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CN-118218010-B - Preparation method of hydroxylase-like bionic monoatomic nano-enzyme and application of hydroxylase-like bionic monoatomic nano-enzyme in electrochemical dopamine sensing

CN118218010BCN 118218010 BCN118218010 BCN 118218010BCN-118218010-B

Abstract

The invention provides a preparation method of hydroxylase-like bionic monoatomic nano-enzyme and application thereof in electrochemical dopamine sensing. The preparation method comprises the steps of mixing zinc acetate, copper nitrate solution and 2-methylimidazole solution, standing and ageing to obtain a copper doped ZIF-8 precursor 1, dispersing the precursor 1 in urea solution, applying negative pressure environment, centrifugally washing to obtain a urea filled copper doped ZIF-8 precursor 2, calcining at high temperature under the protection of argon, pickling, freeze-drying to obtain hydroxylase-like bionic monoatomic nano-enzyme, and ultrasonically mixing the nano-enzyme with a reduced graphene oxide solution to obtain the nano-enzyme composite material. The material designed by the invention has an active site similar to that of dopamine beta-hydroxylase. Can specifically catalyze the hydroxylation of beta-carbon atoms of dopamine under a proper potential. Meanwhile, a stronger electrochemical signal is generated, so that specific electrochemical detection of dopamine is realized, and obvious distinction between norepinephrine and epinephrine is effectively realized.

Inventors

  • ZHAO HUIMIN
  • CHEN BO
  • XING YIFEI

Assignees

  • 大连理工大学

Dates

Publication Date
20260508
Application Date
20240119

Claims (8)

  1. 1. The preparation method of the hydroxylase-like bionic monoatomic nano-enzyme is characterized by comprising the following steps: 1) Uniformly mixing a copper nitrate aqueous solution and a zinc acetate aqueous solution, slowly adding the mixture into a 2-methylimidazole aqueous solution, stirring at a speed of 200 rpm, centrifuging and washing at 8000 rpm after the reaction is completed, and freeze-drying to obtain a precursor 1; 2) Soaking the precursor 1 in 1mol/L urea solution, fully dispersing by ultrasonic, applying negative pressure in a vacuum oven for 30min, centrifugally washing at 8000 revolutions/min, and freeze-drying to obtain a precursor 2; 3) The precursor 2 is calcined at high temperature under the protection of argon, the product is immersed in 3mol/L hydrochloric acid solution for pickling, centrifugal washing is carried out at 8000 revolutions/min after full reaction, and freeze drying is carried out, thus obtaining the dopamine beta-hydroxylase-like bionic monoatomic nano-enzyme; 4) Adding the dopamine beta-hydroxylase-like bionic monoatomic nano-enzyme synthesized in the step 3) into the reduced graphene oxide solution, and carrying out ultrasonic treatment for 1h to uniformly mix the two to obtain the hydroxylase-like bionic monoatomic nano-enzyme solution.
  2. 2. The method according to claim 1, wherein, In the step (1) of the process, The concentration of the 2-methylimidazole aqueous solution is 0.2-2 mol/L, the copper nitrate aqueous solution is 0.1-0.5 mmol/L, and the zinc acetate aqueous solution is 10-30 mmol/L; the volume ratio of the copper nitrate aqueous solution to the zinc acetate aqueous solution is 1 (1-5), and the volume ratio of the metal salt mixed solution to the 2-methylimidazole aqueous solution is 1 (3-10); the reaction time of the precursor 1 is 6-24 hours.
  3. 3. The method according to claim 1, wherein, In the step 2), the concentration of the precursor 1 dispersed in the urea solution is 5-20 g/L.
  4. 4. The method according to claim 1, wherein, In the step 3) of the method, The high-temperature calcination temperature is 800-1000 ℃, the temperature rising speed is 5 ℃ per minute, and the calcination time is 0.5-3 h; the pickling time is 6-24 hours.
  5. 5. The method according to claim 1, wherein, In the step 4) of the method, The concentration of the added nano enzyme is 0.5-2 mg/mL; the concentration of the reduced graphene oxide solution is 1mg/mL, and the solvent is a mixed solution of ethanol and water with the volume ratio of 1:1.
  6. 6. The application of the hydroxylase-like bionic monoatomic nano-enzyme obtained by the preparation method of any one of claims 1 to 5 in electrochemical detection of dopamine, which is characterized by comprising the following steps: 1) Dripping hydroxylase-like bionic monoatomic nano enzyme solution on the surface of a screen printing electrode, and naturally airing to prepare a nano enzyme composite material modified electrode; The working electrode and the counter electrode of the screen printing electrode are made of carbon paste, and the reference electrode is made of silver/silver chloride paste; 2) And (3) under the condition of recording fixed potential by using an electrochemical workstation, modifying electrochemical response signals of the electrode to dopamine with different concentrations in phosphate buffer solution, and obtaining a standard curve and a linear equation according to the change of the response signals.
  7. 7. The use according to claim 6, wherein, In the step 1), the modification amount of the nano enzyme solution is 0.25-1.25 mu L/mm 2 .
  8. 8. The use according to claim 6 or 7, characterized in that, In the step 2) of the process, the process is carried out, The electrochemical measurement technology is a current-time curve method, and the applied potential is +0.7V to +1.2V; the concentration of the phosphate buffer solution is 0.1mol/L, and the pH value is 5-9; the concentration of the dopamine is 0.05-16.7 mu mol/L.

Description

Preparation method of hydroxylase-like bionic monoatomic nano-enzyme and application of hydroxylase-like bionic monoatomic nano-enzyme in electrochemical dopamine sensing Technical Field The invention belongs to the technical field of biosensing, and relates to a preparation method of a bionic monoatomic nano-enzyme composite material with dopamine beta-hydroxylase-like activity and application of the bionic monoatomic nano-enzyme composite material in electrochemical dopamine sensing, which can be particularly used for quantitatively monitoring the concentration of dopamine released by living cells. Background Dopamine is an important excitatory neurotransmitter involved in motor regulation and executive functions such as learning, motor control and memory consolidation, and is also an important biomarker for neurological diseases such as parkinson's disease, depression and schizophrenia. Therefore, the rapid and real-time detection of dopamine is of great clinical significance. Among the various dopamine detection methods, electrochemical sensing methods are attracting attention because of their simplicity in operation, low cost and high time resolution of in vivo and in vitro monitoring. It is reported that most of the current detection mechanisms of dopamine electrochemical sensors are based on electrochemical signals generated by the o-phenolic hydroxyl group on dopamine when oxidized into o-benzoquinone group. However, the o-phenolic hydroxyl groups are also present in other small biomolecules, such as norepinephrine and epinephrine. The simultaneous presence of these substances in the detection system affects the quantitative detection of dopamine. Effective differentiation of dopamine from other catecholamines remains a technical challenge. Through research on in vivo dopamine metabolic pathways, it is found that dopamine beta-hydroxylase can catalyze beta-hydroxylation of dopamine to generate norepinephrine, has stronger specificity for dopamine, and other catecholamine neurotransmitters do not interact with the enzyme. Thus, the enzyme is a potential recognition element for dopamine-specific detection. However, the enzyme is difficult and very expensive to produce on a large scale, and its catalytic activity is sensitive to environmental factors, limiting its use. Nanoenzymes are nanomaterials with enzyme-like properties. Compared with natural enzymes, the nano-enzyme has the advantages of high catalytic activity, low cost, simple and convenient preparation and mass production, recoverability and the like, and has been widely applied to the aspects of catalysis, sensing, disease treatment, environmental management and the like. For nanoenzymes, the design of the active site is critical to catalytic performance. The inspiration is drawn from the space structure of the natural enzyme active site, and the nano enzyme with similar enzyme catalytic activity is synthesized, so that better catalytic effect can be obtained. Disclosure of Invention The invention provides a synthetic method of a bionic monoatomic nano-enzyme composite material with a similar coordination structure and catalytic activity to dopamine beta-hydroxylase. On the basis of the material, a modified electrode for detecting dopamine with high specificity and high sensitivity is developed. The modified electrode can be used for high-sensitivity detection of dopamine released by living cells, and can effectively shield interference of other catecholamine neurotransmitters. In order to solve the above object, the present invention provides the following technical solutions: A preparation method of hydroxylase-like bionic monoatomic nano-enzyme, which comprises the following steps: 1) Uniformly mixing a copper nitrate aqueous solution and a zinc acetate aqueous solution, slowly adding the mixture into a 2-methylimidazole aqueous solution, stirring at a speed of 200 rpm, centrifuging and washing at 8000 rpm after the reaction is completed, and freeze-drying to obtain a precursor 1; Wherein the concentration of the 2-methylimidazole aqueous solution is 0.2-2 mol/L, the concentration of the copper nitrate aqueous solution is 0.1-0.5 mmol/L, and the concentration of the zinc acetate aqueous solution is 10-30 mmol/L; wherein, the volume ratio of the copper nitrate aqueous solution to the zinc acetate aqueous solution is 1 (1-5), and the volume ratio of the metal salt mixed solution to the 2-methylimidazole aqueous solution is 1 (3-10); Wherein the reaction time of the precursor 1 is 6-24 hours; 2) Soaking the precursor 1 in 1mol/L urea solution, fully dispersing by ultrasonic, applying negative pressure in a vacuum oven for 30min, centrifugally washing at 8000 revolutions/min, and freeze-drying to obtain a precursor 2; the concentration of the precursor 1 dispersed in the urea solution is 5-20 g/L; 3) The precursor 2 is calcined at high temperature under the protection of argon, the product is immersed in 3mol/L hydrochloric acid solution for