 500 U / mL). The procedure was followed as described in group ④ of Example 3: 10 μL of each concentration of AChE solution was incubated with 10 μL of ATCh (4 mM) and 80 μL of PBS at 37°C for 30 minutes; then 100 μL of pSD@MnO2 probe was added and incubated for 60 minutes; after centrifugation, the supernatant was collected and uniformly irradiated with 365 nm UV light for 5 minutes; the fluorescence intensity F was measured. The fluorescence value of the sample without AChE (0 U / mL) was used as the background F0.

[0037] The results are as follows Figure 4 As shown, the fluorescence intensity F exhibits a good linear relationship with the logarithm of AChE concentration in the range of 0.05 to 100 U / mL, with the linear equation being Y = 20.05X + 414.39 (R²). 2= 0.9956), correlation coefficient R 2 >0.99. Calculated at a signal-to-noise ratio of 3 (S / N=3), the limit of detection (LOD) of the method is as low as 0.03 U / mL. This result demonstrates that, based on DNAzyme cascade amplification and optically controlled synchronous triggering, the probe of this invention achieves highly sensitive detection of AChE.

[0038] Example 5: Screening of AChE inhibitors in Cistanche deserticola extract using probes Preparation of Cistanche deserticola extract: A eutectic solvent system of choline chloride and lactic acid (molar ratio of 1:2) was constructed, and Cistanche deserticola extract was obtained under the following conditions: ultrasonic temperature of 42℃, solvent water content of 30%, material-liquid ratio of 1:40 g / mL, and extraction time of 35 minutes.

[0039] Inhibitor screening experiment: Experimental and control groups were set up in 96-well plates. For the experimental group, each well was supplemented with: 10 μL of different concentrations of Cistanche deserticola extract solution (final concentrations of 0, 10, 20, 50, 100, 120, 160, and 200 μg / mL) and 10 μL of AChE working solution (final concentration 20 U / mL). The mixture was then pre-incubated at 37°C for 30 minutes. The positive control group was treated with different concentrations of donepezil solution (final concentrations of 0.02 and 0.2 μM) instead of the extract. The blank control group was treated with 10 μL of PBS instead of the extract.

[0040] Activity assay: After pre-incubation, add 10 μL of ATCh substrate solution (final concentration 1.2 mM) to all wells and react at 37°C for 30 minutes. Then add 100 μL of pSD@MnO2 probe working solution to each well, and make up the total volume of each well to 200 μL with PBS. Incubate at 37°C in the dark for 60 minutes. Transfer the reaction solution in the wells to centrifuge tubes, centrifuge at 10,000 rpm for 10 minutes, and take 200 μL of supernatant into a new black 96-well plate. Place the entire plate under 365 nm UV light for 5 minutes. Immediately detect the fluorescence intensity of each well using a fluorescence microplate reader (Ex / Em = 640 / 670 nm), with 6 replicates for each concentration.

[0041] Data analysis: Plot a dose-response curve with inhibitor concentration on the x-axis and inhibition rate on the y-axis. Figure 5 ).

[0042] like Figure 5 As shown in Figure A, under conditions without UV light illumination, the system exhibits almost no fluorescence signal response, indicating that the constructed sensing system maintains a stable "off" state before photoactivation, verifying the feasibility of the light control strategy and the spatiotemporal controllability of the signal. Figure 5In sample B, after UV light activation, the fluorescence signal decreased in a gradient with increasing concentration of Cistanche deserticola extract, indicating that the extract has concentration-dependent inhibitory activity against AChE. Further comparison... Figure 5 C and Figure 5 As shown in Figure D, the inhibitory effect of 80 μg / mL Cistanche deserticola extract after UV light activation was comparable to that of 0.01 μM donepezil, indicating that this natural product exhibits inhibitory potential similar to that of synthetic drugs at lower concentrations. These results confirm that this photoactivated sensing system can be effectively used for the quantitative evaluation and efficacy comparison of AChE inhibitory activity in Cistanche deserticola extract.

[0043] The above embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any equivalent structural transformations or direct / indirect applications made based on the concept of the present invention and utilizing the description and drawings of the present invention are included within the scope of protection of the present invention.

Claims

1. A photoactivated manganese ion-bridged deoxyribonuclease probe, characterized in that, The probe is pSD@MnO2, composed of manganese dioxide nanosheets and a functional nucleic acid assembly adsorbed on its surface; the functional nucleic acid assembly comprises: (a) An S-chain whose sequence contains an Mn 2+ Ion-dependent deoxyribozyme cleavage sites, and labeled with quenching groups and fluorescent groups at their 5' and 3' ends, respectively; (b) pSD probe, which is formed by partial complementary hybridization of a pD chain with the S chain; the sequence of the pD chain contains a photocleavage site that can be specifically cleaved by ultraviolet light, and the fragment that hybridizes with the S chain can be released after the site is cleaved. In the probe, due to the fluorescence filtering effect of MnO2 and the proximity effect of the quenching groups, the fluorescence is quenched. When the catalytic products of acetylcholinesterase are present in the system, the MnO2 nanosheets are decomposed, releasing Mn2. 2+ and the aforementioned functional nucleic acid assembly; activated by ultraviolet light irradiation and Mn 2+ With the assistance of deoxyribozymes, the S-chain is cyclically cleaved, thereby restoring and amplifying the fluorescence signal.

2. The photoactivated manganese ion-bridged deoxyribozyme probe according to claim 1, characterized in that, The nucleotide sequence of the S chain is shown in SEQ ID NO:

1.

3. The photoactivated manganese ion-bridged deoxyribozyme probe according to claim 1, characterized in that, The nucleotide sequence of the pD chain is shown in SEQ ID NO:

2.

4. A method for preparing the photoactivated manganese ion-bridged deoxyribozyme probe of claim 1, characterized in that, Includes the following steps: (1) Probe assembly: Mix the S chain with a concentration of 8.0-12.0 μM and the pD chain with a concentration of 1.5-2.5 μM in a buffer solution at a volume ratio of 1:1, heat at 90-95℃ for 5-15 minutes to denature it, then slowly cool to 4℃ and let stand for 0.5-1.5 hours to allow the two to hybridize through complementary base pairing to form a pSD probe; (2) Composite: The pSD probe mixture obtained in step (1) is mixed with a manganese dioxide nanosheet dispersion with a concentration of 150-250 μg / mL at a volume ratio of 1:1, and incubated at 20-25℃ in the dark for 10-14 hours, so that the pSD probe and free S chain are adsorbed on the surface of manganese dioxide nanosheets through π-π stacking and electrostatic interaction. (3) Purification: Centrifuge the mixture after incubation in step (2) at 8000-12000 rpm for 8-12 minutes, discard the supernatant containing unadsorbed nucleic acid, and resuspend the resulting precipitate in phosphate buffer at pH 7.2-7.6 to obtain the purified pSD@MnO2 composite probe.

5. A method for detecting acetylcholinesterase activity, characterized in that, The use of the probe according to any one of claims 1-3 includes the following steps: (1) Enzymatic reaction: The acetylcholinesterase in the sample to be tested reacts with its substrate acetylthiocholine to generate thiocholine; (2) Probe dissociation and substance release: The reaction system of step (1) is incubated with the composite probe. The reducing properties of thiocholine decompose the manganese dioxide nanosheets, and Mn is released simultaneously. 2+ and the adsorbed S chain and pSD probe; (3) Separation and photoactivation: The system after incubation in step (2) is subjected to solid-liquid separation, the supernatant is taken, and the supernatant is irradiated with ultraviolet light to break the pD chain at the photo-cleaving site; (4) Signal generation and detection: After illumination in step (3), the system in Mn 2+ In its presence, activated deoxyribonuclease cleaves the S chain, resulting in enhanced fluorescence signal. The activity of acetylcholinesterase is quantified by detecting the fluorescence intensity.

6. The detection method according to claim 5, characterized in that, In step (3), the solid-liquid separation method is centrifugation; the wavelength of the ultraviolet light irradiation is 365 nm, and the irradiation time is 1-10 minutes.

7. A method for screening acetylcholinesterase inhibitors, characterized in that, The detection method based on claim 5 specifically includes: (A) Pre-incubate natural plant extracts of different concentrations with a solution of acetylcholinesterase with fixed activity; (B) Add the substrate acetylthiocholine to the mixture in step (A) to initiate the reaction, and then proceed with the steps (2) to (4) of claim 5, and measure the fluorescence intensity F in the presence of each concentration of the sample to be tested; (C) Set up a negative control without the test sample and measure its fluorescence intensity value F0; (D) Calculate the inhibition rate at each concentration according to the formula: Inhibition rate = [1 - (F / F0)] × 100%, and use this to evaluate the inhibitory activity of the sample to be tested.

8. The screening method according to claim 7, characterized in that, The natural plant extracts include Cistanche deserticola extract.

9. Use of the probe according to any one of claims 1-3 in the preparation of a kit for detecting acetylcholinesterase activity or screening for acetylcholinesterase inhibitors.

10. A kit for detecting acetylcholinesterase activity or screening its inhibitors, characterized in that, It comprises the probe as described in any one of claims 1-3, the acetylcholinesterase substrate acetylthiocholine, and a reaction buffer.

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