A method for detecting acetylcholinesterase based on cobalt-doped graphitic carbon nitride photoresponsive nanozymes

Abstract

This invention discloses a method for detecting acetylcholinesterase based on a cobalt-doped graphitic carbon nitride photoresponsive nanozyme, comprising the following steps: first, preparing a Co / g-C3N4 nanozyme; then, mixing acetylcholinesterase with buffer and acetylthiocholine, incubating, and subsequently adding buffer, Co / g-C3N4, and TMB; irradiating with a xenon lamp, centrifuging, and recording the ultraviolet-visible absorption spectrum of the supernatant. This method uses a photoresponsive nanozyme, eliminating the need for unstable hydrogen peroxide, and is extremely simple to operate. The catalytic colorimetric reaction of the photoresponsive nanozyme can be precisely controlled by switching the light source on and off, resulting in more accurate detection results. It also provides guidance for the preparation of highly catalytically active g-C3N4-based photoresponsive nanozymes.

Description

Technical Field

[0001] This invention belongs to the field of biological enzyme detection, specifically relating to a method for detecting acetylcholinesterase based on cobalt-doped graphitic carbon nitride photoresponsive nanoenzymes. Background Technology

[0002] Acetylcholinesterase is an important enzyme in the biological nervous system, primarily regulating the transmission of nerve impulses. Furthermore, acetylcholinesterase participates in many physiological processes, such as tumorigenesis, amyloid fibrosis formation, apoptosis, inflammation regulation, cell growth, and stem cell differentiation. Abnormal expression of acetylcholinesterase is associated with many neurodegenerative diseases in humans. Therefore, detecting acetylcholinesterase activity is of great significance for the diagnosis of neurological diseases. Currently, colorimetric methods, fluorescence methods, chemiluminescence methods, electrochemical methods, and liquid chromatography-tandem mass spectrometry are widely used for the detection of acetylcholinesterase. Among these, colorimetric methods have attracted widespread attention due to their intuitive phenomena and ease of operation.

[0003] Nanozymes, particularly peroxide nanozymes and oxidative nanozymes, have provided new opportunities for the colorimetric detection of acetylcholinesterase. Most of these are based on the colorimetric reaction catalyzed by peroxide or oxidative nanozymes, while the thiocholine generated by the hydrolysis of the substrate by acetylcholinesterase inhibits this reaction. However, the colorimetric reaction catalyzed by peroxide nanozymes requires the addition of hydrogen peroxide, and its termination requires the addition of an additional acid. In contrast, the colorimetric reaction catalyzed by oxidative nanozymes does not require hydrogen peroxide, making the operation simpler; especially photoresponsive oxidative nanozymes, whose catalyzed colorimetric reactions can often be precisely controlled by switching the light source on and off, resulting in more accurate detection results. However, currently reported photoresponsive oxidative nanozymes are relatively few in number, mainly consisting of noble metal nanoclusters, metal-organic frameworks, and covalent organic frameworks, which generally suffer from high cost and complex synthesis. Although graphitic carbon nitride (g-C3N4) nanosheets possess photoresponsive oxidase-like activity, their activity is relatively low. Summary of the Invention

[0004] To address the aforementioned issues, this invention prepares a cobalt-doped graphitic carbon nitride (Co / g-C3N4) nanozyme with high catalytic activity by doping cobalt onto g-C3N4 nanosheets. The photoresponsive oxidase-like activity of this nanozyme enables the detection of acetylcholinesterase. The detection mechanism is as follows: Under visible light irradiation, the Co / g-C3N4 photoresponsive nanozyme catalyzes the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) to generate blue oxidized TMB (oxTMB), which exhibits a strong absorption peak at 652 nm. Acetylcholinesterase hydrolyzes thioacetylcholine to generate thiocholine, inhibiting this colorimetric reaction and leading to a decrease in absorbance at 652 nm. Based on the change in absorbance at 652 nm with acetylcholinesterase activity, the detection of acetylcholinesterase can be achieved.

[0005] This invention is achieved through the following technical solution:

[0006] Preparation and characterization of Co / g-C3N4 photoresponsive nanozymes: 10 g of urea and 0.05 g of cobalt(III) acetylacetonate were added to a mortar and ground evenly. The resulting powder was then transferred to a covered porcelain boat and calcined at 550 °C for 2 h under a flowing nitrogen atmosphere at a heating rate of 5 °C / min. Afterward, the powder was washed several times with ultrapure water and dried in a vacuum drying oven. 0.3 g of the sample was added to 30 mL of 5 M HNO3 solution and refluxed at 100 °C for 24 h. Finally, the sample was washed with ultrapure water until the pH of the supernatant was close to neutral. The preparation of g-C3N4 nanosheets was similar to that of the Co / g-C3N4 photoresponsive nanozymes, except that cobalt(III) acetylacetonate was not required. The morphology, structural composition, and catalytic activity of the prepared Co / g-C3N4 photoresponsive nanozymes were characterized using transmission electron microscopy, Fourier transform infrared spectroscopy, X-ray diffraction spectroscopy, kinetics, and specific activity tests.

[0007] Acetylcholinesterase assay: 10 μL of acetylcholinesterase with different activities was mixed with 80 μL Tris-HCl buffer (10 mM, pH=7.4) and 10 μL 5 mM acetylthiocholine, and incubated at 37 ℃ for 30 min. Then, 700 μL acetate buffer (pH 3.6), 100 μL Co / g-C3N4 (0.3 mg / mL), and 100 μL TMB (5 mM) were added sequentially to a centrifuge tube. The tube was then irradiated with a xenon lamp for 3 min and centrifuged at 11000 rpm for 2 min. Finally, the supernatant was collected and the UV-Vis absorption spectrum was recorded.

[0008] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0009] (1) This method uses photoresponsive nanozymes, which do not require the use of unstable hydrogen peroxide and are relatively easy to operate.

[0010] (2) This method utilizes the catalytic colorimetric reaction of Co / g-C3N4 photoresponsive nanozymes, which can be precisely controlled by switching the light source on and off, making the detection results more accurate.

[0011] (3) The catalytic activity of Co / g-C3N4 photoresponsive nanozymes is significantly improved compared with g-C3N4, which provides guidance for the preparation of g-C3N4-based photoresponsive nanozymes with high catalytic activity. Attached Figure Description

[0012] Figure 1 TEM images of g-C3N4 (A, B) and Co / g-C3N4 (D, E), and HAADF-STEM and EDS elemental mappings of g-C3N4 (C) and Co / g-C3N4 (F);

[0013] Figure 2 XRD (A) and Fourier transform infrared (B) spectra of g-C3N4 and Co / g-C3N4;

[0014] Figure 3 (A) Absorption spectra of g-C3N4-TMB and Co / g-C3N4-TMB under illumination or no illumination, with the inset showing the solution color captured by a camera; (B) Specific activity of g-C3N4 and Co / g-C3N4; (C) Steady-state kinetics of g-C3N4 and Co / g-C3N4, with the inset showing the corresponding Lineweaver-Burk plots of g-C3N4 and Co / g-C3N4;

[0015] Figure 4 (A) Absorption spectra of the Co / g-C3N4 / acetylthiocholine / TMB system with different acetylcholinesterase activities; (B) Relationship between ∆A and acetylcholinesterase activity; (C) Linear calibration graph of acetylcholinesterase activity from 0.1 to 15 mU / mL; (D) Absorbance of the system at 652 nm when other enzymes are added alone or simultaneously with acetylcholinesterase. Detailed Implementation

[0016] The following is in conjunction with the appendix Figure 1 -Appendix Figure 4 The embodiments further illustrate the technical solutions of the present invention. Unless otherwise specified, the technical means used in the specific embodiments of the present invention are all methods known to those skilled in the art. Where specific techniques or conditions are not specified in the embodiments, they are all conventional methods or performed according to the techniques or conditions described in the literature in the field, or according to the product manual. Where the manufacturer is not specified, they are all conventional products that can be purchased through legitimate channels.

[0017] Example 1

[0018] (1) Preparation of g-C3N4 and Co / g-C3N4 photoresponsive nanozymes: 10 g of urea and 0.05 g of cobalt(III) acetylacetonate were added to a mortar and ground evenly; then the resulting powder was transferred to a covered porcelain boat and calcined at 550 °C for 2 h in a flowing nitrogen atmosphere at a heating rate of 5 °C / min. After that, it was washed several times with ultrapure water and dried in a vacuum drying oven. 0.3 g of the above sample was added to 30 mL of 5 M HNO3 solution and refluxed at 100 °C for 24 h. Finally, it was washed with ultrapure water until the pH of the supernatant was close to neutral. The preparation of g-C3N4 nanosheets was similar to that of Co / g-C3N4 photoresponsive nanozymes, except that cobalt(III) acetylacetonate was not required.

[0019] (2) Morphology and structural composition characterization of g-C3N4 and Co / g-C3N4 photoresponsive nanozymes: The morphology of g-C3N4 and Co / g-C3N4 was characterized by transmission electron microscopy. g-C3N4 exhibited a typical sheet-like structure ( Figure 1 A and 1B). High-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) and X-ray energy dispersive spectroscopy (EDS) elemental mapping of g-C3N4 show uniform distribution of C, N, and O. Figure 1 C). Co / g-C3N4 exhibits a coiled, sheet-like structure. Figure 1 D and 1E), C, N, O and Co are evenly distributed ( Figure 1 F). Furthermore, no nanoparticle accumulation was found on the surface of g-C3N4. The crystal structure of these samples was then investigated by X-ray diffraction (XRD). Figure 2 A). g-C3N4 exhibits two typical peaks at 13.0º and 27.5º, corresponding to the (100) and (002) planes, respectively, belonging to the interlayer stacking structure of in-plane repeating 3-s-triazine units and conjugated aromatic systems. Compared with g-C3N4, both the (100) and (002) peaks of Co / g-C3N4 are weaker, which may be due to the distortion of the planar structure around the Co center in Co / g-C3N4. Furthermore, no diffraction peaks of cobalt were observed, proving that cobalt does not exist in the form of nanoparticles. The chemical structure of g-C3N4 was further investigated by Fourier transform infrared spectroscopy. Figure 2 B). With 810 cm -1 The absorption band centered on this element belongs to the typical stretching vibration of the triazine element. (1200-1700 cm⁻¹) -1 Several vibrational peaks within this range belong to the stretching vibrations of carbon and nitrogen in aromatic heterocycles. Among them, those located at 1238, 1317, and 1407 cm⁻¹ are... -1 The peaks are due to CN stretching vibrations, while those located at 1580 and 1640 cm⁻¹ are due to CN stretching vibrations. -1The peak is due to C=N stretching vibration. 3400cm -1 The broad absorption bands in the vicinity correspond to the stretching vibration modes of NH and OH. The characteristic peaks of Co / g-C3N4 are basically consistent with those of g-C3N4, indicating that cobalt doping does not significantly alter the basic composition of g-C3N4. The above results demonstrate the successful preparation of Co / g-C3N4.

[0020] (3) Evaluation of the catalytic activity of g-C3N4 and Co / g-C3N4 photoresponsive nanozymes: The catalytic activity of g-C3N4 and Co / g-C3N4 photoresponsive oxidases was evaluated using TMB as the oxidase substrate. Figure 3 A). Under visible light irradiation, Co / g-C3N4 catalyzes the oxidation of colorless TMB to deep blue oxidized TMB (oxTMB), exhibiting a strong absorption peak at 652 nm. Although g-C3N4 can also catalyze TMB oxidation under visible light, the absorbance of oxTMB is much lower, indicating that g-C3N4 has lower photoresponsive oxidase-like activity. Conversely, without visible light irradiation, blue oxTMB cannot be generated. Furthermore, without the addition of Co / g-C3N4, TMB is hardly oxidized under visible light irradiation. These results indicate that Co / g-C3N4 possesses photoresponsive oxidase-like activity, and its catalytic activity is significantly enhanced compared to g-C3N4. Next, the specific activities of g-C3N4 and Co / g-C3N4 were compared. Figure 3 B). The specific activities of g-C3N4 and Co / g-C3N4 were 0.059 U / mg and 0.37 U / mg, respectively, further confirming that Co / g-C3N4 possesses higher photoresponsive oxidase catalytic activity. Its catalytic activity was investigated in detail using steady-state kinetics. Compared with g-C3N4, Co / g-C3N4 exhibits a lower Michaelis constant (Km) and a higher maximum reaction rate (…). v max () Figure 3 (C and 3D), indicating that Co / g-C3N4 has better affinity and higher catalytic activity for TMB.

[0021] (4) Detection of acetylcholinesterase: 10 μL of acetylcholinesterase with different activities was mixed with 80 μL Tris-HCl buffer (10 mM, pH=7.4) and 10 μL 5 mM acetylthiocholine, and incubated at 37 ℃ for 30 min. Then, 700 μL acetate buffer (pH 3.6), 100 μL Co / g-C3N4 (0.3 mg / mL) and 100 μL TMB (5 mM) were added to the centrifuge tube in sequence. The tube was then irradiated with a xenon lamp for 3 min and centrifuged at 11000 rpm for 2 min. Finally, the supernatant was collected and the UV-Vis absorption spectrum was recorded. Figure 4A). Linear fitting was performed using ∆A and the activity of acetylcholinesterase ( Figure 4 (B and 4C) The linear range of acetylcholinesterase was found to be 0.1–15 mU / mL, with a detection limit of 0.04 mU / mL. Furthermore, the effects of other enzymes on the detection system were determined, and the results showed that these enzymes, added alone or simultaneously with acetylcholinesterase (B and 4C), showed positive results. Figure 4 D) It has virtually no impact on the detection system, proving that the method has high selectivity and good anti-interference ability.

Claims

1. A method for preparing a Co / g-C 3N 4 photo-responsive oxidase nanoreactor, characterized in that, The method comprises the following steps: The urea and cobalt (III) acetylacetonate are ground uniformly, calcined in a nitrogen atmosphere, refluxed and washed to obtain Co / g-C3N4 photoresponsive nanoscale enzyme; The adding amount of urea and cobalt (III) acetylacetonate is 10 g and 0.05 g respectively. Calcination is at 550 °C for 2 h with a heating rate of 5 °C / min. o C / min of 550 °C for 2 h.

2. The preparation method of a Co / g-C3N4 photoresponsive oxidase nanoreactor according to claim 1, characterized in that, The specific operation steps of refluxing are as follows: 0.3 g of sample is taken and added into 30 mL of 5 M HNO3 solution, 100 o C refluxing for 24 h.

3. A method for detecting acetylcholinesterase based on cobalt-doped graphite phase carbon nitride photoresponsive oxidized nanozyme, characterized in that, The method comprises the following steps: mixing acetylcholinesterase, Tris-HCl buffer and acetylthiocholine, incubating, then adding acetic acid buffer, Co / g-C3N4 prepared by the preparation method in claim 1 and TMB in sequence, irradiating by a xenon lamp, centrifuging, taking supernatant and recording ultraviolet-visible absorption spectrum.

4. The method according to claim 3, wherein the cobalt-doped graphite-phase carbon nitride-based photoresponsive oxidase nanoreactor for detecting acetylcholinesterase is characterized by, The adding amount of acetylcholinesterase, Co / g-C3N4, acetylthiocholine and TMB is 10 μL, 100 μL, 10 μL and 100 μL respectively, the concentration of Co / g-C3N4 is 0.3 mg / mL, the concentration of acetylthiocholine is 15 mM and the concentration of TMB is 5 mM.

5. The method according to claim 3, wherein the cobalt-doped graphite phase carbon nitride-based photoresponsive oxidase nanoreactor for detecting acetylcholinesterase is characterized by, The incubation temperature is 37 ℃ and the incubation time is 30 min.

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