PH-mediated gold nanocluster for realizing different fluorescence emissions and preparation method and application thereof

Abstract

The invention discloses a pH-mediated gold nanocluster for realizing different fluorescence emissions and a preparation method and application thereof.The method comprises the following steps that HAuCl4 and a surfactant are mixed, deionized water is added under violent stirring, then the pH is adjusted, stirring is conducted at the temperature of 20-90 DEG C, and a gold nanocluster solution is obtained, the surfactant is one of egg white, pepsin and glutathione. When the pH value of the gold nanocluster solution is in a range of 1.0-5.5, the gold nanocluster solution presents strong yellow fluorescence, when the pH value is in a range of 8.0-9.0, the gold nanocluster solution presents blue fluorescence, when the pH value is in a range of 9.0-10.0, the gold nanocluster solution presents orange red fluorescence, and when the pH value is in a range of 11.0-12.5, the gold nanocluster solution presents red fluorescence. Ultraviolet-visible spectrum and photoluminescence spectrum test results show that the size of the gold nanocluster depends on the pH value of a reaction solution. And the size of the gold nanocluster is increased along with the increase of the pH value of the reaction solution. The red fluorescent gold nanocluster developed by the invention is used for detecting metal ions.

Description

A pH-mediated gold nanocluster achieving different fluorescence emission, its preparation method and application Technical Field

[0001] This invention relates to the fields of nanomaterial preparation and optical sensing technology, specifically to a gold nanocluster that achieves different fluorescence emission through pH mediation, its preparation method, and its application. Background Technology

[0002] The development of nanomaterials science has provided new tools for highly sensitive and specific analytical detection. Among them, gold nanoclusters (AuNCs), as a star material with a size within 2 nanometers, have shown great potential in the field of analytical sensing due to their unique quantum size effect, tunable fluorescence emission, excellent biocompatibility, and photostability. Compared with traditional organic fluorescent dyes and quantum dots, AuNCs have larger Stokes shifts, stronger resistance to photobleaching, and richer surface chemistry, making them easier to functionalize.

[0003] In practical analytical testing, the target substances extend far beyond heavy metal ions. Substances requiring rapid and accurate detection in today's society also include: bioactive molecules, such as disease biomarkers (proteins, DNA, and RNA), neurotransmitters, hormones, and blood glucose, whose detection is crucial for early diagnosis and health monitoring; environmental and food safety pollutants, such as pesticide residues, veterinary antibiotics, mycotoxins, illegal food additives, explosives and their derivatives; and other chemical substances, such as reactive oxygen species / nitrogenous substances (ROS / RNS), pH values, and specific anions (such as CN). - F - )wait.

[0004] Among these, the detection of heavy metal ions has significant practical implications in areas such as environmental monitoring, food safety, and public health. Mercury (Hg) 2+ ), lead (Pb) 2+ ), chromium (Cr) 3+ Heavy metal ions, such as those found in atomic absorption spectrometry (AAS) and inductively coupled plasma mass spectrometry (ICP-MS), are highly toxic, easily accumulate, and are difficult to degrade, posing a serious threat to ecosystems and human health. Currently, while methods such as atomic absorption spectrometry (AAS) and inductively coupled plasma mass spectrometry (ICP-MS) offer high sensitivity, they are expensive, complex to operate, and difficult to implement for rapid on-site screening. Therefore, there is an urgent need to develop a highly selective and sensitive fluorescence sensing platform for on-site detection of heavy metal ions.

[0005] However, developing dedicated sensors for each of these substances is not only time-consuming and labor-intensive, but also faces a common core challenge: how to achieve high selectivity and high sensitivity in complex real-world sample matrices, while resisting interference from other coexisting substances. Traditional methods such as enzyme-linked immunosorbent assay (ELISA) and chromatography-mass spectrometry (GC-MS), while accurate, have limitations such as high cost, long processing time, and the need for professional personnel, making them difficult to popularize in field or home settings.

[0006] Against this backdrop, visual or instrumental sensing strategies based on fluorescence color changes are gaining popularity due to their intuitiveness, fast response, and ease of integration with portable devices. The success of this strategy heavily relies on the availability of high-performance fluorescent probes, especially those capable of emitting different, stable, and bright fluorescent colors. The advantages of multicolor fluorescent probes include: multiple detection—different colored probes can respond simultaneously or separately to different target analytes, enabling parallel analysis of multiple substances on a single platform; ratiometric and self-calibration—detection using the intensity ratio of two or more emission wavelengths effectively overcomes interference from factors such as probe concentration and environmental fluctuations, improving accuracy; and visualization and simplification—obvious color changes can be directly discerned by the naked eye or simple imaging devices, reducing reliance on expensive and complex instruments.

[0007] Therefore, developing a universal method for the simple, controllable, and reproducible preparation of nanoprobes with different fluorescence colors (especially those with significant color differences, such as from yellow to red) is a key material foundation for building the next generation of high-performance, multifunctional fluorescence sensing platforms.

[0008] Currently, existing technologies for preparing gold nanoclusters with different fluorescent colors mainly rely on ligand exchange or the use of different ligands. By changing the type of protecting ligand, the electronic structure and surface environment of the cluster can be altered, thereby modulating the fluorescence. However, this requires the pre-synthesis or acquisition of multiple ligands, and the relationship between ligands and fluorescence color is often difficult to predict and precisely control.

[0009] Controlling core size and composition: Synthesizing different atomic numbers (such as Au) through stringent thermodynamic control. 25 Au 38 This method involves complex synthesis conditions, poor reproducibility, and difficulty in achieving continuous and controllable color transitions from one color to another. Complex multi-step synthesis or post-modification processes result in long workflows, low yields, and are not conducive to large-scale preparation and widespread application. These methods share common characteristics: complex operation, indirect control, or difficulty in achieving "leapfrog" changes in fluorescence color within the same simple system.

[0010] Of particular note is the immense potential of pH, an extremely simple, inexpensive, and easily precisely controllable physicochemical parameter, in materials synthesis. While previous studies have reported that pH may affect the fluorescence intensity of certain AuNCs, a method for directly and controllably preparing gold nanoclusters with significantly different visual fluorescence colors (e.g., bright yellow and bright red) within the same ligand system using pH as a single, decisive variable has not been systematically reported before. The realization of this method will greatly simplify the preparation process of multicolor fluorescent nanoprobes, reduce their production costs, and provide a novel paradigm for the rational design and control of material optical properties through a single fundamental variable (pH).

[0011] In summary, developing a universal method for the simple preparation of gold nanoclusters with different fluorescent colors using pH value as the sole control switch, and exploring the application potential of such materials in the detection of a wide range of analytes from heavy metal ions to biomolecules, is an innovative topic that fills current technological gaps and promotes the development of the field of nanofluorescence sensing. It has significant scientific value and broad application prospects. Summary of the Invention

[0012] The purpose of this invention is to provide a method for preparing gold nanoclusters with different fluorescence emission mediated by pH, and its application. This method allows for the controllable preparation of AuNCs with different fluorescence colors simply by adjusting the pH.

[0013] In one aspect of the present invention, a method for preparing gold nanoclusters with different fluorescence emissions mediated by pH is proposed. According to an embodiment of the present invention, the method includes the following steps: mixing HAuCl4 with a surfactant, adding deionized water under vigorous stirring, adjusting the pH, and stirring at 20-90°C to obtain a gold nanocluster solution, wherein the surfactant is one of egg white, pepsin, and glutathione.

[0014] Tetrachloroauric acid (HAuCl4) provides the gold (Au) element required for synthesis, making it the sole metal source for forming gold nanoclusters. HAuCl4 dissociates into tetrachloroaurate ions ([AuCl4] ions) upon dissolving in water. - A surfactant, [AuCl4], is added to the subsequent reaction. - Au 3+ Gold atoms (Au) that need to be reduced to zero valence 0 These Au 0 Atoms aggregate, nucleate, and grow, eventually forming gold nanoclusters. Their concentration directly affects the yield and concentration of the final clusters.

[0015] Surfactants: 1. Egg white: In an alkaline environment of pH 9.0-10.0, proteins undergo controlled partial denaturation, unraveling their compact structure. This process is crucial, as it exposes the reducing amino acid residues—tyrosine (Tyr) and tryptophan (Trp)—that were originally encased within the hydrophobic interior of the protein. The phenolic hydroxyl and indole groups of these residues, under heating conditions of 20-40°C, act as electron donors to release Au. 3+ Restore to Au 0 .

[0016] 2. Pepsin: At pH 8.0-9.0, key amino acids in the peptide chain are in an optimal ionization state, enabling them to effectively bind Au through coordinate bonds. 3+ Ions form precursor complexes, which possess suitable amphiphilicity or charge distribution to drive self-assembly into ordered nanostructures.

[0017] 3. Glutathione: Glutathione (GSH), a classic small-molecule thiol ligand, functions primarily through strong Au-S bonds to achieve atomic-level precision control over gold clusters, and its mode of action fundamentally changes with pH. Under strong acid conditions: It mainly stabilizes ultra-small gold nuclei, forming yellow fluorescent clusters. Under strong alkaline conditions: OH... - Together with GSH, they form an unstable precursor.

[0018] In addition, the method for preparing gold nanoclusters with different fluorescence emissions mediated by pH according to the above embodiments of the present invention may also have the following additional technical features:

[0019] In some embodiments of the present invention, when the surfactant is glutathione, the gold nanocluster solution exhibits yellow fluorescence emission when the pH value is between 1.0 and 5.5 under 365 nm ultraviolet light irradiation; and exhibits red fluorescence emission when the pH value is between 11.0 and 12.5.

[0020] In some embodiments of the present invention, the molar ratio of HAuCl4, glutathione, and deionized water is 1:1.2-3. The Au content in the mixed system... 3+ The initial molar concentration is 0.5 mM to 5 mM, and an appropriate amount of deionized water is added to meet this concentration requirement.

[0021] In some embodiments of the present invention, when the pH of the gold nanocluster solution is ≤10.5, the stirring speed is 300-700 rpm and the stirring time is 18-30 h; when the pH of the gold nanocluster solution is ≥11.0, the stirring speed is 300-700 rpm and the stirring time is 2-12 h.

[0022] In some embodiments of the present invention, the speed of the vigorous stirring is 500-1200 rpm.

[0023] In some embodiments of the present invention, the pH adjustment is performed using sodium hydroxide or hydrochloric acid.

[0024] In some embodiments of the present invention, the concentration of sodium hydroxide is 0.1-2 mol / L, and the concentration of hydrochloric acid is 0.1-1 mol / L.

[0025] In some embodiments of the present invention, when the surfactant is egg white, the pH value of the gold nanocluster solution is between 9.0 and 10.0, and it exhibits orange-red fluorescence emission under 365 nm ultraviolet light irradiation.

[0026] In some embodiments of the present invention, when the surfactant is pepsin, the pH value of the gold nanocluster solution is between 8.0 and 9.0, and it exhibits blue fluorescence emission under 365 nm ultraviolet light irradiation.

[0027] In another aspect of the invention, a gold nanocluster prepared by the aforementioned method is provided. According to embodiments of the invention, the pH value of the gold nanocluster solution is between 11.0 and 12.5, and it exhibits red fluorescence emission under 365 nm ultraviolet light irradiation.

[0028] In another aspect of the invention, an application of the aforementioned gold nanoclusters is proposed, wherein, according to an embodiment of the invention, the gold nanoclusters are used for the detection of Hg. 2+ content.

[0029] Strong red fluorescent AuNCs prepared at pH 12.0 were used for Hg 2+ The detection showed that these glutathione (Glu)-protected red fluorescent AuNCs were effective against Hg. 2+ It exhibits good selectivity and high sensitivity, attributed to Hg 2+ -Au and thiols-Hg 2+ The strong interaction between them. The red fluorescent AuNCs detect Hg. 2+ The linear range is 0-20 µM, and the detection limit is 1.6 nM (0.32 ppb), exhibiting a wide linear range and a low detection limit.

[0030] Compared with the prior art, the beneficial effects of the present invention are:

[0031] 1) This invention utilizes AuNCs with different fluorescent colors (such as yellow, blue, orange-red, and red) obtained through pH adjustment to construct a multicolor fluorescence sensing array. Combined with chemometric methods, it enables the detection of various heavy metal ions (such as Zn). 2+ Cu 2+ Cr 3+The accurate identification and concentration differentiation of (etc.) significantly improves detection efficiency and practicality.

[0032] 2) This invention prepares Glu-AuNCs with strong yellow and red fluorescence emission under strongly acidic (pH 1.0-5.5) and strongly alkaline (pH 11.0-12.5) environments, respectively. The size of the gold clusters still depends on the pH of the reaction solution; the size of the AuNCs increases with increasing pH of the reaction solution. The strongly red fluorescent AuNCs prepared using a reaction solution at pH 12.0 are used for Hg... 2+ The detection showed good selectivity and high sensitivity. Simultaneously, the detection of Hg by red fluorescent Glu-AuNCs was tested. 2+ Its practical applicability allowed for the successful detection of Hg in tap water and lake water samples. 2+ content.

[0033] 3) The indicator used in this invention is derived from commercial analytical reagents. The entire preparation process does not involve complex synthesis steps, which simplifies the operation process, lowers the threshold for use, and is conducive to its promotion and use in daily testing scenarios.

[0034] 4) The gold nanoclusters constructed in this invention enable the distinguishing detection of metal ions in actual samples, and are suitable for rapidly determining the safety of environmental water sources. Attached Figure Description

[0035] Figure 1 is a flowchart of the preparation of Glu-AuNCs in Example 1 of the present invention;

[0036] Figure 2 shows digital photographs of Glu-AuNCs solutions prepared in different pH ranges (1.0 - 13.0) in Example 1 of the present invention, observed by the naked eye (top) and irradiated by a 365 nm ultraviolet lamp (bottom);

[0037] Figure 3 shows the (a) UV-Vis spectrum and (b) normalized PL spectrum of Glu-AuNCs solutions prepared in Example 1 of the present invention at pH 2.0, 11.0, 12.0 and 12.5.

[0038] Figure 4 shows transmission electron microscopy (TEM) and high-resolution transmission electron microscopy (HRTEM) images of Glu-modified gold nanoclusters in (a) pH 2.0 and (b) pH 12.0 samples. Inset: Interplanar spacing of gold nanoclusters measured by ImageJ software.

[0039] Figure 5 is a schematic diagram of the synthesis and detection of gold nanoclusters in Example 2 of the present invention;

[0040] Figure 6 shows the TEM characterization results of the gold nanoclusters protected by egg white in Example 2 of the present invention;

[0041] Figure 7 shows the TEM characterization results of pepsin-protected gold nanoclusters in Example 3 of the present invention;

[0042] Figure 8 shows the lattice diagram of the egg white-protected gold nanoclusters in Example 2 of the present invention under TEM characterization.

[0043] Figure 9 shows the lattice diagram of the pepsin-protected gold nanoclusters in Example 3 of the present invention under TEM characterization.

[0044] Figure 10 shows the fluorescence emission, excitation spectrum, ultraviolet absorption spectrum, and color observed by the naked eye of the gold nanoclusters protected by egg white in Example 2 of the present invention.

[0045] Figure 11 shows the fluorescence emission, excitation spectrum, ultraviolet absorption spectrum, and color observed by the naked eye of the gold nanoclusters protected by pepsin in Example 3 of the present invention.

[0046] Figure 12 shows the fluorescence emission, excitation spectrum, ultraviolet absorption spectrum, and color observed by the naked eye of Glu-AuNCs synthesized in the reaction solution at pH 12.0 in Example 1 of this invention.

[0047] Figure 13 shows the fluorescence emission spectrum of Glu-AuNCs aqueous solution with 10 heavy metal ions in Example 1 of the present invention (a) and the corresponding ratio of relative fluorescence intensity (F0 / F) (b).

[0048] Figure 14 shows different concentrations of Hg in Example 1 of the present invention (a). 2+ The light emission spectra of quenched Glu-AuNCs to varying degrees (λ) ex = 336 nm), (b) in Hg 2+ The relative fluorescence intensity (F0 / F) varies with Hg within the detection concentration range of 0-20 μM. 2+ A graph showing the linear increase in concentration.

[0049] Figure 15 shows the linear discriminant analysis (LDA) diagram of various metal ions in Example 5 of the present invention;

[0050] Figure 16 shows a clustering heatmap analysis of the detection of multiple metal ions in Example 5 of the present invention;

[0051] Figure 17 shows Zn concentrations at different levels in Example 6 of the present invention. 2+ Cluster heatmap analysis of the detected clusters;

[0052] Figure 18 shows different concentrations of Pb in Example 6 of the present invention. 2+ Cluster heatmap analysis of the detected clusters;

[0053] Figure 19 is an LDA diagram of the anti-interference detection of heavy metal ions by gold nanoclusters in Example 7 of the present invention. Detailed Implementation

[0054] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0055] Example 1

[0056] As shown in Figure 1, a method for preparing gold nanoclusters (Glu-AuNCs) that achieve different fluorescence emissions mediated by pH includes the following steps:

[0057] 0.5 mL of 20 mM HAuCl4 was mixed with 0.15 mL of 100 mM Glu, and then 1.35 mL of deionized water was added while stirring vigorously (1000 rpm). Glu-AuNCs solutions with pH values ​​of 1.0, 1.5, 2.0, 3.0, 4.0, 5.0, 5.5, 6.0, 7.0, 8.0, 8.0, 9.0, 9.5, 10.0, 10.5, 10.0, 11.5, 12.0, and 13.0 were prepared by adding different amounts of 0.1–2 M sodium hydroxide (NaOH) and 0.1–1 M hydrochloric acid (HCl). The reaction mixture was then brought to a final volume of 5 mL (25... 0 C). A solution containing Glu-AuNCs with pH ≤ 10.5 was stirred (500 rpm) for 24 hours at 60 °C, and a solution containing Glu-AuNCs with pH ≥ 11.0 was stirred (500 rpm) for 4 hours. The prepared Glu-AuNCs with different fluorescence emissions were stored at 4 °C for later use.

[0058] The prepared gold nanoclusters were subjected to performance testing:

[0059] (1) Spectroscopic determination.

[0060] As shown in Figure 2, the prepared Glu-AuNCs solutions exhibited different visual and photoluminescent colors. Specifically, within the pH range of 1.0-5.5, the solution appeared light yellow and showed strong yellow emission under ultraviolet light (365 nm). In the pH range of 6.0-10.5, there was no obvious fluorescence emission. In the pH range of 11.0-12.5, the color of the solution changed from light yellow to dark brown and showed strong red emission, especially the sample at pH 12.0.

[0061] Figure 3 shows the UV-Vis absorption and normalized photoluminescence (PL) spectra of Glu-AuNCs. As shown in Figure 3a, the UV-Vis absorption peaks exhibit a significant red shift with increasing pH, indicating that the size of Glu-AuNCs increases with increasing solution pH. The characteristic fluorescence peaks of AuNCs depend on their particle size. According to the spherical Jellium model, smaller AuNCs have shorter emission wavelengths. Figure 3b shows the normalized PL spectra of Glu-AuNCs. At the excitation wavelength (λ... ex The emission peak wavelength (λ) of AuNCs prepared from a sample with a pH of 2.0 and a wavelength of 365 nm is [not specified]. em The wavelength (λ) was 612 nm. When the pH of the reaction solution increased from 2.0 to 12.0, the λ of the prepared AuNCs decreased. em The redshift of approximately 100 nm further illustrates that the particle size of AuNCs increases with increasing pH. Figure 4 shows high-resolution transmission electron microscopy (TEM) images of Glu-AuNCs prepared in reaction solutions at pH 2 and pH 12.0. Approximately 100 particles were selected, and their average diameters were determined using a statistical method, yielding values ​​of 0.65 ± 0.14 nm and 1.55 ± 0.25 nm, respectively. The inset of Figure 4 shows a high-resolution (HRTEM) image of the AuNCs in the figure, with a lattice fringe spacing of approximately 0.23 nm, corresponding to the (111) lattice spacing of face-centered cubic (fcc) Au.

[0062] (2) Test the response of red fluorescent Glu-AuNCs at pH 12.0 to different metal ions.

[0063] As shown in the inset of Figure 12, the prepared Glu-AuNCs solution is deep yellow, as indicated by the blue line in Figure 12, with a UV-Vis absorption peak at 540 nm. When irradiated under a UV lamp (λ=365 nm), Glu-AuNCs emits strong red fluorescence, as shown in the inset of Figure 12, with its fluorescence excitation peak at 336 nm (red line in Figure 12) and its emission peak at 690 nm (black line in Figure 12), respectively.

[0064] The quenching effect of adding representative metal ions on the red fluorescence of Glu-AuNCs (pH 12.0). 50 μL of water and 50 μL of 10 metal ions (Ca) were added. 2+ Cr 3+ Mg 2+ Mn 2+ Pb 2+ and Zn 2+ 30 μM; Co 2+ Cu 2+ Ni2+ and Hg 2+ 5 μM ions were added dropwise to 950 μL of Glu-AuNCs solution and mixed thoroughly. The mixture was incubated at 25°C for approximately 5 min, and then the fluorescence spectra were measured using a PerkinElmer LS55 spectrometer. Figure 13a shows the fluorescence spectra after the addition of 10 metal ions (Ca... 2+ Cr 3 + Mg 2+ Mn 2+ Pb 2+ and Zn 2+ 30 μM; Co 2+ Cu 2+ Ni 2+ and Hg 2+ The fluorescence spectrum of the Glu-AuNCs solution after reaching 5 μM was observed. Clearly, only Hg... 2+ This allows for the significant quenching of the red fluorescence of Glu-AuNCs, and the reaction is very rapid (completed within 1 minute). Hg 2 + The quenching of Glu-AuNCs fluorescence is attributed to Hg 2+ The strong interaction between Hg and Glu-AuNCs 2+ via Hg 2+ -Au and Glu-Hg 2+ The interaction rapidly quenches the fluorescence of Glu-AuNCs.

[0065] (3) Test the different fluorescence quenching effects of various ions on Glu-AuNCs at pH 12.0.

[0066] The quenching effect of adding representative metal ions on the red fluorescence of Glu-AuNCs (pH 12.0) was investigated using the Glu-AuNCs solution prepared in Example 1. 50 μL of water and 50 μL of 10 metal ions (Ca) were added. 2+ Cr 3+ Mg 2+ Mn 2+ Pb 2+ and Zn 2+ 30 μM; Co 2+ Cu 2+ Ni 2+ and Hg 2+A 5 μM solution of Hg ions was added dropwise to 950 μL of Glu-AuNCs solution and mixed thoroughly. The mixture was incubated at 25°C for approximately 5 min, and then the fluorescence spectrum was measured using a PerkinElmer LS55 spectrometer. The peak values ​​of the fluorescence spectra were recorded, and the (F0 / F) value for each metal ion was calculated. Figure 13b shows the corresponding fluorescence intensity ratio (F0 / F) when 10 representative metal ions were added. Clearly, only Hg... 2+ Only then can the red fluorescence of Glu-AuNCs be significantly quenched, and the reaction rate is very fast (completed within 1 min). Experiments have shown that Hg 2+ It forms stable complexes with Glu (also known as GSH), such as [Hg(GS)2]. 4- [Hg(GS)3] 7- and [Hg(GS)4] 10- 。

[0067] (4) Glu-AuNCs at pH 12.0 were tested in the detection of Hg. 2+ Sensitivity at that time.

[0068] The sensitivity of red fluorescent Glu-AuNCs for recognition was tested, and the detection of Hg by red fluorescent Glu-AuNCs was evaluated. 2+ Sensitivity. 50 μL of water and 50 μL of Hg at different concentrations were used. 2+ Ionic solutions (0.05 μM, 0.7 μM, 3 μM, 6 μM, 10 μM, 15 μM, 20 μM, 30 μM, 50 μM, and 500 μM) were added dropwise to 950 μL of a Glu-AuNCs solution at pH 12.0 and mixed thoroughly. The mixture was incubated at 25°C for approximately 5 min, and then the fluorescence spectra were measured using a PerkinElmer LS55 spectrometer. As shown in Figure 14a, red fluorescent Glu-AuNCs detected Hg in the concentration range of 0–500 μM. 2+ At that time, its fluorescence intensity was gradually quenched.

[0069] (5) Testing red fluorescent Glu-AuNCs (pH 12.0) in the detection of low concentrations of Hg 2+ Feasibility at that time.

[0070] 50 μL of water and 50 μL of Hg of different concentrations were added. 2+Ionic solutions (0.05 μM, 0.7 μM, 3 μM, 6 μM, 10 μM, 15 μM, 20 μM) were added dropwise to 950 μL of Glu-AuNCs (pH 12.0) solution and mixed thoroughly. The mixtures were incubated at 25°C for approximately 5 min, and then fluorescence spectra were measured using a PerkinElmer LS55 spectrometer. Hg concentrations in the 0-20 μM range were detected. 2+ At that time, red fluorescent Glu-AuNCs did not detect Hg. 2+ The ratio of relative fluorescence intensities (F0 / F) increases with Hg. 2+ The concentration increases linearly (as shown in Figure 14b). Through linear fitting, F0 / F vs [Hg] was obtained. 2+ Good linear relationship (R) 2 = 0.996), and its linear equation is Y = 0.04X + 1.04. Table 1 lists the corresponding calculation parameters and methods, and calculates the Hg detection efficiency of Glu-AuNCs. 2+ The limit of detection (LOD) was 1.6 nM (0.32 ppb), which is significantly lower than the limit for Hg in drinking water defined by the World Health Organization (WHO). 2+ The maximum allowable content is 6 ppb and the US Environmental Protection Agency (EPA) allows a content of 2 ppb.

[0071] As can be seen from Table 1, compared with the reported fluorescence materials for detecting Hg 2+ Compared with other methods, the red fluorescent Glu-AuNCs developed in this invention for detecting Hg 2+ It exhibits a wide linear range and a relatively low detection limit. Fluorescent silver nanoclusters (Glu-AgNCs) synthesized using the same ligand Glu were applied to Hg... 2+ Compared to the detection of Hg by the synthesized red fluorescent Glu-AuNCs, 2+ It features higher sensitivity and a wider linear detection range, with the detection limit reduced from 5 nM to 1.6 nM and the linear detection range extended from 0-125 nM to 0-20 µM.

[0072] Table 1. Equations and parameters for calculating the limit of detection (LOD)

[0073]

[0074] (6) The actual sample was detected using red fluorescent Glu-AuNCs (pH 12.0).

[0075] Table 2. Detection of Hg in tap water and lake samples using red fluorescent Glu-AuNCs. 2+ content value

[0076]

[0077] The prepared red fluorescent Glu-AuNCs (pH 12.0) were used to detect Hg in the actual sample. 2+ Hg content was determined by collecting tap water and lake samples from Dalongshan, Anqing. Inductively coupled plasma atomic emission spectrometry (ICP) did not detect Hg in the tap water and lake samples. 2+ Different concentrations of Hg were added to the collected tap water and lake water. 2+ The actual sample to be tested is obtained. The Hg content in the actual sample is then detected. 2+ The method for determining Hg levels is similar to that used for deionized water. Red fluorescent Glu-AuNCs were used to detect Hg in tap water and lake samples. 2+ The concentration values ​​are shown in Table 2. Hg was detected using red fluorescent Glu-AuNCs. 2+ The concentrations and dosages were very close (as shown in Table 2), and the recoveries ranged from 84.26% to 115.33%. Therefore, the prepared red fluorescent Glu-AuNCs sensor can be successfully used for Hg detection in real samples. 2+ The test demonstrated high accuracy and precision.

[0078] (7) Identification of Hg using red fluorescent Glu-AuNCs (pH 12.0) 2+ .

[0079] Hg detection using Glu-AuNCs 2+ Hg was detected using Glu-AuNCs 2+ The tests were conducted. Ten representative metal ions (such as Ca) were detected using Glu-AuNCs solution. 2+ Cr 3+ Mg 2+ Mn 2+ Pb 2+ Zn 2+ Co 2+ Cu 2+ Ni 2+ and Hg 2+ 50 μL of water and 50 μL of each of the 10 metal ions were added dropwise to 950 μL of Glu-AuNCs solution and mixed thoroughly. The final detection concentrations were: 30 μM Ca 2+ Cr 3+ Mg 2+ Mn 2+ Pb 2+ Zn 2+ , and 5 μM Co 2+Cu 2+ Ni 2+ and Hg 2+ The mixture was incubated at 25°C for approximately 5 minutes, and then the fluorescence spectrum was measured using a PerkinElmer LS55 spectrometer. The fluorescence intensity ratio (F0 / F) was then calculated based on the measured data to determine whether the solution contained Hg. 2+ ion.

[0080] Example 2

[0081] As shown in Figure 5, the preparation method of protein-protected gold nanoclusters (EALB-AuNCs) includes the following steps:

[0082] Take 2.5 mL (10 mM) tetrachloroauric acid solution (HAuCl4) and 2.5 mL (50 mg / mL) egg white solution, stir vigorously at room temperature, and add 90 mL of water. o After incubating in an oil bath at 4°C for 5 minutes, add 0.8 mL (1.0 M) NaOH solution and incubate for another 15 minutes. When the solution turns golden yellow, the preparation of protein-protected gold nanoclusters (EALB-AuNCs) is complete. The prepared gold nanoclusters can be incubated at 4°C for 5 minutes. o Store in refrigerator C.

[0083] Because the gold nanoclusters protected by egg white exhibit strong fluorescence, the synthesized gold nanoclusters were diluted 10-fold for ease of subsequent research: 100 μL of the synthesized gold nanoclusters protected by egg white was transferred to a 2 mL test tube, and then 900 μL of deionized water was added to make a total volume of 1000 μL. The 10-fold diluted gold nanoclusters were then characterized using TEM.

[0084] Example 3

[0085] The preparation method of protein-protected gold nanoclusters (EALB-AuNCs) differs from that in Example 2 only in that egg white is replaced with pepsin; all other operations are the same. When the solution turns dark blue, it indicates that the pepsin-protected gold nanoclusters have been successfully prepared. The pepsin-protected gold nanoclusters were characterized using TEM.

[0086] Figures 6-9 show the characterization of gold nanoclusters protected by two surfactants. Figures 6 and 7 are TEM images of gold nanoclusters protected by egg white and pepsin, respectively. Figures 8 and 9 are high-resolution TEM images (lattice diagrams) of gold nanoclusters protected by egg white and pepsin, respectively. It can be seen that the lattice spacing of the two gold nanoclusters is 0.2 nm.

[0087] Images of gold nanoclusters were acquired using a digital camera; the emission, excitation, and UV-Vis absorption spectra of the gold nanoclusters were characterized using a fluorescence spectrometer and a UV-Vis spectrophotometer. Figure 10 shows the emission, excitation, and UV absorption spectra of gold nanoclusters protected by egg white. It can be observed that the fluorescence color of gold nanoclusters protected by egg white is orange-red at pH 9.0-10.0. Figure 11 shows the emission, excitation, and UV absorption spectra of gold nanoclusters protected by pepsin. It can be observed that the fluorescence color of gold nanoclusters encapsulated by pepsin is blue at pH 8.0-9.0.

[0088] Example 4

[0089] When using pH-mediated detection of egg white-protected gold nanoclusters, the optimal H2O2 addition concentration is:

[0090] At room temperature, 100 μL of hydrogen peroxide (H2O2) of different concentrations (0.6M, 0.8M, 1.0M, 1.2M, 1.4M) was taken, and 100 μL of deionized water, five concentrations of H2O2, and 800 μL of the gold nanoclusters protected by egg white that exhibited orange-red fluorescence prepared in Example 2 were added sequentially to 2 mL test tubes (to make the total volume 1000 μL). After the same reaction time (10 min), the changes in emission peaks of all sensors after mixing with the ion solution to be tested were examined, and the sensor formulation with fast reaction and sensitive detection was selected.

[0091] In this embodiment, the preferred result is: mixing 100 μL of deionized water, 100 μL of 1.0 M H2O2, and 800 μL of gold nanoclusters protected by egg white to obtain a gold nanocluster sensor.

[0092] Example 5

[0093] Application of pH-mediated egg white-protected gold nanoclusters in metal ion detection:

[0094] To maintain the total volume, when detecting metal ions, the 100 µL of deionized water in Example 4 was replaced with 100 µL of metal ion solution. 100 µL of aqueous solutions of different metal ions, 100 µL of H₂O₂, and 800 µL of the orange-red fluorescent egg white-protected gold nanoclusters prepared in Example 2 were sequentially added to a 2 mL test tube (total volume 1000 µL). After reacting for 10 min, the mixture in the test tube was added to a fully transparent cuvette and characterized using a fluorescence spectrometer and a UV-Vis spectrophotometer.

[0095] There are 10 types of metal ion aqueous solutions. The concentrations of the 10 metal ion aqueous solutions are shown in Table 3. The concentrations of each metal ion aqueous solution in Table 3 are the upper limits of the wastewater discharge concentration.

[0096] Using the emission peak corresponding to the optimal emission wavelength of 640nm as the signal (FL intensity au), the emission peaks corresponding to the emission wavelengths of 425nm and 450nm (FL intensity au) are then extracted to construct a three-channel sensor.

[0097] Table 3 Upper Limits of Wastewater Discharge Concentrations for Various Heavy Metal Ions

[0098]

[0099] To ensure greater accuracy, each experiment was repeated three times. Emission peaks (FLintensity au) measured at three different emission wavelengths were imported into SPSS multivariate data analysis software for classification and statistical analysis. The classification results were then subjected to linear discriminant analysis (LDA) and cluster analysis to distinguish and detect eight metal ions at different concentrations. The LDA results are shown in Figure 15, and the cluster analysis results are shown in Figure 16. The results indicate that different metal ions have different effects on the gold nanocluster sensor, and the differences in data can effectively distinguish the solutions of the eight metal ions.

[0100] This demonstrates that the differential responses of different substances to this pH-mediated gold nanoclusters can be well applied to substance detection.

[0101] Example 6

[0102] pH-mediated quantitative detection of analyte concentration using egg white-protected gold nanoclusters:

[0103] We selected two readily available metal ions, which are also two substances that testing departments frequently need to test: Zn. 2+ and Pb 2+ 100 μL of aqueous solutions of heavy metal ions (Zn) of different concentrations were prepared. 2+ Aqueous solutions of Pb 2+ The following solutions were added sequentially to 2 mL test tubes: an aqueous solution of H₂O₂, 100 µL of H₂O₂, and 800 µL of the orange-red fluorescent egg white-protected gold nanoclusters prepared in Example 2. After reacting for 10 min, the mixture in the test tubes was added to a fully transparent cuvette, and then characterized using a fluorescence spectrometer. Data processing of the mixture was performed according to steps a and b.

[0104] a. Metal ion detection methods

[0105] To maintain a constant total volume, when detecting metal ions, the 100µL deionized water was replaced with 100µL of metal ion solution. 100µL of aqueous solutions of different metal ions, 100µL of H2O2, and 800µL of gold nanoclusters encapsulated in egg white were sequentially added to a 2mL test tube (to make the total volume 1000µL). After reacting for 10 minutes, the mixture in the test tube was added to a fully transparent cuvette and characterized using a fluorescence spectrometer and a UV-Vis spectrophotometer.

[0106] There are 10 types of metal ion aqueous solutions. The concentrations of the 10 types of metal ion aqueous solutions are shown in Table 3. Table 3 shows the upper limit of wastewater discharge concentration for each heavy metal ion.

[0107] b. Selection of sensing channels

[0108] (1) Signal acquisition: Images of gold nanoclusters were acquired using a digital camera; emission, excitation and ultraviolet-visible absorption spectra of the gold nanoclusters and the metal ion aqueous solution were characterized using a fluorescence spectrometer and an ultraviolet-visible spectrophotometer.

[0109] (2) Signal processing: The emission peak corresponding to the optimal emission wavelength of 640nm is used as the signal (FL intensity au), and the emission peaks corresponding to the emission wavelengths of 425nm and 450nm (FL intensity au) are extracted to construct a three-channel sensor.

[0110] The measured fluorescence data were output as cluster heatmaps, as shown in Figures 17 and 18. The results show that metal ions of different concentration gradients have different effects on gold nanoclusters. Furthermore, the images reveal that the three sets of data for each concentration cluster together well without any overlap, indicating that these gold nanoclusters have high sensitivity to the concentration of the substance.

[0111] Example 7

[0112] Interference testing of pH-mediated egg white-protected gold nanocluster sensor:

[0113] Three ions that are relatively important in the human body were selected for anti-interference performance testing. 100µL of 100µL of each of the three common metal ions (K+, K ... + Ca 2+ Na +Aqueous solution, 100 µL H₂O₂, and 800 µL of the orange-red fluorescent egg white-protected gold nanoclusters prepared in Example 2 were sequentially added to a 2 mL test tube. After reacting for 10 min, the mixture in the test tube was added to a fully transparent cuvette, and then characterized using a fluorescence spectrometer. The treatment of the mixture was performed according to steps a and b. The LDA results of the interference test are shown in Figure 19. From the three-dimensional LDA plot, it can be seen that the interfering ions and the ions to be detected are well distinguished, with no overlap. This indicates that the pH-mediated gold nanocluster sensor has good anti-interference performance and can be well applied to the detection of analytes even in the presence of interfering substances.

[0114] Example 8

[0115] Application of pH-mediated pepsin-protected gold nanocluster sensors in metal ion detection:

[0116] To maintain a constant total volume, when detecting metal ions, 100 µL of aqueous solutions of different metal ions, 100 µL of H2O2, and 800 µL of the pepsin-protected gold nanoclusters with blue fluorescence prepared in Example 3 were added sequentially to a 2 mL test tube (to make the total volume 1000 µL). After reacting for 10 min, the mixture in the test tube was added to a fully transparent cuvette and characterized using a fluorescence spectrometer and a UV-Vis spectrophotometer.

[0117] Using the emission peak corresponding to the optimal emission wavelength of 640nm as the signal (FL intensity au), the emission peaks corresponding to the emission wavelengths of 425nm and 450nm (FL intensity au) are then extracted to construct a three-channel sensor.

[0118] To ensure greater accuracy, each experiment was repeated three times. The emission peaks (FLintensity au) measured at three different emission wavelengths were imported into SPSS multivariate data analysis software for classification and statistical analysis. The classification results were then subjected to linear discriminant analysis (LDA) and cluster analysis to distinguish and detect eight metal ions at different concentrations.

[0119] The above description is merely an example and illustration of the present invention. Those skilled in the art can make various modifications or additions to the specific embodiments described, or use similar methods to replace them, as long as they do not deviate from the structure of the present invention or exceed the scope defined in the claims, all of which should fall within the protection scope of the present invention.

Claims

1. A method for preparing gold nanoclusters with different fluorescence emissions mediated by pH, characterized in that, Includes the following steps: HAuCl4 is mixed with a surfactant, and deionized water is added under vigorous stirring. The pH is then adjusted, and the mixture is stirred at 20-90°C to obtain a gold nanocluster solution. The surfactant is one of egg white, pepsin, or glutathione.

2. The method for preparing gold nanoclusters with different fluorescence emissions mediated by pH according to claim 1, characterized in that: When the surfactant is glutathione, the gold nanocluster solution exhibits yellow fluorescence emission when the pH value is between 1.0 and 5.5 under 365 nm ultraviolet light irradiation, and red fluorescence emission when the pH value is between 11.0 and 12.

5.

3. The method for preparing gold nanoclusters with different fluorescence emission mediated by pH according to claim 2, characterized in that: The molar ratio of HAuCl4 to glutathione is 1:1.2-3.

4. The method for preparing gold nanoclusters with different fluorescence emission mediated by pH according to claim 2, characterized in that: When the pH of the gold nanocluster solution is ≤10.5, the stirring speed is 300-700 rpm and the stirring time is 18-30 h; when the pH of the gold nanocluster solution is ≥11.0, the stirring speed is 300-700 rpm and the stirring time is 2-12 h.

5. The method for preparing gold nanoclusters with different fluorescence emissions mediated by pH according to claim 1, characterized in that: The stirring speed is 500-1200 rpm.

6. The method for preparing gold nanoclusters with different fluorescence emission mediated by pH according to claim 1, characterized in that: The pH adjustment is performed using sodium hydroxide or hydrochloric acid.

7. The method for preparing gold nanoclusters with different fluorescence emissions mediated by pH according to claim 6, characterized in that: The concentration of sodium hydroxide is 1 mol / L, and the concentration of hydrochloric acid is 1 mol / L.

8. The method for preparing gold nanoclusters with different fluorescence emission mediated by pH according to claim 1, characterized in that: When the surfactant is egg white, the pH value of the gold nanocluster solution is between 9.0 and 10.0, and it exhibits orange-red fluorescence emission under 365 nm ultraviolet light irradiation; when the surfactant is pepsin, the pH value of the gold nanocluster solution is between 8.0 and 9.0, and it exhibits blue fluorescence emission under 365 nm ultraviolet light irradiation.

9. A gold nanocluster prepared by the preparation method according to any one of claims 1-8, characterized in that: The pH value of the gold nanocluster solution is between 11.0 and 12.5, and it exhibits red fluorescence emission under 365 nm ultraviolet light.

10. An application of the gold nanoclusters according to claim 9, characterized in that: The gold nanoclusters are used to detect Hg. 2+ content.

Images