CN110243777A - It is a kind of to improve reaction microenvironment using CTAB and carry out that copper ion is qualitative and the detection method of quantitative detection - Google Patents

Abstract

Improve reaction microenvironment the present invention relates to a kind of using CTAB and carry out that copper ion is qualitative and the detection method of quantitative detection, this method utilizes catalytic action of the copper ion to hydrogen peroxide, under alkaline condition, in the hybrid reaction system of solution to be measured, TMB and hydrogen peroxide, CTAB is added as micellar, whether method observation solution turns yellow by visual observation, realize the qualitative analysis to copper ion in solution to be measured, absorbance value using the solution after ultraviolet specrophotometer measurement reaction at 405nm later, realizes the quantitative detection to copper ion in solution to be measured；The present invention stablizes TMB and its reaction product greatly in alkaline environment because of the presence of CTAB, and chromogenic reaction is greatly stablized, the phenomenon that all no discovery does not become muddy or fades in 48 hours after solution colour developing, which also has very strong anti-interference ability.

Description

A method for qualitative and quantitative detection of copper ions by using CTAB to improve the reaction microenvironment Detection method

technical field

The invention belongs to the field of analysis and detection, and in particular relates to a detection method for qualitative and quantitative detection of copper ions by using CTAB to improve the reaction microenvironment.

Background technique

Copper ions (Cu ²⁺ ) are essential trace elements for the human body and play an important role in various physiological processes. The lack of Cu ²⁺ in the human body may affect enzyme activity and cell metabolism, while high levels of Cu ²⁺ can cause severe gastrointestinal disorders, liver damage to the kidneys, cause various neurological diseases, and seriously affect human health. At present, all countries have clear limits on the concentration of Cu ²⁺ in drinking water. China's standard for drinking water stipulates that the content of copper ions should not exceed 20 μmol/L. Although there are many detection methods for Cu ²⁺ at present, there is still an urgent need for a simple, rapid, environmentally friendly and highly sensitive and specific Cu ²⁺ quantitative method for effective assessment of environmental and human health risks.

Contents of the invention

One of the objectives of the present invention is to provide a simple, rapid and intuitive detection method for qualitative analysis of copper ions by using CTAB to improve the reaction microenvironment.

The second object of the present invention is to provide a detection method that utilizes CTAB to improve the reaction microenvironment and carry out quantitative detection of copper ions with simple method, strong selectivity and high sensitivity.

The object of the present invention is achieved through the following technical scheme: a kind of detection method that utilizes CTAB to improve the reaction microenvironment to carry out copper ion qualitative analysis, utilizes the catalysis of copper ion to hydrogen peroxide, under alkaline condition, in test solution, 3 , 3',5,5'-Tetramethylbenzidine (TMB) and hydrogen peroxide mixed reaction system, add cetyltrimethylammonium bromide (CTAB) as a micelle, by visual method Observe whether the solution turns yellow, and realize the qualitative analysis of copper ions in the solution to be tested.

A detection method for quantitative detection of copper ions by using CTAB to improve the reaction microenvironment, using the catalytic effect of copper ions on hydrogen peroxide, under alkaline conditions, in the solution to be tested, 3,3',5,5'-tetra In the mixed reaction system of methylbenzidine (TMB) and hydrogen peroxide, add cetyltrimethylammonium bromide (CTAB) as micelle agent, utilize the solution after the ultraviolet spectrophotometer to measure reaction at 405nm place The absorbance value is used to realize the quantitative detection of copper ions in the solution to be tested.

Compared with the prior art, the present invention has the advantages of:

(1) In the prior art, various enzyme-catalyzed reactions and enzyme-like catalyzed reactions based on TMB-H ₂ O ₂ are basically carried out under acidic conditions. Under normal conditions, the color reaction is extremely difficult under alkaline conditions. conduct. In the present invention, the introduction of CTAB can solve many problems encountered in the TMB-H ₂ O ₂ experimental system: 1) the existence of CTAB makes TMB and its reaction products extremely stable in alkaline environment; 2) Effectively inhibit the hydrolysis of divalent copper ions; 3) Provide a relatively stable alkaline environment to accelerate the oxidation of divalent copper ions to monovalent copper ions by hydrogen peroxide, and at the same time make TMB be quickly reduced, and the positively charged A bright yellow color is evident in the environment. In the absence of CTAB, the color reaction cannot proceed.

(2) In the prior art, various enzyme-catalyzed reactions and enzyme-like catalyzed reactions based on TMB-H ₂ O ₂ are basically carried out under acidic conditions, and the test must be carried out immediately after the solution develops color, otherwise it will be damaged by the solution. The absorbance value decreases rapidly, resulting in inaccurate detection results. After adding cetyltrimethylammonium bromide in the present invention, the color reaction is greatly stabilized, and no turbidity or discoloration is found within 48 hours after the solution develops color.

(3) Since the reaction of other metal ions with hydrogen peroxide is passivated in the alkaline microenvironment, the detection method has a strong anti-interference ability. The experiment can quickly detect copper ions without adding additional masking agents.

(4) The detection method for copper ion quantitative detection by using CTAB to improve the reaction microenvironment of the present invention has simple operation, high detection sensitivity and good selectivity. It has the advantages of simplicity, quickness, low cost, sensitivity and stability, etc. Therefore, the copper ion detection method proposed by the present invention is expected to be further applied in the detection of actual samples such as the environment and food.

Description of drawings

Figure 1 is a diagram of the color development state of each solution after the reaction of standard solutions of copper ions with different concentrations in Example 1 of the present invention.

Fig. 2 is a diagram showing the variation of the ultraviolet absorption spectrum of each solution with the concentration of copper ions in Example 1 of the present invention.

FIG. 3 is a standard curve graph corresponding to FIG. 2 .

Fig. 4 is a detection response comparison diagram and a color development state diagram under different quaternary ammonium salt surfactants.

Fig. 5 is a comparison chart of absorbance values and a display state chart of different substances contained in the solution of Example 3.

Fig. 6 is a selectivity test diagram for different metal ions in the present invention.

Detailed ways

The content of the present invention will be described in detail below in conjunction with the accompanying drawings and embodiments of the description:

The inventors of the present invention have found in scientific research practice that: 1) adding a small amount of CTAB to the TMB-H ₂ O ₂ reaction system under alkaline buffer conditions can significantly promote the reaction of copper ions catalyzing TMB-H ₂ O ₂ , making the solution It turns yellow quickly in a short time, and the higher the concentration of copper ions, the darker the color of the solution. It shows that under alkaline conditions, in the nanomicelle microenvironment provided by cetyltrimethylammonium bromide, the Fenton-like reactivity of copper ions is significantly enhanced. 2) The conventional TMB-H ₂ O ₂ color development system faces the problem of easy fading, but in the color development system of the present invention, due to the micellar stabilization effect of CTAB, the solution color can remain stable for a long time, making the detection results more stable and reliable .

Therefore, the present invention utilizes the above points to find that a quick and easy, highly sensitive and anti-interference copper ion qualitative and quantitative detection method is constructed:

A detection method for qualitative analysis of copper ions by using CTAB to improve the reaction microenvironment, using copper ions to catalyze hydrogen peroxide, under alkaline conditions, in the solution to be tested, 3,3',5,5'-Tetra In the mixed reaction system of methylbenzidine (TMB) and hydrogen peroxide, add cetyltrimethylammonium bromide (CTAB) as micelle, observe whether the solution turns yellow by visual method, realize the solution to be tested Qualitative analysis of copper ions in the

The described detection method of utilizing CTAB to improve the reaction microenvironment to carry out the qualitative analysis of copper ions comprises the following steps: after the solution to be tested is fully shaken and mixed with the phosphate buffer containing CTAB, TMB and H ₂ O ₂ , then at room temperature Let it stand for 5-30 minutes, then observe whether the solution turns yellow, if the solution turns yellow, it means that there are copper ions in the solution to be tested.

Wherein, the pH value of the phosphate buffer solution is 7.5-9.0.

After the solution to be tested is fully shaken and mixed with the phosphate buffer containing CTAB, TMB and H2O2, in the formed mixture, the concentration of CTAB is _0.3-0.9mg /mL, the concentration of TMB is _0.15-0.5mM , H The concentration of ₂ O ₂ is 0.15-2 mM.

A detection method for quantitative detection of copper ions by using CTAB to improve the reaction microenvironment, using the catalytic effect of copper ions on hydrogen peroxide, under alkaline conditions, in the solution to be tested, 3,3',5,5'-tetra In the mixed reaction system of methylbenzidine (TMB) and hydrogen peroxide, add cetyltrimethylammonium bromide (CTAB) as micelle agent, utilize the solution after the ultraviolet spectrophotometer to measure reaction at 405nm place The absorbance value is used to realize the quantitative detection of copper ions in the solution to be tested.

The described detection method that utilizes CTAB to improve reaction microenvironment to carry out copper ion quantitative detection, it comprises the following steps:

(1) Establishment of the standard curve: A series of standard solutions of copper ions with different concentrations were mixed with phosphate buffer containing CTAB, TMB and H ₂ O ₂ and then left to stand at room temperature for 5-30 min; Afterwards, measure the absorbance values of the above-mentioned solutions at 405nm at room temperature with an ultraviolet spectrophotometer, establish a copper ion concentration-absorbance value standard curve accordingly, and further obtain a linear regression equation between the absorbance value and the copper ion concentration;

(2) Detection of the solution to be tested: After fully shaking and mixing the solution to be tested with the phosphate buffer containing CTAB, TMB and H ₂ O ₂ , let it stand at room temperature for 5-30min; Measure the absorbance value of the solution at 405 nm at room temperature, put the absorbance value into the linear regression equation obtained in step (1), and calculate the concentration of copper ions in the test solution.

Wherein, the pH value of the phosphate buffer solution is 7.5-9.0.

In step (1), after the copper ion standard solution and the phosphate buffer containing CTAB, TMB and H ₂ O ₂ are fully shaken and mixed, in the formed mixed solution, the concentration of CTAB is 0.3-0.9mg/mL, and the concentration of TMB is 0.15-0.5mM, and the concentration of H ₂ O ₂ is 0.15-2mM.

In step (2), after the solution to be tested is thoroughly shaken and mixed with the phosphate buffer containing CTAB, TMB and H ₂ O ₂ , in the formed mixed solution, the concentration of CTAB is 0.3-0.9 mg/mL, and the concentration of TMB is _0.15-0.5mM , the concentration of H2O2 is _0.15-2mM .

Below in conjunction with specific embodiment, the present invention is described in more detail:

Embodiment one:

(1) Using secondary water as a solvent, prepare a series of 500 μL copper ion standard solutions with concentrations of 2, 4, 8, 16, 20, 40, 60, 80, 120, 160, 200, 240, and 300 μM respectively, and then Add 500 μL of phosphate buffer (20mM, pH 8.0 ₎ containing 1mg/mL CTAB, _0.9mM TMB, and 1mM H2O2 to each copper ion standard solution, shake and mix well, let stand for 10min, and observe the copper ion The color of the solution after the reaction corresponding to the standard solution (see Figure 1 for the specific color development state); the sample tubes of the solution after the reaction corresponding to the 13 copper ion standard solutions are respectively numbered a, b, c, d, e, f , g, h, i, j, k, l, m, as can be seen from Fig. 1, the solution color in the sample tube a to m is all yellow, and wherein, the solution color in the sample tube a to d is slightly yellow, The color of the solution in the sample tubes e, f, g, and h is light yellow, while the color of the solution in the sample tubes i and j is yellow, and the color of the solution in the sample tubes k, l, m is dark yellow. It can be seen that, with the increase of copper ion concentration, the solution color of the whole reaction system becomes darker and darker.

(2) detect respectively the visible light absorbance value of the solution at 405nm after the reaction corresponding to the above-mentioned each copper ion standard solution with a UV spectrophotometer at room temperature (the UV-Vis absorption spectrum of solutions a to m varies with the concentration of copper ions as shown in Figure 2 As shown), the copper ion concentration-absorbance value standard curve (as shown in Figure 3) is obtained by this method, and the linear regression equation y=0.0093x+0.0537 of further obtaining the absorbance value and the copper ion concentration, R ² =0.9968, wherein, y is the absorbance value, and x is the concentration of copper ions in the reaction solution. The detection sensitivity of the detection method can reach 1 μM, and the detection linear range is 20-100 μM.

(3) Determination of the solution to be tested: the solution to be tested is treated in a manner similar to the above step (1). Take 500 μL of the solution to be tested and add it to 500 μL of phosphate buffer (20mM, pH 8.0) containing 1mg/mL CTAB, 0.9mM TMB, and 1mM H ₂ O ₂ , and fully shake and mix. After standing for 10min, the solution becomes slightly Yellow; Afterwards, the solution obtained detects that the absorbance value at its 405nm place is 0.108 at room temperature through a UV-visible spectrophotometer again, and this value is substituted in the linear regression equation that step (2) establishes, and the copper in the reaction solution is obtained through calculation The ion concentration is 5.839 μM. Since the test solution is diluted by 2 times during the detection process, the actual concentration of copper ions in the test solution is 11.68 μM.

The specific calculation method is: 0.108=0.0093x+0.0537, the solution is x=5.839 μM, and the copper ion concentration in the solution to be tested is 11.68 μM.

(4) Detection of copper ions in river water: filter the river water of a certain river with a 0.22 μm microporous membrane, and then process it according to the method of the above step (3), the final result is calculated by the linear regression equation obtained in step (2), The concentration of copper ions in the river water was obtained to be 25 μM.

Embodiment two: in order to explore the effect played by CTAB in the detection method of the present invention, the inventor of the present invention adopts the method of the present invention to measure the copper ion standard solution of the same concentration, and the assay method is specifically:

①Take 500 μL copper ion standard solution with a concentration of 200 μM, add it to 500 μL phosphate buffer (20 mM, pH 8.0) containing 0.9 mM TMB and 1 mM H ₂ O ₂ , shake and mix thoroughly, and let stand for more than 20 min; then observe the solution For discoloration, the absorbance value of the solution reaction product at 405 nm was detected at room temperature by an ultraviolet spectrophotometer.

②Take 500 μL of copper ion standard solution with a concentration of 200 μM, add it to 500 μL of phosphate buffer (20 mM, pH 8.0) containing 1 mg/mL CTAB, 0.9 mM TMB, and 1 mM H ₂ O ₂ , shake and mix thoroughly, and then Leave it for 10 minutes; then observe the discoloration of the solution, and then detect the absorbance value of the reaction product of the solution at 405 nm at room temperature with an ultraviolet spectrophotometer.

③Take 500 μL copper ion standard solution with a concentration of 200 μM and add it to 500 μL phosphate buffer (20 mM, pH 8.0) containing 1 mg/mL dodecyltrimethylammonium bromide, 0.9 mM TMB, 1 mM H ₂ O ₂ , and fully oscillate and mix, and stand still for more than 20 minutes; then observe the discoloration of the solution, and then detect the absorbance value of the solution reaction product at 405 nm at room temperature with an ultraviolet spectrophotometer.

④ Take 500 μL of copper ion standard solution with a concentration of 200 μM, add it to 500 μL of phosphate buffer (20 mM, pH 8.0) containing 1 mg/mL dodecylpyridinium bromide, 0.9 mM TMB, and 1 mM H ₂ O ₂ , and Fully oscillate and mix, and let it stand for more than 20 minutes; then observe the discoloration of the solution, and then detect the absorbance value of the solution reaction product at 405 nm at room temperature with an ultraviolet spectrophotometer.

The test results are shown in Figure 4. Figure 4 is a comparison chart of the absorbance values of solutions containing no surfactant and different types of cationic surfactants. The solution that turns yellow and has the largest absorbance value is the reaction after adding 1mg/mL CTAB solution. From the results shown in Figure 4, it can be seen that the presence of CTAB can significantly enhance the reaction of copper ions catalyzing the oxidation of TMB by hydrogen peroxide, while the absence of CTAB and the other two quaternary ammonium salt surfactants have no such effect.

Embodiment 3: In order to explore the color development when different substances are contained in the detection method of the present invention and to determine the principle of the reaction, the inventors of the present invention measured solutions containing different substances. The measurement method is specifically:

① Take 1000 μL of phosphate buffer (10mM, pH 8.0), add 10 μL of 0.1M H ₂ O ₂ and 10 μL of 0.1M TMB, shake and mix thoroughly, and let it stand for 20 minutes; then observe the discoloration of the solution, and then test it by UV spectrophotometry Measure the absorbance value of the solution reaction product at 405 nm at room temperature.

② Take 1000 μL of phosphate buffer solution (10mM, pH 8.0), add 10 μL of 0.1M H ₂ O ₂ and 10 μL of 0.1M TMB, continue to add 10 μL of 10 mM copper ion standard solution, shake and mix thoroughly, and let stand for 20 minutes; Afterwards, the discoloration of the solution was observed, and the absorbance value of the reaction product of the solution at 405 nm was detected by an ultraviolet spectrophotometer at room temperature.

③Take 1000 μL of phosphate buffer (10mM, pH 8.0), add 10 μL of 0.1M H ₂ O ₂ , 10 μL of 0.1M TMB and 100 μL of 10 mg/mL CTAB, shake and mix thoroughly, and let it stand for 20 minutes; then observe the color of the solution Then, the absorbance value of the solution reaction product at 405 nm was detected at room temperature by an ultraviolet spectrophotometer.

④ Take 1000 μL of phosphate buffer (10mM, pH 8.0), add 10 μL of 0.1M H ₂ O ₂ , 10 μL of 0.1M TMB and 100 μL of 10mg/mL CTAB, continue to add 10 μL of 10mM copper ion standard solution, shake fully After mixing, let it stand for 20 minutes; then observe the discoloration of the solution, and then detect the absorbance value of the reaction product of the solution at 405 nm at room temperature with an ultraviolet spectrophotometer.

⑤Take 1000 μL of phosphate buffer (10mM, pH 8.0), add 10 μL of 0.1M TMB and 100 μL of 10mg/mL CTAB, continue to add 10 μL of 10mM copper ion standard solution, shake and mix thoroughly, and let stand for 20 minutes; then observe the discoloration of the solution , and then detect the absorbance value of the solution reaction product at 405 nm at room temperature by an ultraviolet spectrophotometer.

⑥Take 1000 μL of phosphate buffer (10mM, pH 8.0), add 100 μL of 10mg/mL CTAB, continue to add 10 μL of 10mM copper ion standard solution, shake and mix thoroughly, and let it stand for 20 minutes; then observe the discoloration of the solution, and then test it by UV spectrophotometry Measure the absorbance value of the solution reaction product at 405 nm at room temperature.

⑦Take 1000μL phosphate buffer (10mM, pH 8.0), add 10μL 0.1M H ₂ O ₂ , 10μL 0.1M TMB and 100μL 10mg/mL CTAB, continue to add 10μL 1mM copper ion standard solution, shake fully After mixing, let it stand for 20 minutes; then observe the discoloration of the solution, and then detect the absorbance value of the reaction product of the solution at 405 nm at room temperature with an ultraviolet spectrophotometer.

The test results are shown in Figure 5, which is a comparison chart of the absorbance values of different substances contained in the solution. From the results shown in Figure 5, it can be seen that only the reaction solution in step ④ and step ⑦ develops color, and the color of the reaction solution in step ④ is darker than that in step ⑦, indicating that only the TMB-H ₂ O ₂ reaction in the presence of CTAB After adding copper ions to the system, the solution can undergo a color reaction, and the displayed color is closely related to the concentration of copper ions.

Embodiment four: selectivity test experiment:

In order to explore the situation that the detection method of the present invention responds to different metal ions, the inventor of the present invention has used this method to measure different metal ion solutions, and the measurement method is specifically:

Take Cu ²⁺ standard solution, 1.0mM Fe ³⁺ standard solution, 1.0mM Fe ²⁺ standard solution, 1.0mM Pb ²⁺ standard solution, 1.0mM Zn ²⁺ standard solution, 1.0mM Mg ²⁺ standard solution, 1.0mM K ⁺ standard solution, 1.0mM Na ⁺ standard solution, 1.0mM Cr ³⁺ standard solution, 1.0mM Cd ²⁺ standard solution, 1.0mM Mn ²⁺ standard solution, Each 500 μL of 1.0 mM Ag ⁺ standard solution and 1.0 mM Co ²⁺ standard solution was added to 500 μL of phosphate buffer (20 mM, pH 8.0) containing 1 mg/mL CTAB, 0.9 mM TMB, and 1 mM H ₂ O ₂ , Shake and mix well, and let stand for 10 minutes; then observe the discoloration of each solution, and then detect the absorbance value of the reaction product of each solution at 405 nm at room temperature with an ultraviolet spectrophotometer.

The test results are shown in FIG. 6 , which is the ultraviolet absorption diagram and color development diagram of the selectivity test for different metal ions in Example 4. It can be seen from the test results that only Cu ²⁺ shows yellow color, and other metal ions do not show color, which shows that the detection method of the present invention has good selectivity.

The above is only a preferred embodiment of the present invention, and it should be pointed out that for those of ordinary skill in the art, some changes, improvements and modifications can also be made without departing from the concept of the present invention. These changes, improvements and modifications It should also be regarded as the protection scope of the present invention.

Claims (9)

1. A detection method that utilizes CTAB to improve the reaction microenvironment to carry out copper ion qualitative analysis is characterized in that: utilize copper ion to the catalysis of hydrogen peroxide, under alkaline condition, in test solution, TMB and hydrogen peroxide In the mixed reaction system, CTAB was added as a micelle agent, and whether the solution turned yellow was visually observed to realize the qualitative analysis of copper ions in the solution to be tested. 2. utilize CTAB to improve reaction microenvironment according to claim 1 and carry out the detection method of copper ion qualitative analysis, it is characterized in that: it comprises the following steps: test solution and contain CTAB, TMB and H ₂ O phosphate buffered _saline After fully shaking and mixing the solution, let it stand at room temperature for 5-30 minutes, and then observe whether the solution turns yellow. If the solution turns yellow, it means that there are copper ions in the solution to be tested. 3. The detection method for qualitative analysis of copper ions by utilizing CTAB to improve the reaction microenvironment according to claim 2, characterized in that: the pH value of the phosphate buffer is 7.5-9.0. 4. Utilize CTAB according to claim ₂ to improve the reaction microenvironment to carry out the detection method of copper ion qualitative analysis, it is characterized in that: solution to be tested contains CTAB, TMB and H ₂ O phosphate buffer saline buffered saline fully shakes and mixes , in the formed mixed solution, the concentration of CTAB is 0.3-0.9mg/mL, the concentration of TMB is 0.15-0.5mM, and the concentration of H ₂ O ₂ is 0.15-2mM. 5. A detection method utilizing CTAB to improve the reaction microenvironment to carry out copper ion quantitative detection is characterized in that: utilize copper ion to the catalysis of hydrogen peroxide, under alkaline condition, in test solution, TMB and hydrogen peroxide In the mixed reaction system, CTAB was added as a micelle agent, and the absorbance value of the reacted solution at 405 nm was measured by an ultraviolet spectrophotometer to realize the quantitative detection of copper ions in the solution to be tested. 6. utilize CTAB according to claim 5 to improve the reaction microenvironment to carry out the detection method of copper ion quantitative detection, it is characterized in that: it comprises the following steps: (1) Establishment of the standard curve: A series of copper ion standard solutions with different concentrations were mixed with phosphate buffer containing CTAB, TMB and H ₂ O ₂ , and then left to stand at room temperature for 5-30 minutes; , measure the absorbance value of above-mentioned each solution at 405nm place respectively at room temperature with ultraviolet spectrophotometer, establish copper ion concentration-absorbance value standard curve accordingly, further obtain the linear regression equation of absorbance value and copper ion concentration; (2) Detection of the solution to be tested: After fully shaking and mixing the solution to be tested with the phosphate buffer containing CTAB, TMB and H ₂ O ₂ , let it stand at room temperature for 5-30min; Measure the absorbance value of the solution at 405 nm at room temperature, put the absorbance value into the linear regression equation obtained in step (1), and calculate the concentration of copper ions in the test solution. 7. The detection method for quantitatively detecting copper ions by using CTAB to improve the reaction microenvironment according to claim 6, characterized in that: the pH value of the phosphate buffer is 7.5-9.0. 8. Utilize CTAB according to claim 6 to improve the reaction microenvironment to carry out the detection method of copper ion quantitative detection, it is characterized in that: in step (1), copper ion standard solution and containing CTAB, TMB and H ₂ O phosphoric _acid After the salt buffer solution is fully shaken and mixed, in the formed mixed solution, the concentration of CTAB is 0.3-0.9 mg/mL, the concentration of TMB is 0.15-0.5 mM, and the concentration of H ₂ O ₂ is 0.15-2 mM. 9. Utilize CTAB according to claim 6 to improve the reaction microenvironment to carry out the detection method of copper ion quantitative detection, it is characterized in that: in step ( ₂ ), test solution and containing CTAB, TMB and H ₂ O phosphate After the buffer solution is fully shaken and mixed, in the formed mixed solution, the concentration of CTAB is 0.3-0.9 mg/mL, the concentration of TMB is 0.15-0.5 mM, and the concentration of H ₂ O ₂ is 0.15-2 mM.