Method for detecting acetylcholin esterase based on cobalt-doped graphite phase carbon nitride photoresponse nano-enzyme

Abstract

The invention discloses a method for detecting acetylcholin esterase based on cobalt-doped graphite-phase carbon nitride photoresponse nano-enzyme, which comprises the following steps: firstly preparing Co / g-C3N4 nano-enzyme, then mixing acetylcholin esterase with a buffer solution and acetylthiocholine, incubating, sequentially adding the buffer solution, Co / g-C3N4 and TMB, irradiating with a xenon lamp, centrifuging, and detecting the acetylcholin esterase based on the cobalt-doped graphite-phase carbon nitride photoresponse nano-enzyme. Taking supernate and recording an ultraviolet-visible absorption spectrum. The method adopts the photoresponse nano-enzyme, does not need to use unstable hydrogen peroxide, and is extremely simple and convenient to operate. The catalytic chromogenic reaction of the photo-responsive nano-enzyme can be accurately regulated and controlled by switching on and off the light source, so that the detection result is more accurate. And meanwhile, guidance is provided for preparation of the g-C3N4-based photoresponse nano enzyme with high catalytic activity.

Description

Technical Field

[0001] The invention belongs to the field of biological enzyme detection, and specifically relates to a method for detecting acetylcholinesterase based on cobalt-doped graphite phase carbon nitride light-responsive nanoenzyme. Background Art

[0002] Acetylcholinesterase is an important enzyme in the biological nervous system, which mainly regulates the transmission of nerve impulses. In addition, acetylcholinesterase is involved in many physiological processes, such as tumorigenesis, amyloid fiber formation, apoptosis, inflammation regulation, cell growth and stem cell differentiation. Abnormal expression of acetylcholinesterase is associated with many neurodegenerative diseases in humans. Therefore, the detection of acetylcholinesterase activity is of great significance for the diagnosis of neurological diseases. At present, colorimetry, fluorescence, chemiluminescence, electrochemical method and liquid chromatography-tandem mass spectrometry are widely used for the detection of acetylcholinesterase. Among them, colorimetry has attracted widespread attention in the detection of acetylcholinesterase because of its intuitive phenomenon and simple operation.

[0003] Nanozymes, especially peroxidase nanozymes and oxidase nanozymes, provide a new opportunity for the colorimetric detection of acetylcholinesterase. Most of them are based on the color development reaction catalyzed by peroxidase nanozymes or oxidase nanozymes, and the thiocholine generated by the hydrolysis of the substrate by acetylcholinesterase inhibits the color development reaction. Among them, the color development reaction catalyzed by peroxidase nanozymes requires the addition of hydrogen peroxide, and the termination of the reaction requires the additional addition of acid. The color development reaction catalyzed by oxidase nanozymes does not require the participation of hydrogen peroxide, and the operation is simpler; especially the light-responsive oxidase nanozymes, the color development reaction catalyzed by them can mostly be precisely controlled by the switch of the light source, making the detection results more accurate. However, there are few types of light-responsive oxidase nanozymes reported so far, mainly precious metal nanoclusters, metal organic framework materials, covalent organic framework materials, etc., which generally have the disadvantages of high cost and complex synthesis. Although graphene phase carbon nitride (g-C3N4) nanosheets have light-responsive oxidase-like activity, their activity is low. Summary of the invention

[0004] In view of the above problems, the present invention prepares cobalt-doped graphite phase carbon nitride (Co / g-C3N4) nanozymes with high catalytic activity by doping cobalt in g-C3N4 nanosheets, and uses its light-responsive oxidase-like activity to detect acetylcholinesterase. The detection mechanism is as follows: Under visible light conditions, Co / g-C3N4 photoresponsive nanozymes catalyze the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) to generate blue oxidized TMB (oxTMB), which has a strong absorption peak at 652 nm; acetylcholinesterase hydrolyzes thioacetylcholine to generate thiocholine, inhibiting the color development reaction, resulting in a decrease in the absorbance of the system at 652 nm. Based on the change in the absorbance of the system at 652 nm with the activity of acetylcholinesterase, the detection of acetylcholinesterase can be achieved.

[0005] The present invention is achieved through the following technical solutions: Preparation and characterization of Co / g-C3N4 photoresponsive nanozyme: 10 g urea and 0.05 g 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 in a flowing nitrogen atmosphere at a heating rate of 5 °C / min. Afterwards, it was washed several times with ultrapure water and dried in a vacuum 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 nanozyme, except that cobalt (III) acetylacetonate was not required. The morphology, structural composition and catalytic activity of the prepared Co / g-C3N4 photoresponsive nanozyme were characterized by transmission electron microscopy, Fourier transform infrared spectroscopy and X-ray diffraction spectroscopy, kinetics and specific activity tests.

[0006] Determination 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 °C for 30 min. Then 700 μL acetate buffer (pH 3.6), 100 μL Co / g-C3N4 (0.3 mg / mL) and 100 μL TMB (5mM) were added to the centrifuge tube in sequence. Then, the tube was irradiated with a xenon lamp for 3 min and centrifuged at 11000 rpm for 2 min. Finally, the supernatant was taken to record the UV-visible absorption spectrum.

[0007] Compared with the prior art, the present invention has the following beneficial effects: (1) This method uses light-responsive nanozymes and does not require the use of unstable hydrogen peroxide, making the operation relatively simple.

[0008] (2) This method utilizes the catalytic color development reaction of Co / g-C3N4 photoresponsive nanozyme, which can be precisely controlled by switching the light source, making the detection results more accurate.

[0009] (3) The catalytic activity of Co / g-C3N4 photoresponsive nanozyme is significantly improved compared with that of g-C3N4, which provides guidance for the preparation of g-C3N4-based photoresponsive nanozymes with high catalytic activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figure 1 TEM images of g-C3N4 (A, B) and Co / g-C3N4 (D, E), HAADF-STEM and EDS element mapping images of g-C3N4 (C) and Co / g-C3N4 (F); Figure 2 XRD (A) and Fourier transform infrared spectra (B) of g-C3N4 and Co / g-C3N4; Figure 3 (A) Absorption spectra of g-C3N4-TMB and Co / g-C3N4-TMB in the absence of light or under illumination, and the inset is the solution color captured by the camera; (B) Specific activity of g-C3N4 and Co / g-C3N4; Steady-state kinetics determination of g-C3N4 (C) and Co / g-C3N4 (D), the inset is the corresponding Lineweaver-Burk plot of g-C3N4 and Co / g-C3N4; Figure 4 (A) Absorption spectra of the Co / g-C3N4 / acetylthiocholine / TMB system when different acetylcholinesterase activities were introduced; (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 detection system at 652 nm when other enzymes were added alone or simultaneously with acetylcholinesterase. DETAILED DESCRIPTION

[0011] The following is combined with Figure 1 -Attached Figure 4 The technical scheme of the present invention is further described in the following embodiments. Unless otherwise specified, the technical means used in the specific embodiments of the present invention are methods known to those skilled in the art. If no specific technology or conditions are specified in the embodiments, they are all conventional methods or carried out according to the technology or conditions described in the literature of the field, or according to the product instructions. If no manufacturer is specified, they are all conventional products that can be purchased through regular channels.

[0012] Example 1 (1) Preparation of g-C3N4 and Co / g-C3N4 photoresponsive nanozymes: 10 g urea and 0.05 g 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 in a flowing nitrogen atmosphere at a heating rate of 5 °C / min. Afterwards, it was washed several times with ultrapure water and dried in a vacuum 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.

[0013] (2) Characterization of the morphology and structural composition 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 exhibits a typical flake 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 that C, N, and O are uniformly distributed ( Figure 1 C). Co / g-C3N4 presents a curled sheet structure ( Figure 1 D and 1E), C, N, O and Co are evenly distributed ( Figure 1 F). In addition, no nanoparticles were found on the surface of g-C3N4. Then, the crystal structures of these samples were studied by X-ray diffraction (XRD). Figure 2 A). g-C3N4 has two typical peaks at 13.0º and 27.5º, corresponding to the (100) and (002) planes, respectively, belonging to the interlayer stacking structure of the repeating 3-s-triazine units and the conjugated aromatic system in the plane. Compared with g-C3N4, the (100) and (002) peaks of Co / g-C3N4 are both weaker, which may be due to the distortion of the planar structure around the Co center in Co / g-C3N4. In addition, no diffraction peak of cobalt was observed, proving that cobalt does not exist in the form of nanoparticles. The chemical structure of g-C3N4 was further studied by Fourier transform infrared spectroscopy ( Figure 2 B). At 810 cm -1 The absorption band centered at 1200-1700 cm belongs to the typical stretching vibration of triazine unit. -1 The multiple vibration peaks in the range belong to the stretching vibration of carbon and nitrogen in the aromatic heterocyclic ring. Among them, the peaks at 1238, 1317 and 1407 cm -1 The peaks at 1580 and 1640 cm -1 The peak is C=N stretching vibration. 3400cm -1The broad absorption bands near the surface correspond to the stretching vibration modes of NH and OH. The characteristic peaks of Co / g-C3N4 and g-C3N4 are basically consistent, indicating that cobalt doping has no significant change in the basic composition of g-C3N4. The above results indicate that Co / g-C3N4 was successfully prepared.

[0014] (3) Evaluation of the photoresponsive oxidase catalytic activity of g-C3N4 and Co / g-C3N4: TMB was used as the oxidase substrate to evaluate the photoresponsive oxidase catalytic activity of g-C3N4 and Co / g-C3N4. Figure 3 A). Under visible light irradiation, Co / g-C3N4 catalyzes the oxidation of colorless TMB to dark blue oxidized state TMB (oxTMB), which has a strong absorption peak at 652 nm. Although g-C3N4 can also catalyze the oxidation of TMB under visible light, the absorbance of oxTMB is much lower, indicating that the light-responsive oxidase-like activity of g-C3N4 is low. Conversely, without visible light irradiation, blue oxTMB cannot be generated. In addition, when Co / g-C3N4 is not added, TMB is hardly oxidized under visible light conditions. These results indicate that Co / g-C3N4 has light-responsive 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, which further confirmed that Co / g-C3N4 had higher photoresponsive oxidase catalytic activity. Its catalytic activity was studied in depth using steady-state kinetics. Compared with g-C3N4, Co / g-C3N4 had 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 for TMB and higher catalytic activity.

[0015] (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 °C 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. Then, the tube was irradiated with a xenon lamp for 3 min and centrifuged at 11000 rpm for 2 min. Finally, the supernatant was taken to record the UV-visible absorption spectrum ( Figure 4 A). Linear fitting was performed using ∆A and acetylcholinesterase activity ( Figure 4 B and 4C), the linear range of acetylcholinesterase was 0.1-15 mU / mL, and the detection limit was 0.04 mU / mL. In addition, the effects of other enzymes on the detection system were determined, and the results showed that these enzymes were added alone or simultaneously with acetylcholinesterase ( Figure 4 D) has little effect on the detection system, proving that this method has high selectivity and good anti-interference ability.

Claims

1. A method for preparing a Co / g-C3N4 light-responsive nanozyme, characterized in that: The following steps are involved: Urea and cobalt (III) acetylacetonate were ground evenly, calcined in a nitrogen atmosphere, refluxed and washed to obtain Co / g-C3N4 photoresponsive nanozyme.

2. The method for preparing a Co / g-C3N4 light-responsive nanozyme according to claim 1, characterized in that: The added amounts of urea and cobalt (III) acetylacetonate were 10 g and 0.05 g, respectively.

3. The method for preparing a Co / g-C3N4 light-responsive nanozyme according to claim 1, characterized in that: Calcination is 5 o The calcinations were carried out at 550 °C for 2 h at a heating rate of C / min.

4. The method for preparing a Co / g-C3N4 light-responsive nanozyme according to claim 1, characterized in that: The specific operation steps of reflux are as follows: take 0.3 g sample, add it to 30 mL of 5 M HNO3 solution, o C and reflux for 24 h.

5. A method for detecting acetylcholinesterase based on cobalt-doped graphene-phase carbon nitride light-responsive nanozyme, characterized in that: The method comprises the following steps: mixing acetylcholinesterase with Tris-HCl buffer and acetylthiocholine, incubating, sequentially adding acetate buffer, Co / g-C3N4 prepared by the preparation method of claim 1 and TMB, irradiating with a xenon lamp, centrifuging, taking the supernatant and recording the ultraviolet-visible absorption spectrum.

6. The method for detecting acetylcholinesterase based on cobalt-doped graphene-phase carbon nitride light-responsive nanozyme according to claim 5, characterized in that: The added amounts of acetylcholinesterase, Co / g-C3N4, acetylthiocholine, and TMB were 10 μL, 100 μL, 10 μL, and 100 μL, respectively. The concentrations of Co / g-C3N4, acetylthiocholine, and TMB were 0.3 mg / mL, 15 mM, and 5 mM, respectively.

7. The method for detecting acetylcholinesterase based on cobalt-doped graphene-phase carbon nitride light-responsive nanozyme according to claim 5, characterized in that: The incubation temperature was 37°C and the time was 30 min.

Images