Application of a gold nanocluster / long-afterglow nanorod-based ratiometric fluorescent probe in mercury ion detection

Abstract

The application aims to provide an application of a ratio fluorescent probe based on gold nanoclusters / long afterglow nanorods in mercury ion detection, and belongs to the technical field of fluorescent detection, which comprises synthesis of gold nanoclusters, preparation of long afterglow nanorods, and preparation and application of the ratio fluorescent probe. The ratio fluorescent probe comprises red light emitting gold nanoclusters and green light emitting long afterglow nanorods, and the fluorescent emission of the two materials has obvious spectral and color distinction. The responses of the gold nanoclusters and the long afterglow nanorods to mercury ions are completely opposite, mercury ions can quench the luminescence of the gold nanoclusters, while the luminescence of the long afterglow nanorods is enhanced to a certain extent. The response difference of the two fluorescent signals to different concentrations of mercury ions is utilized, and the ratio processing of the fluorescent intensity can be used for ratio fluorescent detection of mercury ions, and obvious composite fluorescent color change can be observed under the same excitation light irradiation, which is convenient for visual analysis of mercury ions.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of fluorescence detection, and particularly relates to application of a gold nanocluster / long-afterglow nanorod-based ratio fluorescence probe in mercury ion detection. BACKGROUND

[0002] Mercury ion is a highly toxic and widely distributed heavy metal ion, which causes serious harm to the environment and human health. Mercury ion mainly enters the ecological system through industrial emissions. Since it is not metabolized, it is accumulated in the human body through the food chain, causing serious damage to the human kidney function, immune system and central nervous system, and therefore it is of great significance to detect mercury ion for monitoring human health and preventing environmental pollution. At present, many analysis methods have been used for the detection of mercury ion, such as atomic absorption spectrometry, atomic emission spectrometry, etc. However, these analysis techniques rely on large and expensive instruments and require the operation of professional technicians. Therefore, it is imperative to develop a simple, sensitive and accurate mercury ion determination method.

[0003] Gold nanoclusters are a new type of fluorescent material composed of several to dozens of atoms. Due to its size of less than 2 nm, it is close to the Fermi wavelength of electrons, and is affected by the quantum confinement effect, so gold nanoclusters exhibit “molecular-like” fluorescence properties. Compared with ordinary fluorescent dyes, gold nanoclusters have better light stability, larger stokes shift and longer fluorescence lifetime; in addition, simple preparation process, excellent stability and biocompatibility promote the application of gold nanoclusters in biological sensing. Mercury ions can cause fluorescence quenching of gold nanoclusters. Based on this fluorescence quenching property, researchers have obtained gold nanoclusters stabilized by different ligands by changing the ligand in the preparation process of gold nanoclusters. In addition to classic ligands such as glutathione and bovine serum albumin, other polypeptide or protein ligands have also been used for the synthesis and application research of gold nanoclusters. For example, in 2016, Zhang et al. prepared a new type of gold nanocluster using N-acetyl-L-cysteine as a protective agent and a reducing agent for the fluorescence detection of mercury ions (Sens. Actuators, B 2016, 235, 386-393); in 2022, Yuan et al. prepared gold nanoclusters using a polypeptide containing cysteine-cysteine-tyrosine as the core sequence and conducted mercury ion detection research (Colloids Surf., B 2022, 219, 112820).

[0004] Compared with single-signal fluorescence detection, the design of ratio fluorescence can effectively eliminate the influence of external environment, instrument and other factors on the detection process, and improve the signal-to-noise ratio and the accuracy of detection. Since mercury ions can quench the fluorescence of gold nanoclusters, gold nanoclusters can be used as a fluorescence response unit for mercury ion detection, and another fluorescent material with different emission wavelengths can be introduced to realize ratio fluorescence detection of mercury ions. In 2018, Xie et al. introduced carbon quantum dots based on gold nanoclusters. Gold nanoclusters and carbon quantum dots can be excited by a 360 nm ultraviolet lamp, but mercury ions have no effect on the luminescence of carbon quantum dots, thereby constructing a ratio fluorescence probe for mercury ion analysis (Sens. Actuators, B 2018, 259, 1082-1089); In 2019, Wu et al. constructed a ratio fluorescence probe based on red-emitting gold nanoclusters and blue-emitting metal-organic framework materials, in which red gold nanoclusters can respond to mercury ions, and metal-organic framework materials act as internal fluorescent signals (Analyst, 2019, 144, 2523-2530). The design of the above ratio fluorescence probe relies on the fluorescence quenching response of mercury ions to gold nanoclusters, while the other fluorescent material remains stable in fluorescence state as a detection internal standard. However, an ideal ratio fluorescence probe not only needs to contain two kinds of fluorescent materials that can be excited by the same wavelength but have different emission wavelengths, more importantly, the response of the two materials to mercury ions should have opposite trends, that is, with the addition of mercury ions, the fluorescence intensity of one fluorescent material decreases, and the fluorescence intensity of the other fluorescent material increases. Therefore, it is very urgent to develop a sensitive and reliable new ratio fluorescence probe for the detection of mercury ions. SUMMARY

[0005] In view of the needs and field blank of the above research methods, the application provides an application of a ratio fluorescence probe based on gold nanoclusters / long-afterglow nanorods in mercury ion detection, which is simple, sensitive and has good fluorescence response to mercury ions.

[0006] The application adopts the following technical scheme:

[0007] The application provides an application of a ratio fluorescence probe based on gold nanoclusters / long-afterglow nanorods in mercury ion detection, wherein the ratio fluorescence probe comprises red-emitting gold nanoclusters and green-emitting long-afterglow nanorods.

[0008] Further, the ratio fluorescence analysis method is used to detect mercury ions, the response of the red-emitting gold nanoclusters to mercury ions is a negative response, and the response of the green-emitting long-afterglow nanorods to mercury ions is a positive response.

[0009] Further, the preparation method of the ratio fluorescence probe comprises the following steps:

[0010] The first step is to prepare red luminescent gold nanoclusters by reacting tetrachloroauric acid and bovine serum albumin through a hydrothermal method;

[0011] The second step is to prepare manganese-doped zinc germanate long afterglow nanorods by reacting zinc nitrate, sodium germanate and manganese nitrate through a hydrothermal method.

[0012] The third step is to mix the gold nanoclusters obtained in the first step and the long afterglow nanorod dilution solution obtained in the second step, and the ratio fluorescent probe is obtained.

[0013] Further, in the first step, the concentration of the tetrachloroauric acid aqueous solution is 10 mM, the concentration of the bovine serum albumin solution is 50 mg / mL, the volume ratio of the tetrachloroauric acid solution to the bovine serum albumin solution is 1:1, and the hydrothermal reaction temperature is 37 DEG C.

[0014] Further, in the second step, the molar ratio of zinc nitrate to sodium germanate is 1:1, the doping amount of manganese nitrate is 0.5% of the molar amount of sodium germanate, the hydrothermal reaction temperature is 220 DEG C, and the reaction time is 6 h.

[0015] Further, in the third step, the dilution multiple of the long afterglow nanorod is 50 times, and the volume ratio of the gold nanocluster solution to the diluted long afterglow nanorod solution is 1:1.

[0016] Further, the detection process of the mercury ion is as follows: different concentrations of mercury nitrate solution are mixed with 40 μL of the ratio fluorescent probe solution, then ultrapure water is added to make the total volume of the detection system 200 μL, after sufficient reaction, the fluorescence signal intensity is detected by using a fluorescence instrument; at the same time, the visual fluorescence analysis of the system after reaction is carried out by using a portable ultraviolet lamp.

[0017] Further, the excitation wavelength used in the fluorescence instrument detection and the irradiation wavelength of the ultraviolet lamp are both 254 nm.

[0018] The gold nanoclusters and the long afterglow nanorods can be excited by ultraviolet light, and the mercury ion has a quenching effect on the fluorescence of the gold nanoclusters, but can enhance the luminescence of the long afterglow nanorods. Both materials can respond to mercury ions, but the response trends are opposite, which conforms to the design concept of ideal ratio fluorescence.

[0019] The detection principle of the present application is as follows: Figure 1The red luminescent gold nanoclusters were prepared by using tetrachloroauric acid (HAuCl4•4H2O) as raw material and bovine serum albumin (BSA) as protective agent and reducing agent, and the green luminescent long afterglow nanorods (PLNPs) were further prepared by using a hydrothermal method. The ratio fluorescent probe can be obtained by mixing the gold nanoclusters and the long afterglow nanorods according to a proper volume ratio. Both the gold nanoclusters and the long afterglow nanorods can be excited by 254 nm ultraviolet light, and when no mercury ions exist in the solution system, the gold nanoclusters emit red light and the long afterglow nanorods emit green light. When mercury ions are added, the mercury ions can quench the red luminescence of the gold nanoclusters, but can enhance the luminescence of the long afterglow nanorods to a certain extent. Thus, the ratio fluorescent detection of mercury ions can be realized by recording the fluorescence intensities of the gold nanoclusters and the long afterglow nanorods for ratio fluorescence processing, and further, the visualized images of the response of the ratio fluorescent probe to mercury ions can be obtained under ultraviolet light irradiation, and with the increase of the concentration of mercury ions, the color of the reaction solution changes from orange to yellow to green.

[0020] The beneficial effects of the present application are as follows:

[0021] 1. The preparation method of the gold nanoclusters and the long afterglow nanorods is simple, and the two luminescent materials emit bright light, and the spectrum and color have good distinguishability;

[0022] 2. The ratio fluorescent probe based on the gold nanoclusters and the long afterglow nanorods is easy to store, has good stability, and can eliminate the influence of external environment such as environment on the detection process. Since the fluorescence responses of the gold nanoclusters and the long afterglow nanorods to mercury ions are completely different, the mercury ions can quench the fluorescence of the gold nanoclusters but can enhance the luminescence of the long afterglow nanorods, which meets the ideal ratio fluorescent design concept.

[0023] 3. The response of the ratio fluorescent probe of the present application to mercury ions has good ratio fluorescent response, high detection specificity, simple and convenient operation process, and further, the fluorescence visualized analysis of mercury ions can be realized through the color change of fluorescence. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 Schematic diagram of the ratio fluorescent probe based on gold nanoclusters / long afterglow nanorods for mercury ion detection.

[0025] Figure 2 Fluorescence spectrum and fluorescence actual image of gold nanoclusters.

[0026] Figure 3 Fluorescence characterization and morphology characterization of long afterglow nanorods.

[0027] Figure 4 Spectral characterization of the ratio fluorescent probe based on gold nanoclusters / long afterglow nanorods.

[0028] Figure 5Fluorescence response spectra of gold nanoclusters and long afterglow nanorods to mercury ions.

[0029] Figure 6 Fluorescence spectra, linear fitting and fluorescence visualization images of a gold nanocluster / long afterglow nanorod-based ratio fluorescent probe to mercury ions.

[0030] Figure 7 Verification of specific response of a gold nanocluster / long afterglow nanorod-based ratio fluorescent probe to mercury ions. DETAILED DESCRIPTION

[0031] In order to make the above features and advantages of the present application more clear and easy to understand, the embodiments of the present application will be further described in detail below with reference to the accompanying drawings.

[0032] Example 1

[0033] Synthesis of a gold nanocluster / long afterglow nanorod-based ratio fluorescent probe:

[0034] (1) Synthesis of red luminescent gold nanoclusters (AuNCs): 5 mL of an aqueous solution of tetrachloroauric acid (10 mM) was mixed with 5 mL of a bovine serum albumin solution (50 mg / mL) and stirred vigorously at 37 ℃. After 2 min, 500 μL of a sodium hydroxide (1 M) solution was added, and the stirring was continued at 37 ℃ for 12 h to obtain bovine serum albumin-stabilized gold nanoclusters. The synthesized gold nanoclusters were stored in a 4 ℃ refrigerator for later use.

[0035] (2) Synthesis of green luminescent long afterglow nanorods (PLNPs): First, a sodium germanate solution was prepared by weighing 2.0928 g of germanium oxide into a 50 mL flask, adding 30 mL of an aqueous sodium hydroxide solution (2 M), stirring overnight at room temperature, and then diluting to 50 mL. Further, 2 mmol of zinc nitrate, 0.005 mmol of manganese nitrate solution, and 300 μL of concentrated nitric acid were added to 11 mL of ultrapure water, stirred and mixed uniformly, and then 2.5 mL of the above-mentioned sodium germanate solution was added dropwise, and the pH of the reaction system was slowly adjusted to 9.5 with concentrated ammonia water. After stirring at room temperature for 1 h, the reaction solution was transferred to an autoclave and reacted at 220 ℃ for 6 h to obtain a white precipitate. The product was washed with ultrapure water for 2-3 times at a centrifugal speed of 10000 rpm. Then, 5 mL of water was added to the washed product and stored at room temperature for later use.

[0036] (3) Synthesis of a gold nanocluster / long afterglow nanorod-based ratio fluorescent probe: First, the above-mentioned long afterglow nanorod solution was diluted 50 times with ultrapure water, and then an equal volume of the gold nanocluster solution and the diluted long afterglow nanorod solution were mixed uniformly to obtain the ratio fluorescent probe, which was stored in a 4 ℃ refrigerator for later use.

[0037] The fluorescence spectrum and fluorescence real image of the gold nanoclusters (AuNCs) are shown in FIG. 5A and FIG. 5B, respectively. The excitation wavelength was set to 254 nm during the fluorescence spectrum test. The fluorescence emission peak of the gold nanoclusters was observed at 650 nm. Under the irradiation of a 254 nm portable ultraviolet lamp, bright red fluorescence could be observed. Figure 1

[0038] The spectral characterization and morphology characterization of the long afterglow nanorods (PLNPs) are shown in FIG. 6A and FIG. 6B, respectively. The excitation wavelength was set to 254 nm during the spectral test. The photoluminescence spectrum peak of the long afterglow nanorods was observed at 530 nm. Under the irradiation of a 254 nm portable ultraviolet lamp, bright green luminescence could be observed. Figure 3 Figure 3 B is the transmission electron microscopy characterization of the long afterglow nanorods. As can be seen from the figure, the long afterglow nanorods are in good regular rod shape and have good dispersibility.

[0039] The fluorescence spectrum of the ratio fluorescent probe based on the gold nanoclusters / long afterglow nanorods is shown in FIG. 7. Since both the gold nanoclusters and the long afterglow nanorods can be excited by 254 nm, and the positions of the emission wavelengths are well distinguished, a ratio fluorescent probe can be constructed based on these two materials. From the fluorescence spectrum of the ratio fluorescent probe, it can be observed that under 254 nm excitation, the luminescence peaks of the gold nanoclusters and the long afterglow nanorods are clear, and the positions of the luminescence peaks are the same as when they exist alone. There is no mutual interference between the two, indicating the successful construction of the ratio fluorescent probe. Figure 3

[0040] Example 2

[0041] Application of the ratio fluorescent probe based on the gold nanoclusters / long afterglow nanorods in mercury ion detection:

[0042] (1) Fluorescence response of gold nanoclusters (AuNCs) to mercury ions: Take two centrifuge tubes and add 20 μL of gold nanocluster solution to each. Add an appropriate amount of mercury nitrate solution to one of the centrifuge tubes to make the final concentration of mercury nitrate 50 μM. Then add ultrapure water to the two centrifuge tubes to make the total volume of the reaction system 200 μL. Use a fluorescence spectrometer to perform spectral determination on the two groups of solutions, with the excitation wavelength set to 254 nm.

[0043] (2) Fluorescence response of long afterglow nanorods (PLNPs) to mercury ions: Take two centrifuge tubes and add 20 μL of 50-fold diluted long afterglow nanorods to each. Add an appropriate amount of mercury nitrate solution to one of the centrifuge tubes to make the final concentration of mercury nitrate 50 μM. Then add ultrapure water to the two centrifuge tubes to make the total volume of the reaction system 200 μL. Use a fluorescence spectrometer to perform spectral determination on the two groups of solutions, with the excitation wavelength set to 254 nm. ​​​

[0044] (3) Fluorescence response of the ratio fluorescent probe to mercury ions: 40 μL of the probe was placed in a centrifuge tube, different concentrations of mercury nitrate solution were added, and then ultrapure water was added to make up the reaction system to 200 μL. Then, spectral determination was performed by using a fluorescence spectrometer, the excitation wavelength was set to 254 nm, the fluorescence intensities at 530 nm and 650 nm were recorded, and the fluorescence intensity ratio was processed. Further, fluorescence visualization analysis was performed on different concentrations of mercury ions by using a 254 nm portable ultraviolet lamp as a light source.

[0045] (4) Detection specificity of the ratio fluorescent probe: 40 μL of the probe was placed in a centrifuge tube, appropriate amounts of mercury ions, magnesium ions, manganese ions, iron ions and sodium ions were added, so that the final concentrations of the ions were all 50 μM, and then ultrapure water was added to make up the reaction system to 200 μL. Then, spectral determination was performed by using a fluorescence spectrometer, the excitation wavelength was set to 254 nm, the fluorescence intensities at 530 nm and 650 nm were recorded, and the fluorescence intensity ratio was processed.

[0046] The fluorescence response of the gold nanoclusters and the long afterglow nanorods to mercury ions is shown in Figure 4 The fluorescence response of mercury ions to the gold nanoclusters and the long afterglow nanorods is opposite, and with the addition of the gold nanoclusters, the fluorescence of the gold nanoclusters is obviously quenched, and the luminescence of the long afterglow nanorods is enhanced.

[0047] The fluorescence response of the ratio fluorescent probe based on the gold nanoclusters / long afterglow nanorods to mercury ions is shown in Figure 5 With the addition of mercury ions, the luminescence of the gold nanoclusters is gradually quenched, and the luminescence of the long afterglow nanorods is enhanced to a certain extent. By recording the fluorescence intensities of the gold nanoclusters at 650 nm and the fluorescence intensities of the long afterglow nanorods at 530 nm and processing the ratio, a linear fitting curve can be obtained. It can be observed from the figure that the linear response range of the ratio fluorescent probe of the application to mercury ions is 0-50 μM, and the detection limit is as low as 0.19 μM. Under the irradiation of a 254 nm portable ultraviolet lamp, fluorescence visualization images were collected, when no mercury ions were present, the emission peaks of the gold nanoclusters and the long afterglow nanorods both existed, at this time, the solution was a mixture of red light and green light; with the increase of the concentration of mercury ions, the red luminescence of the gold nanoclusters was continuously quenched, the luminescence of the long afterglow nanorods was enhanced, the color of the reaction solution gradually changed to yellow, and finally to continuously enhanced green.

[0048] The fluorescence response specificity of the ratio fluorescent probe based on the gold nanoclusters / long afterglow nanorods to mercury ions is shown in Figure 6As shown. Only when mercury ions exist, the ratio fluorescence value of gold nanoclusters and long afterglow nanorods is obviously reduced, and the ratio fluorescence intensity of other kinds of metal ions is close to the blank value, which shows that the ratio fluorescence probe of the application has good specificity for detection of mercury ions.

Claims

1. An application of a ratiometric fluorescent probe based on gold nanoclusters / long afterglow nanorods in the detection of mercury ions, wherein the ratiometric fluorescent probe comprises red-emitting gold nanoclusters and green-emitting long afterglow nanorods, and mercury ions are detected by ratiometric fluorescence analysis, wherein the red-emitting gold nanoclusters have a negative response to mercury ions, and the green-emitting long afterglow nanorods have a positive response to mercury ions. Its features are: The method for preparing the ratiometric fluorescent probe includes the following steps: The first step involved preparing red-luminescent gold nanoclusters by reacting tetrachloroauric acid and bovine serum albumin via a hydrothermal method. The second step involves preparing manganese-doped zinc germanate long afterglow nanorods from zinc nitrate, sodium germanate, and manganese nitrate using a hydrothermal method. The third step involves mixing the gold nanoclusters obtained in the first step with the diluent of the long afterglow nanorods obtained in the second step to obtain the ratiometric fluorescent probe.

2. The application of a ratiometric fluorescent probe based on gold nanoclusters / long afterglow nanorods in mercury ion detection according to claim 1, characterized in that: In the first step, the concentration of the tetrachloroauric acid aqueous solution was 10 mM, the concentration of the bovine serum albumin solution was 50 mg / mL, the volume ratio of the tetrachloroauric acid solution to the bovine serum albumin solution was 1:1, and the temperature of the hydrothermal reaction was 37℃.

3. The application of a ratiometric fluorescent probe based on gold nanoclusters / long afterglow nanorods in mercury ion detection according to claim 1, characterized in that: In the second step, the molar ratio of zinc nitrate to sodium germanate is 1:1, the doping amount of manganese nitrate is 0.5% of the molar amount of sodium germanate, the hydrothermal reaction temperature is 220℃, and the reaction time is 6h.

4. The application of a ratiometric fluorescent probe based on gold nanoclusters / long afterglow nanorods in mercury ion detection according to claim 1, characterized in that: In the third step, the long afterglow nanorods are diluted 50 times, and the volume ratio of the gold nanocluster solution to the diluted long afterglow nanorod solution is 1:

1.

5. The application of a ratiometric fluorescent probe based on gold nanoclusters / long afterglow nanorods in mercury ion detection according to claim 1, characterized in that: The detection process of mercury ions is as follows: mercuric nitrate solutions of different concentrations are mixed with 40 μL of ratio fluorescent probe solution, and then ultrapure water is added to make the total volume of the detection system 200 μL. After the reaction is complete, the intensity of the fluorescence signal is detected using a fluorescence instrument; at the same time, a handheld ultraviolet lamp is used to perform visual fluorescence analysis on the system after the reaction.

6. The application of a ratiometric fluorescent probe based on gold nanoclusters / long afterglow nanorods in mercury ion detection according to claim 5, characterized in that: The excitation wavelength and the irradiation wavelength of the ultraviolet lamp used in fluorescence detection are both 254 nm.

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