CN114280024B

CN114280024B - Specific nitrite fluorescent detection method based on copper nanoclusters and oxidation reaction

Info

Publication number: CN114280024B
Application number: CN202111605747.6A
Authority: CN
Inventors: 吕海霞; 范丹阳; 宋昊天
Original assignee: Fuzhou University
Current assignee: Fuzhou University
Priority date: 2021-12-25
Filing date: 2021-12-25
Publication date: 2023-10-20
Anticipated expiration: 2041-12-25
Also published as: CN114280024A

Abstract

The invention discloses a specific nitrite fluorescent detection method based on copper nanoclusters and oxidation reaction, and belongs to the technical field of nanomaterial preparation and nitrite detection. Firstly, preparing copper nanoclusters by using glutathione as a stabilizer and ascorbic acid as a reducing agent through a one-pot method, and constructing a fluorescence sensor for detecting nitrite based on oxidation-reduction reaction. NO under acidic conditions ²⁻ The presence of (C) enables Fe to be ²⁺ Is easily oxidized to Fe ³⁺ By mixing Fe with ²⁺ /Fe ³⁺ Redox process and Fe ³⁺ Fluorescence quenching of Cu NCs combined, NO can be easily achieved ^2‑ Is a quantitative analysis of (a). The sensor can improve the sensitivity and accuracy of nitrite detection, and has better specificity.

Description

Specific nitrite fluorescent detection method based on copper nanoclusters and oxidation reaction

Technical Field

The invention belongs to the technical field of nano material preparation and nitrite detection, and particularly relates to a specific nitrite fluorescent detection method based on copper nanoclusters and oxidation reaction.

Background

Nitrite is one of the most common nitrogen-containing compounds in nature, not only in the natural environment, but also in the human society, being widely used in the food and chemical industry, however, nitrite has been a great threat to human health due to its potential toxicity, and nitrite ions have been considered as a class a inorganic contaminant in drinking water: nitrite ions interact with proteins and are important precursors for generating highly oncogenic N-nitrosamines; excessive intake of nitrite can cause a series of medical problems such as esophageal cancer, infant methemoglobin (blue infant syndrome), spontaneous abortion, congenital defects of the central nervous system, etc.; the U.S. Environmental Protection Agency (EPA) specifies a Maximum Contaminant Level (MCL) of 1 ppm (21.7 μm) for nitrite and a similar guideline value set by the World Health Organization (WHO) of 3 ppm, and therefore, the determination of nitrite content is extremely important for drinking water quality monitoring and clinical diagnosis.

In order to control and detect the use of nitrite, corresponding nitrite detection techniques have been developed, but the accuracy of early nitrite detection methods such as colorimetry and spectrophotometry are susceptible to other substrates in the sample. Accordingly, high precision and versatile methods such as gas chromatography mass spectrometry (GC-MS) and liquid chromatography mass spectrometry (LC-MS) have been developed to overcome the interference of various substrates. However, these detection methods have high cost, poor portability and complex pretreatment, and cannot meet the requirements of on-site rapid detection. In recent years, some novel nitrite sensors, such as electrochemical sensors, fluorescence sensors, and Surface Enhanced Raman Scattering (SERS) sensors, have been successfully used for detection of nitrite using specific physical and chemical interactions between the sensor and nitrite molecules, and have greatly promoted miniaturization of nitrite detection instruments. Among these sensors, the fluorescence sensor is the most widely used analytical tool because of its low cost, high accuracy and high portability.

Disclosure of Invention

The invention aims to provide a specific nitrite fluorescent detection method based on copper nanoclusters and oxidation reaction, which can not only improve the sensitivity and accuracy of nitrite detection, but also has better specificity.

In order to achieve the above purpose, the invention adopts the following technical scheme:

a specific nitrite fluorescent detection method based on copper nanoclusters and oxidation reaction comprises the following steps:

(1) Preparation of copper nanoclusters with glutathione as a stabilizer and ascorbic acid as a reducing agent: 125 μl of CuCl ₂ The solution was mixed with 20ml of GSH solution under vigorous stirring for 5min to form a copper mercaptide complex, at which time the mixed solution became progressively cloudy. Then, 450. Mu.l of freshly prepared AA solution was added to the mixed solution and heated to a certain temperatureStirring was continued for a period of time during which the solution became gradually clear, after which the solution was cooled at room temperature to give a Cu NCs suspension, which was stored at 4 ℃ for further use. The preparation and application routes of Cu NCs are shown in FIG. 1.

(2) Cu NCs are used for constructing a method for detecting nitrite: taking NO with different concentrations ^2- Mixing with acetic acid buffer solution, placing in a centrifuge tube, and adding Fe with a certain concentration into the mixed solution ²⁺ The solution was thoroughly mixed by vortexing, after which 200 μl of the prepared Cu NCs was added to the solution and the mixture was incubated at room temperature for a period of time. Finally, the fluorescence emission spectrum is measured at a certain excitation wavelength, and the detection mechanism is shown in fig. 2.

(3) Establishment of a nitrite detection standard curve: detecting the change of a fluorescence emission peak of the copper nanocluster by using a fluorescence spectrometer, respectively adding nitrite solutions with different concentrations into a specific detection system in sequence, and establishing the relationship between the nitrite concentration c and a fluorescence quenching value delta f to obtain a nitrite detection standard curve.

Wherein, the concentration of the Glutathione (GSH) solution of the synthetic copper nanoclusters in the step (1) is 0.19-0.25 mM, preferably 0.21mM; the concentration of the Ascorbic Acid (AA) is 80-120 mM, preferably 100mM; the synthesis time is 2-6 h, preferably 4h, and the synthesis temperature is 35-75 ℃, preferably 65 ℃.

Wherein, the pH of the acetic acid buffer solution in the step (2) is 3, 4, 5, 6 and 7, preferably 3.

Wherein the Fe in the step (2) ²⁺ The solution concentration is 25, 50, 75, 100, 125, 150, 175, uM, preferably 150 uM;

wherein the incubation time in the step (2) is 5, 10, 15, 20, 25, 30, 35min, preferably 30min.

Wherein, the fluorescence quenching value Δf measured in the step (3) is specifically measured as the difference in fluorescence intensity between the fluorescence intensity value without nitrite and the fluorescence intensity value with nitrite at 432nm under excitation of 365nm excitation light.

The nitrite detection standard curve in the step (3) refers to a standard curve for constructing a relationship between a fluorescence quenching value and a nitrite concentration of a specific detection system.

The invention has the beneficial effects that:

(1) According to the invention, the copper nanocluster is obtained by using the green one-pot method with glutathione as a stabilizer and ascorbic acid as a reducing agent, and the low-cost, high-sensitivity and rapid specific detection of nitrite is successfully realized based on the nanocluster and the oxidation reaction.

(2) The specificity detection system constructed by the invention is realized by taking the optimally designed copper nanocluster fluorescent nanomaterial as a fluorescent probe. NO under acidic conditions ²⁻ The presence of (C) enables Fe to be ²⁺ Is easily oxidized to Fe ³⁺ By mixing Fe with ²⁺ /Fe ³⁺ Redox process and Fe ³⁺ In combination with fluorescence quenching for Cu NCs, the amount of change in fluorescence quenching produced can be linearly related to the change in fluorescence intensity between nitrite concentrations. The detection method has strong fluorescence response to nitrite and specificity to nitrite detection.

(3) The invention successfully establishes a nitrite detection method, and the fluorescence quenching value and the nitrite concentration (5-70 uM) have good linear curve (R) ² = 0.9972）。

Drawings

FIG. 1 shows the preparation and application routes of Cu NCs.

FIG. 2 shows the mechanism of Cu NCs for nitrite detection.

FIG. 3 is a standard curve for different concentrations of nitrite.

FIG. 4 is a graph comparing fluorescence quenching effects of nanoclusters in the presence of different substances.

Detailed Description

In order to make the contents of the present invention more easily understood, the technical scheme of the present invention will be further described with reference to the specific embodiments, but the present invention is not limited thereto.

Example 1

Preparation of copper nanoclusters: 125 μl of CuCl ₂ The solution was mixed with 20ml of 0.21mM GSH solution under vigorous stirring for 5min to form a copper mercaptide complex, at which time the mixed solution became cloudy. Then, 450 μl of freshly prepared 100mM AA solution was added to the mixed solution, heated to 65deg.C and stirring continued for 4 hours, during which time the solution became gradually clear, after which the solution was cooled at room temperature to give a Cu NCs suspension, which was stored at 4deg.C for further use. The preparation and application routes of Cu NCs are shown in figure 1.

Example 2

Copper nanoclusters for detection of nitrite: taking NO ^2- Mixing with acetic acid buffer solution with pH of 3, placing in a centrifuge tube, and adding Fe with concentration of 150 μm into the mixed solution ²⁺ After the solution was thoroughly mixed by vortexing, 200. Mu.l of the prepared Cu NCs was added to the above solution, and the mixture was incubated at room temperature for 30min, the detection mechanism of which is shown in FIG. 2.

Example 3

Establishment of a nitrite detection standard curve: and respectively adding the solutions of nitrite with different concentrations into a specific detection system, measuring the fluorescence intensity value at 432nm under excitation of 365nm excitation light, recording the fluorescence intensity change value of nitrite or not after incubation, and constructing a standard curve of the relationship between the fluorescence intensity quenching amount of the nanoclusters and the nitrite concentration in the detection method, wherein the linear range and the standard curve are shown in figure 3.

Example 4

The specific recognition effect is also an important factor for an aptamer sensor. Thus, the specific recognition function of the detection method was tested, and the selectivity was examined by adding interfering anions, by adding Cl at a concentration of 100. Mu.m ^- 、I ^- 、Ac ^- 、CO ₃ ² 、HCO ³ 、SO ₄ ^2- 、HSO ^3- 、S ₂ O ₅ ^2- 、NO ₃ ^- 、ClO ^4- 、PO ₄ ^3- 、HPO ₄ ^2- And H ₂ PO ^4- Then comparing the fluorescence intensity of nitriteThe degree change value is shown in fig. 4. As can be seen from FIG. 4, even though the concentration of interfering ions is higher than that of nitrite added, the fluorescence quenching degree is smaller than that of NO ^2- This indicates that the detection method has good selectivity.

Claims

1. A specific nitrite fluorescent detection method based on copper nanoclusters and oxidation reaction is characterized by comprising the following steps: firstly, preparing copper nanoclusters by using glutathione as a stabilizer and ascorbic acid as a reducing agent through a one-pot method, and constructing a fluorescence sensor for detecting nitrite based on oxidation-reduction reaction;

the method comprises the following steps:

(1) Preparing copper nanoclusters by taking glutathione as a stabilizer and ascorbic acid as a reducing agent: 125. Mu.L of CuCl ₂ Mixing the solution with 20mL of glutathione solution under the condition of intense stirring for 5min to form a copper mercaptide complex, adding 450 mu L of freshly prepared ascorbic acid solution, heating, continuously stirring, and naturally cooling to obtain Cu NCs suspension;

(2) Copper nanoclusters for detection of nitrite: taking NO with different concentrations ^2- Mixing with acetic acid buffer solution, placing into a centrifuge tube, adding Fe ²⁺ The solution is fully mixed by vortex oscillation, 200 mu L of Cu NCs suspension is added, and fluorescence emission spectrum is measured after incubation at room temperature;

(3) Establishment of a nitrite detection standard curve: and establishing a relation between the nitrite concentration c and the fluorescence quenching value delta f, namely a nitrite detection standard curve.

2. The method according to claim 1, characterized in that: the concentration of glutathione solution in the step (1) is 0.21mM; the concentration of the ascorbic acid solution is 100mM; the synthesis time was 4h and the synthesis temperature was 65 ℃.

3. The method according to claim 1, characterized in that: the pH of the acetic acid buffer solution in the step (2) is 3; fe (Fe) ²⁺ The concentration of the solution is 150 uM; incubation time was 30min.

4. The method according to claim 1, characterized in that: the fluorescence quenching value Deltaf in the step (3) is the fluorescence intensity difference between the fluorescence intensity value without nitrite at 432nm under the excitation of 365nm excitation light and the fluorescence intensity value with nitrite.