Method for continuously and rapidly detecting multiple heavy metals by GSH-AuNCs based on fluorescence'on-off-on '

Abstract

The invention discloses a novel nanogold sensing method for continuously detecting various heavy metals by fluorescence'on-off-on 'based on a gradual detection strategy. Gold nanoclusters (GSH-AuNCs) taking glutathione (GSH) as a reducing agent and a stabilizing agent respond to various heavy metals to cause fluorescence quenching, and a novel GSH-AuNCs visual sensing platform is designed according to the remarkable difference of fluorescence intensity before and after coordination / redox competition, so that four heavy metal ions of Fe (III), Cu (II), Hg (II) and Cr (VI) are successfully and rapidly detected step by step. The method is simple in operation steps, high in detection speed (Fe (III): 3 min, Cu (II): 30 s, Hg (II): 3 min and Cr (VI): 3 min), wide in quantitative range (0.1-200 [mu] M), high in sensitivity (LDDFe (III): 87 nM, LODCu (II): 36 nM, LODHg (II): 32 nM and LODCr (VI): 75 nM) and high in anti-interference capability, and is successfully applied to gradual detection of four heavy metals in actual water and biological fluid (urine and serum).

Description

Technical Field

[0001] This invention belongs to the field of visualization detection technology, specifically relating to a method for detecting four heavy metals in tap water, urine, and serum using GSH-AuNCs as fluorescent probes and employing a stepwise detection strategy. Background Technology

[0002] Heavy metal ions pose a serious threat to human health due to their long half-lives, non-biodegradability, and ability to enter the human body through direct contact or indirect absorption via the food chain or food web. Excessive accumulation of some heavy metals, including iron (Fe), copper (Cu), mercury (Hg), and chromium (Cr), can lead to acute or chronic poisoning, resulting in severe consequences such as neurological disorders, skin and vascular damage, immune system dysfunction, gastrointestinal and kidney dysfunction, and even cancer. Many metal ions play important roles in biological processes and human metabolism, and are essential for maintaining life activities. However, abnormal levels of heavy metals in the body can harm the human body and even lead to some serious diseases. For example, excessive Fe(III) is associated with chronic liver disease, kidney disease, diabetes, esophageal cancer, and colon cancer; Cu(II) is closely related to the occurrence of Alzheimer's disease, Parkinson's disease, Wilson's disease, and cancer; mercury, as a highly toxic pollutant, is considered by the World Health Organization (WHO) to be one of the top ten pollutants with health hazards, posing a huge threat to life systems. Accumulation of Hg(II) may cause Parkinson's disease, Alzheimer's disease, cancer, and other diseases; Cr(VI) is carcinogenic and highly lethal. Exposure to low concentrations of hexavalent chromium may also lead to cognitive impairment, diabetes, genotoxicity, mutagenesis, and even cancer in children. In summary, heavy metal pollution poses a significant threat to human health and safety. However, current traditional methods for heavy metal detection, including inductively coupled plasma mass spectrometry (ICP-MS), atomic fluorescence spectrometry (AFS), and atomic absorption spectrometry (AAS), mostly require large and expensive instruments, sample pretreatment, and are costly, time-consuming, and complex. Furthermore, considering the complexity of the sample detection environment—as real-world samples often contain multiple heavy metals simultaneously—there is an urgent need to develop a simple and rapid detection method that can simultaneously identify and differentiate between multiple heavy metals. Fluorescence methods are favored by researchers due to their high sensitivity and rapid detection. However, most researchers currently construct fluorescence sensor arrays based on materials such as organic fluorophores, lanthanide metal-organic frameworks, quantum dots, covalent organic frameworks, and plasmonic nanostructures for the simultaneous detection of multiple heavy metals. However, these array sensors often suffer from drawbacks such as high toxicity, large data volumes, and complex statistical analysis.

[0003] Fluorescent gold nanoclusters (AuNCs) have stood out due to their excellent optical properties, high surface area, high biocompatibility, controllable electrical properties, and tunable morphology and function. They have attracted widespread attention in the fields of detection and sensing due to their high sensitivity and selectivity, and are considered an ideal choice for sensor design, representing a promising nanomaterial. Among them, thiol-stabilized AuNCs possess excellent properties such as small size, good stability, unique core-shell structure, and large specific surface area, showing significant advantages in analytical detection and biomedical fields. GSH-protected AuNCs exhibit even greater rigidity and stability. This study uses the natural tripeptide glutathione (GSH= γ Using Glu-Cys-Gly as a thiolate ligand model, an oligomer Au(I)−thiolate complex was synthesized. Then, Au(0)@Au(I)-SG NCs (denoted as GSH-AuNCs) with a core-shell structure formed by in-situ generation of Au atoms were generated. Based on the effective quenching of the fluorescence of GSH-AuNCs by Fe(Ⅲ), Cu(Ⅱ), Hg(Ⅱ), and Cr(Ⅵ), a novel visual detection platform for continuous "on-off-on" detection of four heavy metals was constructed using a stepwise detection strategy. The recognition mechanism was explored by calculating the coordination formation energy of the relevant metal complexes using density functional theory. Therefore, it provides a simple, rapid, economical, and environmentally friendly method for identifying multiple heavy metal ions, and has great potential in developing diverse heavy metal sensors. Further application of the visualization detection platform to the continuous visual detection of Fe(III), Cu(II), Hg(II), and Cr(VI) in complex biological fluid samples revealed high anti-interference ability (discovered in anti-interference experiments on 18 metal ions, 13 anions, and 22 common small organic molecules in urine), low detection limits (Fe(III): 87 nM, Cu(II): 36 nM, Hg(II): 32 nM, Cr(VI): 75 nM), and rapid response (Fe(III): 3 min, Cu(II): 30 s, Hg(II): 3 min, Cr(VI): 3 min). Therefore, it breaks through the cumbersome bottleneck of various heavy metal detection methods, providing a rapid, simple, environmentally friendly, economical, sensitive, and accurate visualization detection platform for the early diagnosis and prevention of diseases involving four heavy metals. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a novel nano-gold sensing method for the rapid, simple, environmentally friendly, economical, sensitive, and accurate continuous visual detection of four heavy metals. (1) Add 1% HAuCl4 .3H2O (1.0-10.0 mL) and GSH (0.1 mol / L, 0.5-2.0 mL) and an appropriate amount of ultrapure water were mixed and stirred evenly. The mixture was refluxed at 70 °C overnight and then cooled to room temperature to obtain a pale yellow transparent solution. GSH (10 mmol / L) was then mixed evenly with the above-synthesized pale yellow transparent liquid in a certain proportion and incubated at 37 °C for 2 h. The mixture was then transferred to a dialysis bag and dialyzed for 24 h. Finally, the solution was freeze-dried to obtain a yellow powder, which was stored at 4 °C in the dark for later use. (2) For those including Mg 2+ Pb 2+ Ca 2+ Zn 2+ 18 kinds of metal ions, Cl - ,ClO - NO3 - SO4 2- Anti-interference experiments were conducted on 13 anions and 22 common interfering substances in urine, including amino acids, urea, and uric acid. The prepared GSH-AuNCs were reconstituted in acetate buffer (0.2 mol / L, pH=4.0). 1.5-3.0 mL of GSH-AuNCs was placed in centrifuge tubes, and 0.3-1.0 mL of solutions of the different interfering substances were added (for common, high-content ions including K+). + Ca 2+ Na + Mg 2+ NO3 - Cl - CO3 2- HCO3 - SO4 2- The concentration of the detected ion was 10 times that of the ion (10 mmol / L), and the concentration of other substances was 1 mmol / L. After mixing, the fluorescence spectrum was scanned. Then, 0.2-1.0 mL of sodium citrate (SC), disodium ethylenediaminetetraacetate (EDTA-2Na), cysteine ​​(Cys), and hydroxylamine hydrochloride (HH) were added one after another at a concentration of 10 mmol / L. The fluorescence spectrum was scanned once after each masking reagent was added. (3) The prepared GSH-AuNCs were reconstituted in acetate buffer (0.2 mmol / L, pH=4.0). 1.5-3.0 mL of GSH-AuNCs was placed in centrifuge tubes, and 0.3-1.0 mL of Fe-containing buffer at different concentrations (0.1 μmol / L, 1.0 μM, 5.0 μmol / L, 10.0 μmol / L, 50.0 μmol / L, 100.0 μmol / L, 150.0 μmol / L, 200.0 μmol / L) was added respectively. 3+ Cu 2+ Hg 2+ Cr2O7 2- A mixed solution of four heavy metal ions was prepared, and the fluorescence spectrum was measured using a Cary Eclipse fluorescence spectrophotometer. Then, 0.2–1.0 mL of 10 mM SC, EDTA-2Na, Cys, and HH were added sequentially. The fluorescence spectrum was measured after each addition of a masking reagent. The fluorescence reproducibility (R0) was plotted on the x-axis as concentration. Q2 -R Q1 Plot a linear relationship with y as the vertical axis; (4) Spike analysis was performed on actual samples of tap water, urine, and serum. Serum was deproteinized with 5% trichloroacetic acid and then the supernatant was diluted 10 times with deionized water; urine samples were directly diluted 10 times with deionized water; tap water did not require further treatment and fluorescence was measured using a fluorescence spectrometer. Attached Figure Description

[0005] Figure 1 (a) TEM image of GSH-AuNCs; (b) particle size distribution of GSH-AuNCs; (c) fluorescence spectrum and UV-Vis absorption spectrum of GSH-AuNCs (the inset on the right shows photographs of GSH-AuNCs under natural light and UV irradiation); (d) FTIR spectra of GSH-AuNCs and GSH.

[0006] Figure 2 XPS spectra of GSH-AuNCs: (a) full spectrum; (b) C 1s spectrum; (c) Au 4f spectrum; (d) S 2p spectrum.

[0007] Figure 3 The PL spectra of GSH-AuNCs were scanned at (a) different excitation wavelengths (380 nm-460 nm); (b) the fluorescence spectra of synthesized GSH-AuNCs stored at 4 °C for 30 days; and the fluorescence stability of GSH-AuNCs was measured at (c) different temperatures and (d) different pH values ​​(pH=2-12).

[0008] Figure 4 (a) The fluorescence intensity (E) within 30 min after adding 1 mmol / L Fe(III), Cu(II), Hg(II), and Cr(VI) to GSH-AuNCs (0.2 mol / L, pH=4.0 acetate buffer). m =612 nm); (b) fluorescence intensity within 30 min after adding four masking reagents (SC, EDTA-2Na, Cys and HH) respectively (E = 612 nm); m =612 nm).

[0009] Figure 5 pH dependence of fluorescence quenching of GSH-AuNCs caused by 1 mmol / L Fe(Ⅲ), Cu(Ⅱ), Hg(Ⅱ) and Cr(Ⅵ) (RSD range 1.17%–4.65%, n=3).

[0010] Figure 6 The specificity of (a) addition of SC to Fe(III) (RSD range 0.94%–5.10%, n=3); and the specificity of addition of EDTA-2Na to Cu(II) (RSD range 1.02%–4.93%, n=3) were compared. Figure 7 The specificity of Cys addition for Hg(II) was evaluated (RSD range 1.16%–4.47%, n=3); and the specificity of HH addition for Cr(VI) was evaluated (RSD range 1.31%–5.06%, n=3). Figure 8 The graph shows the linear relationship between the fluorescence recovery rate at 612 nm of Fe(Ⅲ), Cu(Ⅱ), Hg(Ⅱ) and Cr(Ⅵ) solutions of different concentrations and the addition of four "masking agents" SC, EDTA-2Na, Cys and HH.

Claims

1. A detection method for four heavy metals based on novel GSH-AuNCs visualization and stepwise detection, comprising the following steps: (1) Add 1% HAuCl4 . 3H2O (1.0-10.0 mL) and GSH (0.1 mol / L, 0.5-2.0 mL) and an appropriate amount of ultrapure water were mixed and stirred evenly. The mixture was refluxed at 70 °C overnight and then cooled to room temperature to obtain a pale yellow transparent solution. GSH (10 mmol / L) was then mixed evenly with the above-synthesized pale yellow transparent liquid in a certain proportion and incubated at 37 °C for 2 h. The mixture was then transferred to a dialysis bag and dialyzed for 24 h. Finally, the solution was freeze-dried to obtain a yellow powder, which was stored at 4 °C in the dark for later use. (2) For those including Mg 2+ Pb 2+ Ca 2+ Zn 2+ 18 kinds of metal ions, Cl - ,ClO - NO3 - SO4 2- Anti-interference experiments were conducted on 13 anions and 22 common interfering substances in urine, including amino acids, urea, and uric acid. The prepared GSH-AuNCs were reconstituted in acetate buffer (0.2 mol / L, pH=4.0). 1.5-3.0 mL of GSH-AuNCs was placed in centrifuge tubes, and 0.3-1.0 mL of solutions of the different interfering substances were added (for common, high-content ions including K+). + Ca 2+ Na + Mg 2+ NO3 - Cl - CO3 2- HCO3 - SO4 2- The concentration of the detected ion was 10 times that of the ion (10 mmol / L), and the concentration of other substances was 1 mmol / L. After mixing, the fluorescence spectrum was scanned. Then, 0.2-1.0 mL of sodium citrate (SC), disodium ethylenediaminetetraacetate (EDTA-2Na), cysteine ​​(Cys), and hydroxylamine hydrochloride (HH) were added one after another at a concentration of 10 mmol / L. The fluorescence spectrum was scanned once after each masking reagent was added. (3) The prepared GSH-AuNCs were reconstituted in acetate buffer (0.2 mmol / L, pH=4.0). 1.5–3.0 mL of GSH-AuNCs were placed in centrifuge tubes, and 0.3–1.0 mL of Fe-containing buffer at different concentrations (0.1 μmol / L, 1.0 μM, 5.0 μmol / L, 10.0 μmol / L, 50.0 μmol / L, 100.0 μmol / L, 150.0 μmol / L, 200.0 μmol / L) were added respectively. 3+ Cu 2 + Hg 2+ Cr2O7 2- A mixed solution of four heavy metal ions was prepared, and the fluorescence spectrum was measured using a Cary Eclipse fluorescence spectrophotometer. Then, 0.2–1.0 mL of 10 mM SC, EDTA-2Na, Cys, and HH were added sequentially. The fluorescence spectrum was measured after each addition of a masking reagent. The fluorescence reproducibility (R0) was plotted on the x-axis as concentration. Q2 -R Q1 Plot a linear relationship with y as the vertical axis; (4) Spike analysis was performed on actual samples of tap water, urine and serum. Serum was deproteinized with 5% trichloroacetic acid and then the supernatant was diluted 10 times with deionized water; urine samples were directly diluted 10 times with deionized water; tap water did not require further treatment and fluorescence was measured using a fluorescence spectrometer.

2. The method as described in claim 1, characterized in that: In step (1), 1% HAuCl4 . 3H2O is 1-10 mL, 0.1 mol / L LGSH was 0.5-2 mL; dialyzed for 24 h; freeze-dried to obtain a yellow powder.

3. The method as described in claim 1, characterized in that: In step (2), GSH-AuNCs are reconstituted in acetate buffer (0.2 mol / L, pH=4.0); the amount of GSH-AuNCs is 1.5-3 mL; the amount of interfering agent is 0.3-1 mL; and the four masking reagents SC, EDTA-2Na, Cys, and HH are all 10 mmol / L, 0.2-1 mL.

4. The method as described in claim 1, characterized in that: In step (3), 0.3-1 mL of Fe-containing solutions of different concentrations... 3+ Cu 2+ Hg 2+ Cr2O7 2- A mixed solution of four heavy metal ions was prepared; the fluorescence spectrum was measured using a Cary Eclipse fluorescence spectrophotometer.

5. The method as described in claim 1, characterized in that: In step (4), 5% trichloroacetic acid is used for deproteinization treatment, and then the supernatant is diluted 10 times with deionized water; urine samples are directly diluted 10 times with deionized water.

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