Preparation of a ratiometric fluorescent probe and its application in nitrite detection

Abstract

The present invention discloses the preparation of a ratio fluorescent probe and its application in nitrite detection, wherein a ratio fluorescent probe system is constructed by using blue luminescent carbon dots BCDs as an internal reference and gold nanoclusters Au NCs@ew as a sensing unit. 2 ‑ It can be used as a detection reagent in the detection process of NO 2 ‑ Visual qualitative or quantitative detection. The ratiometric fluorescent probe of the present invention has good sensitivity, selectivity and fast response speed. 2 ‑ With the increase of NO concentration, the ratio fluorescent probe provides a continuous fluorescence color change from red to blue. The ratio fluorescent probe of the present invention has an extraordinary detection limit (LOD) of 9.38nM and completes the response within 1s, which means that the ratio fluorescent probe of the present invention is effective in the field and quantitative analysis of NO. 2 ‑ It has great application potential in the analysis of NO 2 ‑ Provides an effective way.

Description

Technical Field

[0001] The invention relates to the preparation of a ratio fluorescent probe and application thereof in nitrite detection, belonging to the fields of rapid detection, equipment sensing platform, high-sensitivity detection, etc. Background Art

[0002] In agriculture, nitrite is abundant in nitrogen and reacts with NH4 + and NO3 - It has a synergistic effect, so it is often used as a plant growth-promoting fertilizer. At the same time, nitrite is widely used as an additive or preservative in food processing because it can inhibit the growth of bacteria and help maintain the color and flavor of jerky. However, it is worth noting that nitrite interferes with the body's oxygen transport system because it irreversibly converts hemoglobin in the blood into methemoglobin, seriously impairing the ability of hemoglobin to exchange oxygen. This hazard is particularly serious for pregnant women and infants. Nitrite can also react with secondary amines and amides in the stomach to form carcinogenic N-nitrosamines. The World Health Organization sets the lethal dose of nitrite in drinking water at 1.0 mg / L. Therefore, it is necessary to quickly detect the nitrite content in biological and environmental systems.

[0003] In recent years, there have been more and more reports on nitrite detection methods, including electrochemical method, colorimetric method, UV-visible absorption method, chemiluminescence method (CL), capillary electrophoresis, spectrophotometry and chromatography. However, these reported methods require cumbersome detection procedures, expensive instruments, or poor visual semi-quantitative ability, or are time-consuming. Compared with the above methods, fluorescent sensors for nitrite detection have the advantages of simplicity, high sensitivity, good selectivity and fast response, and have been widely used.

[0004] With the rapid development of nanotechnology, fluorescent nanomaterials (including quantum dots and metal nanomaterials) have attracted great interest due to their high luminescence, high photochemical stability, large Stokes shift, spectral line width, etc., especially in the field of sensing and detection. However, the synthesis process of most quantum dots requires harsh and complex conditions, especially the toxicity of quantum dots limits their application. Therefore, it is of great significance to develop a reliable, simple, non-toxic and harmless method for preparing fluorescent nanomaterials. Summary of the invention

[0005] The present invention aims to provide a ratiometric fluorescent probe for preparation and its application in nitrite detection. The present invention uses blue luminescent carbon dots (BCDs) as internal reference and gold nanoclusters (AuNCs@ew) as sensing units to construct a ratiometric fluorescent probe system. -With the increase of concentration, the ratio fluorescence probe of the present invention provides a continuous fluorescence color change from red to blue. The ratio fluorescence probe of the present invention has an extraordinary detection limit (LOD) of 9.38nM and completes the response within 1s, with good sensitivity, selectivity and fast response speed. In addition, the ratio fluorescence probe combined with hydrogel can also provide a new type of hydrogel gloves. This intelligent manufacturing equipment can be used for NO2 - Visual and quantitative detection of NO2. The glove equipment has good sensitivity and selectivity as well as fast response speed, which means that the manufacturing equipment can be used in the field and quantitative NO2 - It has great application potential in analysis and provides a fast and intuitive way to detect NO2 - Provides an effective way.

[0006] The preparation method of the ratio fluorescence probe of the present invention is to construct a ratio fluorescence probe system using blue luminescent carbon dots (BCDs) as an internal reference and gold nanoclusters (AuNCs@ew) as a sensing unit, comprising the following steps:

[0007] Step 1: Preparation of BCDs

[0008] 20-30 mL of pulp-free lemon juice was mixed with 10-20 mL of water to form a transparent solution, which was then transferred to a 50-75 mL stainless steel autoclave lined with polytetrafluoroethylene and heated at 180-200°C for 12-24 h. After the reaction was completed, the solution was naturally cooled to room temperature, and the CDs solution was dialyzed with deionized water (dialysis bag of 500 nanometers) for 24-48 h to remove small molecules. The carbon quantum dot solution was freeze-dried to obtain blue carbon dot powder BCDs. It was stored in a refrigerator at 4°C for future use.

[0009] Step 2: Synthesis of AuNCs@ew

[0010] 10.0-20 mL of HAuCl4 solution with a concentration of 5-10 mmol / L and 10.0-20 mL of egg white solution with a concentration of 50-70 mg / mL were vigorously stirred and mixed at room temperature for 5-10 min, and then the pH value of the solution was adjusted to 8-10, and reacted at 37-40°C for 12-24 h; the prepared AuNCs@ew was placed in a vacuum oven at 60-80°C and dried for 12-24 h, and the dry powder was collected. It was prepared into a solution and stored in a refrigerator at a temperature of 4-6°C for future use.

[0011] Step 3: Preparation of ratiometric fluorescent probe

[0012] Blue carbon dot powder BCDs was dissolved into AuNCs@ew solution with a molar ratio of 1:1 to construct a ratiometric fluorescent probe.

[0013] pH value and temperature have a certain influence on the fluorescence intensity of the probe. When pH>8, the fluorescence of the ratio probe gradually increases with the decrease of pH. The fluorescence intensity ratio results show that the ratio probe has a strong fluorescence intensity for NO2 under pH=3-6. - It has better sensitivity. The change of temperature has little effect on the fluorescence intensity of the synthesized ratio fluorescent probe. Adding NO2 - After that, below 50° C., the fluorescence recovery amplitudes of the ratio fluorescence probes are similar. Therefore, considering comprehensively, room temperature is selected as the optimal temperature for the synthesis of the probe in the present invention.

[0014] The application of the ratiometric fluorescent probe of the present invention is to - It is used as a detection reagent in the detection process to achieve NO2 - Visual qualitative or quantitative detection.

[0015] Specifically, under a single wavelength excitation of 365 nm, a standard curve is constructed by the ratio of the blue value to the red value and the concentration of nitrite, and is used for quantitative detection of the sample to be tested.

[0016] Furthermore, the ratio fluorescence probe of the present invention is used to construct a hydrogel glove equipment sensing platform to realize NO2 - Specifically, 2.5-3.5g of poloxamer 407 is dissolved in 7.5-11.5ml of distilled water prepared in advance with a certain proportion of Au NCs@ew and BCDs, and the solution is placed in a 4℃ refrigerator, reacted for 6-8h, and then taken out and evenly smeared / spin-coated on the surface of non-fluorescent experimental gloves, and placed in a 40-60℃ oven for 5-10min to obtain a hydrogel glove equipment sensing platform.

[0017] The ratiometric fluorescent probe of the present invention is a mixture of red-emitting AuNC and blue-emitting CD for NO2 - Visual inspection can quickly detect NO2 in water and pork - , and can perform sensitive and accurate detection without complicated pre-processing.

[0018] The hydrogel gloves have two emission peaks at 461 and 647 nm under single wavelength excitation at 365 nm. The red fluorescence of AuNCs can be - The blue fluorescence of CDs, which is used as background reference, is quenched by NO2 - Not sensitive. With NO2 - The addition of , the change in fluorescence intensity ratio produces a distinct change in fluorescence color from red to blue, which can be easily distinguished by naked eyes under ultraviolet light without the need for complex instruments. The detection limit of the gloves is as low as 9.38nM, which is much lower than the WHO's limit on NO2 in drinking water. -In addition, the hydrogel glove sensing device composed of the ratiometric probe can be applied to the NO2 in real water and pork samples. - Visual fluorescence identification.

[0019] The ratiometric fluorescent probe of the present invention can be used to detect NO2 in water and pork - The hydrogel glove equipped sensing platform combined with a smartphone color recognition system was used to detect NO2 in real samples. - Compared with the traditional instrumental method, the sensing platform of the present invention is simple to operate, time-saving, and does not require a lot of sample pretreatment. - As the concentration increases, the sensor equipment also shows obvious color changes in actual samples. Compared with traditional instrumental analysis methods, this sensor equipment has the advantages of on-site visual and quantitative determination of NO2 - potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to facilitate understanding by those skilled in the art, the present invention is further described below with reference to the accompanying drawings.

[0021] Figure 1 A hydrogel sensing device based on multiple colorimetric responses was used for rapid visual quantitative determination of nitrite residue.

[0022] Figure 2 Synthesis of BCDs and AuNCs@ew probes and NO2 - Illustration of the ratiometric fluorescence detection mechanism.

[0023] Figure 3 Fluorescence spectra of the ratiometric probe solution in response to different concentrations of nitrite.

[0024] Figure 4 (A) Selectivity of the ratio probe solution, (B) Fluorescence photograph, (C) Selectivity and anti-interference of the ratio probe solution.

[0025] Figure 5 (A) Liquid hydrogel and fluorescent color at 4℃, gel hydrogel and fluorescent color at 25℃, excess NO2 added at 25℃ - Post-gel state hydrogel and fluorescent color, (B) preparation of hydrogel gloves and (E) glove display, (C) hydrogel gloves exposed to NO2 that does not exceed the standard - Pork and (F) gloves display, (D) hydrogel gloves exposed to NO2 - Excessive pork and (G) gloves display; (H) with NO2 - As the concentration increases, the fluorescent color of the hydrogel changes. DETAILED DESCRIPTION

[0026] The following embodiments are further descriptions of the content of the present invention as an explanation of the technical content of the present invention, but the essential content of the present invention is not limited to the following embodiments. Ordinary technicians in this field can and should know that any simple changes or replacements based on the essential spirit of the present invention should fall within the scope of protection required by the present invention.

[0027] The present invention aims to provide a hydrogel glove sensing device based on multiple chromaticity responses for rapid visual quantitative determination of nitrite residues, which can quickly detect NO2 in water and pork. - , sensitive and accurate detection can be performed without complicated pre-processing.

[0028] The present invention rapidly detects NO2 - The core of the proposed method is a ratiometric fluorescence system and a hydrogel glove equipped sensing platform for nitrite (NO2 - ), where blue luminescent carbon dots (BCDs) were used as internal references and gold nanoclusters (AuNCs@ew) were used as sensing units. - As the concentration increases, the sensing platform provides a continuous fluorescence color change from red to blue. The probe has an extraordinary detection limit (LOD) of 9.38nM and responds within 1 second, thus achieving NO2 - In addition, the ratiometric fluorescent probe was combined with the hydrogel to develop a new type of hydrogel glove. This smart manufacturing equipment can be used for NO2 - Visual and quantitative detection of NO2. The glove equipment has good sensitivity and selectivity as well as fast response speed, which means that the manufacturing equipment can be used in the field and quantitative NO2 - It has great application potential in analysis and provides a fast and intuitive way to detect NO2 - Provides an effective way.

[0029] Example 1: Preparation of ratiometric fluorescent probe

[0030] 1. Preparation of BCDs

[0031] Mix 20-30 mL of pulp-free lemon juice with 10-20 mL of water to form a transparent solution, and then transfer the mixture to a 50-75 mL stainless steel autoclave lined with polytetrafluoroethylene and heat at 180-200 ° C for 12-24 h. After the reaction is completed, the reactor is naturally cooled to room temperature, and the CDs solution is dialyzed with deionized water (500 nm dialysis bag) for 24-48 h to remove small molecules. The carbon quantum dot solution is freeze-dried to obtain a blue carbon dot powder. Collect the dry powder and store it in a refrigerator at 4 ° C for future use;

[0032] 2. Synthesis of AuNCs@ew

[0033] 10.0~20mL HAuCl4 solution (5~10mmol L -1 ) and 10.0-20 mL of egg white solution (50-70 mg mL -1 ) Mix vigorously at room temperature for 5-10 min. Then, adjust the solution to slightly alkaline and react at 37-40 °C for 12-24 h. Place the prepared AuNCs@ew in a vacuum oven at 60-80 °C for 12-24 h. Collect the dry powder, dissolve it in 50 ml of water, prepare it into a solution, and store it in a refrigerator at 4-6 °C for future use.

[0034] Step 3: Preparation of ratiometric fluorescent probe

[0035] Blue carbon dot powder BCDs was dissolved into Au NCs@ew solution with a molar ratio of 0.1mmol / L:0.1mmol / L to construct a ratiometric fluorescent probe.

[0036] Example 2: Sensing mechanism of ratiometric fluorescence system, characterization of each component, and NO2 - Optimization of response and group ratio

[0037] 1. Ratiometric fluorescence system sensing mechanism

[0038] AuNCs@ew and NO2 - The diazo reaction between S1 and T1 generates diazonium salt. Diazonium salt is an electron-withdrawing group. The n-electron lone pair electron cloud is not coplanar with the π-electron cloud on the benzene ring. The transition from n to π* is a forbidden process. The probability of intersystem crossover transition between S1 and T1 is very high, which induces the fluorescence quenching of Au NCs@ew. Adding NO2 - After that, the red fluorescence at 647nm becomes weaker, and as NO2 - As the concentration increases, the sensor system produces a significant color change from red to blue, which can realize NO2 - This mode improves detection efficiency and sensitivity compared to the usual single emission wavelength.

[0039] 2. Characterization of components in ratiometric fluorescence system

[0040] Considering that the detection sensitivity of the probe to the analyte is related to its own properties, the structural characteristics and spectral properties of the BCDs and Au NCs@ew probes were studied using FT-IR, UV-vis and fluorescence spectroscopy, respectively. In addition, TEM was used to determine the morphology and dispersion of the BCDs and AuNCs@ew probes.

[0041] 3. The components of the ratio fluorescence system react with NO2- Response

[0042] Since the detection of analytes by fluorescent probes is related to their fluorescence intensity, under 365nm excitation, for BCDs, NO2 - Although the fluorescence intensity is not the highest, since the eyes are very sensitive to blue, BCDs has enough fluorescence brightness to be used as internal standard fluorescence. Through appropriate proportions, a dual emission ratio fluorescent probe was prepared, which emits red fluorescence under ultraviolet light. - After that, the fluorescent probe showed blue fluorescence under ultraviolet light. In addition, fluorescence spectroscopy was also used to study the photostability of fluorescent probes. They all had good photostability within 5 hours. The designed ratiometric fluorescent probe was used for NO2 - The detection is feasible.

[0043] 4. Effects of pH and temperature on fluorescent probes

[0044] The pH value and temperature have a certain influence on the fluorescence intensity of the probe. When pH>8, the fluorescence of the ratio probe gradually increases with the decrease of pH. The fluorescence intensity ratio results show that the ratio probe has a strong effect on NO2 - Have better sensitivity.

[0045] The change of temperature has little effect on the fluorescence intensity of the synthesized ratio fluorescent probe. - After that, below 50° C., the fluorescence recovery amplitudes of the ratio fluorescence probes are similar. Therefore, considering comprehensively, room temperature is selected as the optimal temperature for the synthesis of the probe in the present invention.

[0046] 5. Selectivity and anti-interference ability of ratiometric fluorescence system

[0047] For an excellent fluorescent sensing system, select representative NH4 + , Na + , Zn 2+ , Cu 2+ , Cd 2+ , K + , Fe 3+ , HClO - , SO4 2- , HCO3 - , NO3 - , Cl - These 12 substances were used to evaluate the ratiometric fluorescence sensing system for NO2 - selectivity and anti-interference ability.

[0048] Example 3: Design of sensor based on ratiometric fluorescence system of hydrogel glove equipped sensing platform

[0049] In order to evaluate the applicability of ratiometric fluorescence probes, NO2 - The ratio of blue value to red value was calculated by fluorescence spectrometer to evaluate its good linear relationship R 2 =0.9914, LOD = 9.38nM. The results show that, combined with the smartphone application, the sensor equipment has realized the NO2 - Portable, visual and quantitative monitoring.

[0050] In order to further evaluate its applicability in actual samples, poloxamer 407 was dissolved in distilled water prepared in advance with a certain proportion of AuNCs@ew and BCDs (for the preparation process of AuNCs@ew and BCDs, see Example 1), and the solution was placed in a 4°C refrigerator. After the reaction, the solution was taken out and evenly smeared / spin-coated on the surface of non-fluorescent experimental gloves. After being placed in an oven for a few minutes, the hydrogel glove equipped with a sensing platform was obtained.

[0051] Specifically, 2.5-3.5 g of poloxamer 407 is dissolved in 7.5-11.5 ml of distilled water prepared in advance with a certain proportion of Au NCs@ew and BCDs, the solution is placed in a 4°C refrigerator, reacted for 6-8 hours, taken out, evenly smeared / spin-coated on the surface of non-fluorescent experimental gloves, and placed in an oven at 40-60°C for 5-10 minutes to obtain a hydrogel glove equipped with a sensing platform.

[0052] Our sensing platform was used to detect NO2 in water and pork - The hydrogel glove equipped sensing platform combined with a smartphone color recognition system was used to detect NO2 in real samples. - Compared with the traditional instrumental method, the sensing platform of the present invention is simple to operate, time-saving, and does not require a lot of sample pretreatment. - As the concentration increases, the sensor equipment also shows obvious color changes in actual samples. Compared with traditional instrumental analysis methods, this sensor equipment has the advantages of on-site visual and quantitative determination of NO2 - potential.

Claims

1. An application of a ratiometric fluorescent probe, characterized in that: The ratiometric fluorescent probe is used in NO2 - It is used as a detection reagent in the detection process to achieve NO2 - Visual qualitative or quantitative detection; The ratiometric fluorescent probe is constructed using blue luminescent carbon dots BCDs as an internal reference and gold nanoclusters Au NCs@ew as a sensing unit, and includes the following steps: Step 1: Preparation of BCDs 20-30 mL of pulp-free lemon juice was mixed with 10-20 mL of water to form a transparent solution, which was then transferred to a polytetrafluoroethylene-lined stainless steel autoclave and heated at 180-200°C for 12-24 h. After the reaction was completed, the solution was naturally cooled to room temperature, and the CDs solution was dialyzed with deionized water for 24-48 h to remove small molecules. The carbon quantum dot solution was freeze-dried to obtain blue carbon dot powder BCDs, which was stored at 4°C for later use. Step 2: Synthesis of Au NCs@ew 10.0-20 mL of HAuCl4 solution with a concentration of 5-10 mmol / L and 10.0-20 mL of egg white solution with a concentration of 50-70 mg / mL were stirred and mixed at room temperature for 5-10 min, and then the pH value of the system was adjusted and reacted at 37-40°C for 12-24 h; the prepared Au NCs@ew was placed in a vacuum oven at 60-80°C and dried for 12-24 h, and the dry powder was collected and prepared into a solution, which was stored at 4-6°C for later use; Step 3: Preparation of ratiometric fluorescent probe Blue carbon dot powder BCDs was dissolved into Au NCs@ew solution to construct a ratiometric fluorescent probe.

2. The use according to claim 1, characterized in that: In step 1, a dialysis bag with a size of 500 nanometers is used for dialysis.

3. The use according to claim 1, characterized in that: In step 2, the pH value of the system is adjusted to 8-10.

4. The use according to claim 1, characterized in that: In step 3, the molar ratio of blue carbon dot powder BCDs and Au NCs@ew is 1:

1.

5. The use according to claim 1, characterized in that: Under single wavelength excitation of 365 nm, a standard curve was constructed by comparing the ratio of blue value to red value with the concentration of nitrite and used for quantitative detection of the samples to be tested.

6. The use according to claim 1 or 5, characterized in that: The pH of the detection system is 3-6.

Images