Colorimetric detection method based on functionalized copper cysteine nano-enzyme

Abstract

The invention belongs to the field of biosensor detection, and particularly discloses a colorimetric detection method based on functionalized copper cysteine nano-enzyme, which comprises the following steps: preparing a specific functionalized copper cysteine nano-enzyme composite material; and constructing a detection reaction system containing hydrogen peroxide, tetramethyl benzidine, an acetate buffer solution and a target solution, and determining the concentration of the target by using a colorimetric analysis technology. Particularly, CuCl2. 2H2O is used as a copper source, PEG-4000 is used as polyethylene glycol, reaction is performed under nitrogen protection and alkalescence conditions, and then the reaction product reacts with chloroauric acid and trisodium citrate to prepare the composite material. According to the method, hydrogen peroxide and tetramethyl benzidine are taken as substrates, high-sensitivity detection of a target object (such as dopamine) is realized by measuring an absorption peak value of a reaction system at 652nm, and a standard curve is drawn so as to accurately measure the concentration of a solution to be measured. The method is simple and convenient to operate and high in sensitivity, and has a wide application prospect in detection of biological molecules such as dopamine.

Description

Technical Field

[0001] The invention belongs to the field of biosensor detection, and specifically discloses a colorimetric detection method based on functionalized copper cysteine ​​nanozyme. Background Art

[0002] Nanozymes, as an emerging class of enzyme mimics, are gradually becoming the shining star in the field of biosensors with their excellent stability, versatility and significant cost-effectiveness. They can not only adapt to extreme or complex detection environments, showing strong potential to replace traditional natural enzyme reagents, but also greatly simplify the experimental operation process and promote the popularization and application of biological detection technology. In many dimensions such as early diagnosis of diseases, real-time monitoring of bioactive substances, and precise detection of intracellular microenvironments, the application of nanozymes has shown incomparable advantages and broad prospects.

[0003] However, although the existing nanozyme technology has made significant progress, it still faces some key challenges and shortcomings. On the one hand, how to further enhance the catalytic activity of nanozymes to achieve more sensitive biomolecule detection is an important topic of current research. High sensitivity means that tiny physiological changes in organisms can be captured earlier and more accurately, which is crucial for disease early warning and treatment effect evaluation. On the other hand, enhancing the ability of nanozymes to recognize specific target molecules, that is, improving detection specificity, is also an urgent problem to be solved. In complex biological samples, accurately distinguishing target molecules from interfering substances is crucial to ensure the accuracy of test results.

[0004] In addition, although nanozymes have significantly improved stability compared to natural enzymes, their performance may still be affected under certain extreme conditions (such as extreme pH values, high temperature or high pressure environments), limiting their application in certain special fields. Therefore, developing nanozymes with greater stability and adaptability to broaden their detection range has become another key direction of current research.

[0005] Dopamine, as a key neurotransmitter in the nervous system, plays a vital role in metabolic regulation, maintenance of cardiovascular system function, and information transmission in the central nervous system. Accurate and rapid detection of dopamine content is of great significance for understanding its physiological functions, monitoring related disease states, and evaluating drug efficacy. Therefore, the construction of efficient and sensitive colorimetric sensors based on nanozyme technology for the detection of biomolecules such as dopamine can not only promote the development of biomolecule detection technology, but also bring revolutionary breakthroughs in neuroscience research, clinical diagnosis, and drug development. Summary of the invention

[0006] To solve the above problems, we disclose a colorimetric detection method based on functionalized copper cysteine ​​nanozymes. The present invention prepares copper cysteine ​​nanozymes functionalized with gold nanoparticles, and uses TMB and H 2 O 2 As substrate, the reaction conditions were optimized and the enzyme activity parameters were obtained. 2 O 2 Hydroxyl free radicals are generated, and hydroxyl free radicals often react with TMB to generate blue oxidized TMB. Dopamine inhibits this catalytic reaction, and a colorimetric detection method for dopamine is constructed.

[0007] The present invention includes the following specific technical solutions:

[0008] A colorimetric detection method based on functionalized copper-cysteine ​​nanozyme, the method comprising the steps of preparing a functionalized copper-cysteine ​​nanozyme composite material, constructing a detection reaction system, and determining the concentration of a target substance by colorimetric analysis;

[0009] The functionalized copper-cysteine ​​nanozyme composite material is prepared by reacting a copper source, polyethylene glycol and cysteamine under specific conditions, and further reacting with a gold source and trisodium citrate;

[0010] The detection reaction system comprises hydrogen peroxide, tetramethylbenzidine, acetate buffer solution, target solution and the functionalized copper-cysteine ​​nanozyme composite material, and the concentration of the target is determined by measuring the light absorption characteristics of the reaction system.

[0011] Furthermore, in the above-mentioned colorimetric detection method based on functionalized copper cysteine ​​nanozyme, the copper source is CuCl2·2H2O, and the polyethylene glycol is PEG-4000.

[0012] Furthermore, in the above-mentioned colorimetric detection method based on functionalized copper cysteine ​​nanozyme, the specific conditions include adjusting the pH value of the reaction system to a weakly alkaline range under nitrogen protection.

[0013] Furthermore, in the above-mentioned colorimetric detection method based on functionalized copper cysteine ​​nanozyme, the gold source is chloroauric acid solution, and the trisodium citrate is used as a reducing agent and stabilizer.

[0014] Furthermore, in the above-mentioned colorimetric detection method based on functionalized copper-cysteine ​​nanozyme, the functionalized copper-cysteine ​​nanozyme composite material is separated and purified from the reaction system through centrifugation, washing and drying steps.

[0015] Furthermore, in the above-mentioned colorimetric detection method based on functionalized copper-cysteine ​​nanozyme, hydrogen peroxide and tetramethylbenzidine in the detection reaction system are both used as substrates to react with the functionalized copper-cysteine ​​nanozyme composite material to produce detectable light absorption changes.

[0016] Furthermore, in the above-mentioned colorimetric detection method based on functionalized copper cysteine ​​nanozyme, the target is dopamine, and the method further includes drawing a standard curve of dopamine so as to determine its concentration according to the light absorption characteristics of the solution to be tested.

[0017] Furthermore, in the above-mentioned colorimetric detection method based on functionalized copper cysteine ​​nanozyme, the colorimetric analysis is performed by measuring the absorption peak of the reaction system at 652 nm.

[0018] Furthermore, the above-mentioned colorimetric detection method based on functionalized copper cysteine ​​nanozyme comprises the following specific steps:

[0019] 1) In a 100 mL three-necked flask, 273 mg CuCl 2 ·2H 2 O was added to 50 mL of deionized water at 25 °C, and then 32 mg of PEG-4000 and 381 mg of cysteamine hydrochloride were added under nitrogen protection, and 1 M sodium hydroxide solution was added dropwise to adjust the pH value to weak alkalinity, pH = 7-8;

[0020] 2) Continue stirring and reacting for about 15 to 30 minutes. A dark purple substance appears on the wall of the flask. Then transfer to an oil bath and react at 100°C for 20 minutes.

[0021] 3) Add 25 μL 0.1 g / ml chloroauric acid solution and 20 mg trisodium citrate, heat for 20 min, cool to room temperature, centrifuge at 9000 rpm for 10 min, wash three times with a mixture of deionized water and ethanol, and dry in a vacuum oven at 40 °C overnight to obtain a Cu-Cy-PEG@Au NPs composite material;

[0022] 4) Prepare 10 mM HO 2 O 2 1mM TMB stock solution, 2M acetate buffer solution at pH 4.5, 50μL of each of the three 10× stock solutions, 100μL of dopamine solution, ultrapure water and the above nanozyme solution were added to a final concentration of 200ug / mL to a total volume of 500μL;

[0023] 5) After reacting at 25°C for 15 minutes, measure the light absorption curve and record the absorption peak at 652nm;

[0024] 6) preparing a series of standard dopamine solutions ranging from 0 to 300 μM, measuring according to the method in steps 4) to 5), and drawing a standard curve;

[0025] 7) Add the solution to be tested into the reaction system, perform the test according to the method in steps 4)-5), and calculate the concentration of the solution to be tested from the standard curve.

[0026] The invention discloses application of the colorimetric detection method in detecting dopamine.

[0027] In some embodiments, Cu-Cy-PEG@Au NPs are effective for TMB-H 2 O 2 The catalytic color development ability of the system is affected by pH, and its optimum pH value is 4.5.

[0028] In some embodiments, Cu-Cy-PEG@Au NPs are effective for TMB-H 2 O 2 The catalytic color development ability of the system is affected by temperature, and its optimum temperature is 25℃.

[0029] In some embodiments, Cu-Cy-PEG@Au NPs are effective for TMB-H 2 O 2 The catalytic color development ability of the system reaches a maximum value at 15 minutes, so the reaction time of the catalytic color development of this method is 15 minutes.

[0030] In some embodiments, under optimal conditions, a dopamine standard curve is obtained, which is linearly correlated in the concentration range of 0-100 μM. It is expressed as y=-0.0127x+1.5069, R 2 =0.9959, and its detection limit is 0.04μM.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] The present invention prepares the copper cysteine ​​nanozyme functionalized with gold nanoparticles, and uses TMB and H 2 O 2 As substrate, the reaction conditions were optimized and the enzyme activity parameters were obtained. 2 O 2 Hydroxyl radicals are generated, and hydroxyl radicals often react with TMB to generate blue oxidized TMB. Dopamine inhibits this catalytic reaction, and a colorimetric detection method for dopamine is constructed. In the embodiment, we obtained the colorimetric detection of Cu-Cy-PEG@Au NPs for TMB and H by double reciprocal reaction. 2 O 2The Vmax of both are better than that of HRP, indicating that it performs outstandingly in catalyzing the reactions of the two substrates. This proves that we have obtained a nanozyme with excellent catalytic activity by constructing copper cysteine ​​functionalized with gold nanoparticles, and applied it to the detection of dopamine with a detection limit as low as 0.04 μM. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Effect of Cu-Cy-PEG@Au NPs on TMB-H at different pH values ​​(pH 3.5-pH 7.0) 2 O 2 system

[0034] Catalytic color development ability;

[0035] Figure 2 Effect of Cu-Cy-PEG@Au NPs on TMB-H under different temperature conditions 2 O 2 Catalytic color development ability of the system

[0036] Figure 3 Signal intensity of Cu-Cy-PEG@Au NPs catalytic system at different times;

[0037] Figure 4 Dopamine (0-100 μM) standard curve. DETAILED DESCRIPTION

[0038] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] Example

[0040] A colorimetric detection method based on functionalized copper cysteine ​​nanozyme comprises the following steps:

[0041] 1) In a 100 mL three-necked flask, 273 mg CuCl 2 ·2H 2 O was added to 50 mL of 25°C deionized water, and then 32 mg of PEG-4000 and 381 mg of cysteamine hydrochloride were added under nitrogen protection, and 1 M sodium hydroxide solution was added dropwise to adjust the pH value to weak alkaline (pH = 7-8).

[0042] 2) Continue stirring and reacting for about 15 to 30 minutes. A dark purple substance appears on the wall of the flask. Then transfer to an oil bath and react at 100°C for 20 minutes.

[0043] 3) Add 25 μL 0.1 g / ml chloroauric acid solution and 20 mg trisodium citrate, heat for 20 min, cool to room temperature, centrifuge at 9000 rpm for 10 min, wash three times with a mixture of deionized water and ethanol, and dry in a vacuum oven at 40 °C overnight to obtain a Cu-Cy-PEG@Au NPs composite material.

[0044] 4) Prepare 10 mM HO 2 O 2 (10mM) and 1mM TMB (1mM) stock solutions, 2M pH 4.5 acetate (NaAc-HAc) buffer solution. Take 50μL of each of the three 10× stock solutions, add 100μL of dopamine solution, ultrapure water and the above nanozyme solution (final concentration 200ug / mL) to a total volume of 500μL.

[0045] 5) After reacting at 25°C for 15 minutes, measure the light absorption curve and record the absorption peak at 652 nm.

[0046] 6) Prepare a series of standard dopamine solutions ranging from 0 to 300 μM, perform the assay according to the above method, and draw a standard curve.

[0047] 7) Add the solution to be tested into the reaction system, perform the test according to the above method, and calculate the concentration of the solution to be tested from the standard curve.

[0048] Results and Discussion:

[0049] We obtained the Cu-Cy-PEG@Au NPs for TMB and H by double reciprocal reaction. 2 O 2 The Vmax of 247 amino acids (Table 1) were better than those of HRP, indicating that it performed outstandingly in catalyzing the reactions of these two substrates.

[0050] Table 1. Cu-Cy-PEG@Au NPs corresponding to TMB and H 2 O 2 Catalytic ability

[0051]

[0052] like Figure 1 As shown in Figure 2, Cu-Cy-PEG@Au NPs have a significant effect on the TMB-H 2 O 2 The catalytic color development ability of the system is affected by pH, and its optimum pH value is 4.5.

[0053] like Figure 2 As shown in Figure 2, Cu-Cy-PEG@Au NPs have a significant effect on the TMB-H 2 O 2The catalytic color development ability of the system is affected by temperature, and its optimum temperature is 25℃.

[0054] like Figure 3 As shown in Figure 2, Cu-Cy-PEG@Au NPs have a significant effect on the TMB-H 2 O 2 The catalytic color development ability of the system reaches a maximum value at 15 minutes, so the reaction time of the catalytic color development of this method is 15 minutes.

[0055] like Figure 4 As shown in the figure, under the optimal conditions, a dopamine standard curve was obtained, which was linearly correlated in the concentration range of 0-100 μM. 2 =0.9959, and its detection limit is 0.04μM.

[0056] From the above examples, it can be seen that the present invention prepares the copper cysteine ​​nanozyme functionalized with gold nanoparticles, and uses TMB and H 2 O 2 As substrate, the reaction conditions were optimized and the enzyme activity parameters were obtained. 2 O 2 Hydroxyl free radicals are generated, and hydroxyl free radicals often react with TMB to generate blue oxidized TMB. Dopamine inhibits this catalytic reaction, and a colorimetric detection method for dopamine is constructed.

[0057] The above are only a few preferred embodiments of the present invention, which are described in a more specific and detailed manner, but they cannot be understood as limiting the scope of the present invention. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A colorimetric detection method based on functionalized copper cysteine ​​nanozyme, characterized in that: The method comprises the steps of preparing a functionalized copper-cysteine ​​nanozyme composite material, constructing a detection reaction system, and determining the concentration of a target substance by colorimetric analysis; The functionalized copper-cysteine ​​nanozyme composite material is prepared by reacting a copper source, polyethylene glycol and cysteamine under specific conditions, and further reacting with a gold source and trisodium citrate; The detection reaction system comprises hydrogen peroxide, tetramethylbenzidine, acetate buffer solution, target solution and the functionalized copper-cysteine ​​nanozyme composite material, and the concentration of the target is determined by measuring the light absorption characteristics of the reaction system.

2. The colorimetric detection method according to claim 1, characterized in that The copper source is CuCl2·2H2O, and the polyethylene glycol is PEG-4000.

3. The colorimetric detection method according to claim 1, characterized in that The specific conditions include adjusting the pH value of the reaction system to a weakly alkaline range under nitrogen protection.

4. The colorimetric detection method according to claim 1, characterized in that The gold source is chloroauric acid solution, and the trisodium citrate is used as a reducing agent and a stabilizer.

5. The colorimetric detection method according to claim 1, characterized in that The functionalized copper-cysteine ​​nanozyme composite material is separated and purified from the reaction system through centrifugation, washing and drying steps.

6. The colorimetric detection method according to claim 1, characterized in that: The hydrogen peroxide and tetramethylbenzidine in the detection reaction system are both used as substrates to react with the functionalized copper-cysteine ​​nanozyme composite material to produce detectable light absorption changes.

7. The colorimetric detection method according to claim 1, characterized in that The target substance is dopamine, and the method further comprises drawing a standard curve of dopamine so as to determine its concentration according to the light absorption characteristics of the solution to be tested.

8. The colorimetric detection method according to claim 1, characterized in that The colorimetric analysis is performed by measuring the absorption peak of the reaction system at 652 nm.

9. The colorimetric detection method according to claim 1, characterized in that: The specific steps include: 1) In a 100 mL three-necked flask, add 273 mg of CuCl2·2H2O to 50 mL of deionized water at 25°C, then add 32 mg of PEG-4000 and 381 mg of cysteamine hydrochloride under nitrogen protection, and dropwise add 1 M sodium hydroxide solution to adjust the pH value to weak alkalinity, pH = 7-8; 2) Continue stirring and reacting for about 15 to 30 minutes. A dark purple substance appears on the wall of the flask. Then transfer to an oil bath and react at 100°C for 20 minutes. 3) Add 25 μL 0.1 g / ml chloroauric acid solution and 20 mg trisodium citrate, heat for 20 min, cool to room temperature, centrifuge at 9000 rpm for 10 min, wash three times with a mixture of deionized water and ethanol, and dry in a vacuum oven at 40 °C overnight to obtain a Cu-Cy-PEG@AuNPs composite material; 4) Prepare 10mM H2O2 and 1mM TMB stock solutions, 2M pH 4.5 acetate buffer solution, take 50μL of each of the three 10× stock solutions, add 100μL of dopamine solution, ultrapure water and the above nanozyme solution, the final concentration is 200ug / mL, to a total volume of 500μL; 5) After reacting at 25°C for 15 minutes, measure the light absorption curve and record the absorption peak at 652nm; 6) preparing a series of standard dopamine solutions ranging from 0 to 300 μM, measuring according to the method in steps 4) to 5), and drawing a standard curve; 7) Add the solution to be tested into the reaction system, perform the test according to the method in steps 4)-5), and calculate the concentration of the solution to be tested from the standard curve.

10. Use of the colorimetric detection method according to claims 1 to 9 in detecting dopamine.

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