CN104316580B - A kind of method of nanogold enzyme sensor detection water nitrite - Google Patents

Abstract

A method for detecting nitrite in water by a nano-gold enzyme sensor, comprising the following steps: (1) immersing a bare gold electrode in an ethanol solution of 1,6-hexanedithiol; (2) immersing the bare gold electrode in a nano-gold solution for modification; (3) Immerse the electrode in the phosphate buffer solution of horseradish peroxidase and modify it in the dark; (4) draw the standard curve for nitrite detection; (5) detect and calculate the concentration of nitrite in the solution to be tested. The invention not only utilizes the strong adsorption of the nano-gold particles on the horseradish peroxidase to increase the loading capacity of the horseradish peroxidase on the electrode, but also the nano-gold particles can activate and modify the surface of the electrode, and accelerate the transfer of electrons on the modified electrode , to increase the response current and expand the linear range. By adopting the method of the invention, the detection of nitrite in water has a wide linear range, and the recovery rate of nitrite in water is between 96.5% and 105%.

Description

A method for detecting nitrite in water by nano-gold enzyme sensor

technical field

The invention relates to a method for detecting nitrite in water, in particular to a method for detecting nitrite in water by a nano gold enzyme sensor.

Background technique

When the intake of nitrite is excessive, the normal hemoglobin in the human body is converted into methemoglobin under the action of nitrite, which loses the ability to carry oxygen molecules, resulting in insufficient oxygen supply in the body, which is life-threatening in severe cases. Nitrite intake can be fatal if it reaches 1-3 grams. In addition, the reaction of amines and amides with nitrite can produce carcinogenic nitrosamines, which are related to human esophageal cancer, gastric cancer, and liver cancer.

Although there are many measuring methods for nitrite, each has its own advantages; but each method also has its own disadvantages: the instruments and equipment used by the spectroscopic method are simple, cheap, practical and operable, and are easy to use in grassroots units, but their The sensitivity is relatively low and the lower limit of detection is high; chromatography has high precision and can simultaneously determine multiple components, but the equipment used is complicated and the operation is cumbersome; electrochemical analysis has simple equipment, easy operation, and fast analysis speed, but there is currently a linear range Narrow, poor specificity and other issues.

Wang Mina used the horseradish peroxidase electrochemical sensor to detect NaNO ₂ in water, and the linear range was 8.3×10 ^-4 -1.05×10 ^-2 mol/L (see Wang Mina, Liu Huihong. Immobilized horseradish peroxide Enzyme Electrochemical Determination of Organic Peroxides and Nitrite. Journal of Xiangfan University, 2009, 30(2): 5-9). Ma Chaoyue used nano-manganese dioxide-chitosan composite film to immobilize horseradish peroxidase to prepare an enzyme biosensor to detect NaNO ₂ in water, and the linear range was 1.45×10 ^-7 mol/L-1.45×10 ^{- 3} mol/L (see Ma Chaoyue. Fabrication and Application of Immobilized Horseradish Peroxidase Biosensor. Henan University of Technology, 2011). Zheng Dongyun et al constructed a polybromophenol blue modified glassy carbon electrode to detect NaNO ₂ in water with a linear range of 2.00×10 ^-8 -1.09×10 ^-4 mol/L (see Zheng Dongyun, Liu Xiaojun, Zhu Shanying, et al. Electrochemical Communication Sensitive determination of nitrite in water. China Environmental Monitoring 2014, 30(4):140-145). It can be seen that the linear range of the existing method is 2-4 orders of magnitude.

Contents of the invention

The technical problem to be solved by the present invention is to overcome the deficiencies of the prior art and provide a method for detecting nitrite in water with a gold nanometer enzyme sensor capable of quick response, simple operation, wide linear range, good specificity and high sensitivity.

The technical solution adopted by the present invention to solve the technical problems is a method for detecting nitrite in water by a nano-goldenzyme sensor, comprising the following steps:

(1) Immerse the bare gold electrode in the ethanol solution of 1,6-hexanedithiol (HDT) for 10-15 hours; 1,6-hexanedithiol is modified on the surface of the bare gold electrode by molecular self-assembly; and ultrapure water from the side to wash off the 1,6-hexanedithiol physically adsorbed on the surface of the gold electrode;

In the ethanol solution of 1,6-hexanedithiol (HDT), the concentration of 1,6-hexanedithiol is preferably 0.8-1.2 mmol/L, more preferably 1 mmol/L;

(2) Immerse the bare gold electrode modified with 1,6-hexanedithiol in step (1) into the nano-gold solution, and modify it at 2-6°C for 10-15 hours in the dark; gold nanoparticles;

The concentration of the nano gold solution is preferably 0.1-0.3mmol/L;

(3) Immerse the gold nanometer modified electrode in step (2) in the phosphate buffer solution of horseradish peroxidase (HRP); modify at 2-6°C for 10-15 hours in the dark; HRP is fixed on the gold nanometer by electrostatic adsorption Then wash the electrode with dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer solution, the concentration is 0.08-0.12 mol/L (preferably 0.1 mol/L), the pH is 6.8-7.2, and the electrode can be used for nitrite Horseradish peroxidase biosensor for detection, marked as Au electrode-HDT-nano gold-HRP modified electrode;

In the phosphate buffer solution of horseradish peroxidase, the concentration of horseradish peroxidase is preferably 4-6 mg/mL, more preferably 5 mg/mL; the phosphate buffer solution refers to dipotassium hydrogen phosphate-phosphoric acid Potassium dihydrogen buffer solution, the concentration is preferably 0.08-0.12 mol/L (more preferably 0.1 mol/L), and the pH is preferably 6.8-7.2;

(4) Using the Au electrode-HDT-nanogold-HRP modified electrode prepared in step (3) as the working electrode, the response currents of the HRP modified electrode in different concentrations of nitrite solutions were obtained by cyclic voltammetry, and the nitrite concentration and the corresponding The functional relationship between the response currents is the standard curve for nitrite detection;

⑸Using the Au electrode-HDT-nano gold-HRP modified electrode prepared in step (3) to detect the nitrite solution to be tested, the working potential is E=0.70-0.80V, and the response current obtained is based on the standard curve obtained in step ⑷ The concentration of nitrite in the solution to be tested can be calculated.

The present invention immobilizes 1,6-hexanedithiol molecules on the surface of bare gold electrodes through 1,6-hexanedithiol (HDT) molecular self-assembly technology, and then fixes nano-gold particles on the 1,6- On the hexanedithiol molecule, the horseradish peroxidase is finally fixed on the surface of the gold nanoparticles by electrostatic adsorption, so as to realize the effective fixation of the horseradish peroxidase on the surface of the bare gold electrode. The biosensor of the invention has good sensitivity, low detection limit and good selectivity, and can realize rapid and accurate determination of nitrite.

The present invention has the following advantages:

The invention not only utilizes the strong adsorption of the nano-gold particles on the horseradish peroxidase to increase the loading capacity of the horseradish peroxidase on the electrode, but also the nano-gold particles can activate and modify the surface of the electrode, and accelerate the transfer of electrons on the modified electrode , so that the response current increases, thereby expanding the linear range. By adopting the method of the present invention, the detection of nitrite in water has a wide linear range ranging from 9.06×10 ^-8 mol/L to 3.98×10 ^-3 mol/L, reaching 5 orders of magnitude, and the recovery rate of nitrite in water is between 96.5% and Between 105%.

Description of drawings

Figure 1 shows the cyclic voltammograms of different modified electrodes in 5mmol/L nitrite-phosphate buffer solution. Cyclic voltammograms of nano-modified electrode, Au electrode-HDT-gold nano-HRP modified electrode in phosphate buffer solution with nitrite concentration of 5mmol/L;

Figure 2 shows that when the concentration of nitrite is in the range of 9.06×10 ^-8 -3.98×10 ^-3 mol/L, the response current of nitrite on the Au electrode-HDT-nano gold-HRP modified electrode and the Concentration linear relationship graph.

Detailed ways

The present invention will be described in further detail below in conjunction with specific examples.

Example 1

This embodiment includes the following steps:

(1) Immerse the bare gold electrode in an ethanol solution of 1,6-hexanedithiol (HDT) with a concentration of 1 mmol/L for 12 hours, and then wash off the physical adsorption on the surface of the gold electrode from the side with absolute ethanol and ultrapure water. HDT;

(2) Immerse the bare gold electrode modified with HDT in step (1) into the prepared nano-gold solution (the concentration of the nano-gold solution is 0.25mmol/L), modify it at 4°C for 12 hours in the dark, and then Flush the physisorbed gold nanoparticles from the side with ultrapure water;

(3) Immerse the gold-nano-modified electrode in step (2) in 5 mg/mL HRP phosphate buffer solution (dipotassium hydrogen phosphate-potassium dihydrogen phosphate, concentration 0.1 mol/L, pH 7), at 4°C Modified in the dark for 12 hours, and then rinsed the electrode with a buffer solution (dipotassium hydrogen phosphate-potassium dihydrogen phosphate, 0.1mol/L, pH 7) to obtain a horseradish peroxidase biological substance that can be used for nitrite detection. sensor;

(4) Electrochemical detection of 5 mmol/L nitrite on bare gold electrode, Au electrode-HDT modified electrode, Au electrode-HDT-nano-modified electrode, Au electrode-HDT-nano-gold-HRP modified electrode by cyclic voltammetry The difference in behavior, the composition of phosphate buffer solution (pH=7) with nitrite concentration of 5mmol/L: nitrite concentration 5mmol/L, dipotassium hydrogen phosphate-potassium dihydrogen phosphate concentration 0.1mol/L, KCl concentration 0.1 mol/L, cyclic voltammetry parameter setting: potential scanning range 0-8V, scanning speed 0.1V/s (the results are shown in Figure 1). Figure 1 shows the cyclic voltammograms of different modified electrodes in 5mmol/L nitrite-phosphate buffer solution. Curves a, b, c, and d represent Au electrode, Au electrode-HDT modified electrode, Au electrode-HDT- Cyclic voltammograms of nano-modified electrode, Au electrode-HDT-nano gold-HRP modified electrode in phosphate buffer solution with nitrite concentration of 5mmol/L. From the analysis of Figure 1, it is found that when the Au electrode, Au electrode-HDT modified electrode, Au electrode-HDT-gold nano-gold modified electrode, Au electrode-HDT-nano-gold-HRP modified electrode are subjected to cyclic voltammetry scanning in nitrite solution When , nitrite produced an oxidation peak current on the surface of each electrode and no reduction peak current appeared, indicating that nitrite had an irreversible oxidation reaction on the surface of each electrode. The oxidation peak current of Au electrode-HDT-nanogold-HRP modified electrode surface is the largest, followed by that of bare gold electrode, and the oxidation peak current of Au electrode-HDT modified electrode and Au electrode-HDT-nanogold modified electrode is the smallest.

The above analysis results prove that the introduction of gold nanoparticles not only increases the specific surface area of nitrite oxidation reaction and electron transfer specific surface area to a certain extent, but also effectively protects the catalytic activity of HRP to nitrite. Therefore, under the synergistic effect of gold nanoparticles and HRP, the Au electrode-HDT-gold nano-HRP modified electrode exhibits good electrochemical catalytic activity for nitrite, so that nitrite can be synthesized in the Au electrode-HDT-nano gold-HRP The oxidation peak current on the surface of the modified electrode is the largest, and the Au electrode-HDT-nano gold-HRP modified electrode has better sensitivity and selectivity than the bare gold electrode in the detection of nitrite due to the efficient immobilization of HRP.

4. Using the Au electrode-HDT-nano gold-HRP modified electrode as the working electrode, the response currents of the HRP modified electrode in different concentrations of nitrite solutions were obtained by using the current-time curve method. Analysis and research when the nitrite concentration is 9.06×10 ^-8 mol/L, 2.09×10 ^-7 mol/L, 5.96×10 ^-6 mol/L, 9.06×10 ^-6 mol/L, 3.98×10 ^{- 5} mol/L, 7.94×10 ^-5 mol/L, 2.09×10 ^-4 mol/L, 5.96×10 ^-4 mol/L, 9.06×10 ^-4 mol/L, 2.09×10 ^-3 mol/L, The functional relationship between 3.98×10 ^-3 mol/L and the corresponding response current is used to obtain the standard curve and linear range of nitrite detection; according to the lowest detection limit (LOD) = 3σ/k (σ represents the blank standard Deviation, k represents the slope of the standard curve), and the detection limit of this method is calculated (as shown in Figure 2). Figure 2 shows that under the optimal experimental conditions (working potential 0.76V, pH=7), when the concentration of nitrite is in the range of 9.06×10 ^-8 mol/L to 3.98×10 ^-3 mol/L, the nitrite The response current of the salt on the Au electrode-HDT-nano gold-HRP modified electrode meets the linear relationship with the nitrite concentration: I/μA = -0.0424－2.7791C/mmol/L [NO ₂ ^- ], R ² =0.9996, The lower limit of detection is 6.57×10 ^-8 mol/L (according to LOD=3σ/k).

5. The working potential is selected as E=0.76V, and nitrites of different qualities are directly added to mineral water (without nitrite) according to needs to prepare nitrite-phosphate buffer solutions of different concentrations. Then use the Au electrode-HDT-nano gold-HRP modified electrode to detect the nitrite sample solution to be tested, analyze the obtained current-time curve, and calculate the nitrite concentration in the sample and the standard addition of the sample through the relevant experimental data The recovery rate, the obtained results are listed in Table 1.

Table 1 shows the recovery rate detection of nitrite in mineral water samples. The measured recovery rate is between 96.5% and 105%, which proves that the HRP modified electrode has better selectivity and overcomes the interference of related ions in mineral water.

Claims (4)

1. A kind of 1. method of nanogold enzyme sensor detection water nitrite, it is characterised in that comprise the following steps：(1) will 10-15 hours in the ethanol solution of naked gold electrode immersion 1,6- ethanthiols；Rushed respectively with absolute ethyl alcohol and ultra-pure water from side Wash the 1,6- ethanthiols of gold electrode surfaces physical absorption off；

   (2) will be through step（1）The naked gold electrode of upper 1, the 6- ethanthiols of modification is immersed in nano-Au solution, 2-6 DEG C of lucifuge modification 10-15 hours；The nano Au particle of physical absorption is rinsed out from side with ultra-pure water；

   (3) will be through step（2）The electrode of decorated by nano-gold is immersed in the phosphate buffer solution of horseradish peroxidase；2-6℃ Lucifuge modifies 10-15 hours；HRP is fixed on nano Au particle by electrostatic adsorption, is then with concentration by electrode 0.08-0.12 mol/L, dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer solution that pH is 6.8-7.2 are rinsed well, can use In the horseradish peroxidase biology sensor of nitrite detection, labeled as Au electrode-HDT- nanogold-HRP modified electrodes；

   In the phosphate buffer solution of the horseradish peroxidase, horseradish peroxidase is 4-6 mg/mL, pH 6.8- 7.2；The phosphate buffer solution is dipotassium hydrogen phosphate-potassium dihydrogen phosphate, and concentration is that 0.08-0.12 mol/L, pH are 6.8-7.2;

   (4) with step（3）Au electrode-HDT- nanogold-HRP the modified electrodes of preparation are working electrode, using cyclic voltammetry Obtain response current of the HRP modified electrodes in various concentrations nitrite solution, nitrite concentration and corresponding response electricity Functional relation between stream is the standard curve of nitrite detection；

   (5) with step（3）Au electrode-HDT- nanogold-HRP the modified electrodes of preparation detect to nitrite solution to be measured, Operating potential selects E=0.70-0.80V, and (4) standard curve that obtained response current obtains according to step can be calculated and treated Survey the concentration of nitrite in solution.
2. 2. the method for nanogold enzyme sensor detection water nitrite according to claim 1, it is characterised in that step （1）In, in the ethanol solution of 1, the 6- ethanthiols, 1,6- ethanthiol concentration is 0.8-1.2 mmol/L.
3. 3. the method for nanogold enzyme sensor detection water nitrite according to claim 2, it is characterised in that described In the ethanol solution of 1,6- ethanthiol, 1,6- ethanthiol concentration is 1mmol/L.
4. 4. the method for nanogold enzyme sensor detection water nitrite according to claim 1 or 2, it is characterised in that Step（2）In, the concentration of the nano-Au solution is 0.1-0.3mmol/L.