A double-gold nanocluster nanosatellite fluorescent probe and its application in rapid indication of calcium carbonate precipitation reaction

Abstract

The application discloses a double-gold nanocluster nanosatellite fluorescent probe and application thereof to rapidly indicating a calcium carbonate precipitation reaction. ATT AuNCs and GSH / GSH-Arg-1AuNCs are synthesized by one-pot synthesis, and then the double-gold nanocluster nanosatellite fluorescent probe is self-assembled by hydrogen bond interaction between nitrogen atoms on the surface of green fluorescent ATT AuNCs and guanidino groups on the surface of orange-yellow fluorescent GSH / GSH-Arg-1AuNCs. The probe presents rich fluorescence color changes from green to orange-yellow under different pH values. The theoretical pH change range of the precipitation titration reaction of calcium carbonate is 11-9, the fluorescence color of the double-gold nanocluster nanosatellite changes significantly before and after the stoichiometric point of the precipitation titration reaction, the titration end point can be determined by utilizing the fluorescence color change, and the generation process of the calcium carbonate precipitation is monitored by utilizing fluorescence data.

Description

Technical Field

[0001] This invention belongs to the field of fluorescent probe technology, and specifically relates to a dual-gold nanocluster nanosatellite fluorescent probe and its application in rapidly indicating calcium carbonate precipitation reactions. Background Technology

[0002] Titration analysis is currently a major analytical method due to its simple instrument operation, high accuracy, convenience, and speed. The main precipitation titration methods include argentometric titration, which is further divided into Mohr's method, Volhard's method, and Fajans method based on the principle of different indicators. While there are various suitable indicators for silver halide precipitation, fewer indicators are available for calcium carbonate precipitation analysis due to its high solubility and slow precipitation reaction rate. Therefore, finding suitable indicators for calcium carbonate precipitation is currently crucial.

[0003] Gold nanoclusters (AuNCs) with a size of several to tens of atoms (≤2 nm) have attracted widespread attention in recent years due to their excellent properties such as low toxicity, high photostability, and ease of synthesis. Unlike bulk metals, the band structure of ultraminiature gold nanoclusters transitions from a continuous to a discontinuous state, resulting in their decomposition into discrete energy levels. Molecular-like particles (MNCs) with Fermi-wavelength electrons establish connections between the metal atoms and nanoparticles (NPs). This property endows ultraminiature gold nanoclusters with unique physical and chemical properties, making them promising for a wide range of applications, such as fluorescence biosensing and bioimaging, therapy, catalysis, and pH indication. Summary of the Invention

[0004] The purpose of this invention is to provide a dual-gold nanocluster nanosatellite fluorescent probe and its application in rapidly indicating calcium carbonate precipitation reactions. The dual-gold nanocluster nanosatellite probe of this invention has a particle size of approximately 2 nm, exhibiting good water solubility, dispersibility, excellent biocompatibility, sensitive pH response, and good reversible stability. Furthermore, the preparation method is simple, the raw materials are readily available, and it is suitable for large-scale promotion and development.

[0005] The preparation method of the dual-gold nanocluster nanosatellite fluorescent probe is as follows: Aqueous dispersions of 6-aza-2-thiothymidine gold nanoclusters (ATT AuNCs) and aqueous dispersions of gold nanoclusters co-modified with glutathione and arginine-modified glutathione (GSH / GSH-Arg-1 AuNCs) are mixed and the pH is adjusted to 6.8-7.2. The mixture is reacted at 30-40℃ for 15-25 min, filtered through a 0.22 μm filter membrane, and then precipitated by adding ethanol or isopropanol and centrifuging. The precipitate is dispersed in ultrapure water to obtain the dual-gold nanocluster nanosatellite fluorescent probe.

[0006] The molar ratio of 6-aza-2-thiothymidine gold nanoclusters to gold nanoclusters co-modified with glutathione and arginine-modified glutathione is 1:3-5.

[0007] The synthesis method of the 6-aza-2-thiothymidine gold nanoclusters is as follows: ATT solution with a pH of 10.5-11.5 and HAuCl4 solution are mixed, stirred and reacted in a dark environment, ultrafiltered, and the precipitate is dispersed in ultrapure water to obtain the product.

[0008] The concentration of ATT after mixing is 75-85 mM; the concentration of HAuCl4 is 8-10 mg / mL.

[0009] The method for synthesizing the gold nanoclusters co-modified with glutathione and arginine-modified glutathione is as follows: HAuCl4 solution, GSH solution and GSH-Arg-1 solution are mixed and stirred at 68-72℃ for 20-24h. The mixture is then filtered through a 0.22μm filter membrane, and ethanol or isopropanol is added to precipitate the gold nanoclusters. After centrifugation, the precipitate is dispersed in ultrapure water to obtain the final product.

[0010] After mixing, the concentration of GSH was 95-105 mM, the concentration of HAuCl4 was 18-22 mM, and the molar ratio of GSH to GSH-Arg-1 was 1.4-1.6:8.5.

[0011] The above-prepared double gold nanocluster nanosatellite fluorescent probe is used as a pH indicator.

[0012] The above-prepared double gold nanocluster nanosatellite fluorescent probe is used to indicate the calcium carbonate precipitation reaction.

[0013] The method for indicating calcium carbonate precipitation reaction by the dual-gold nanocluster nanosatellite fluorescent probe is as follows: the dual-gold nanocluster nanosatellite fluorescent probe is added to a solution containing calcium ions or a solution containing carbonate ions, and the titration endpoint is determined by color change under ultraviolet light during the titration process.

[0014] This invention synthesizes ATT AuNCs and GSH / GSH-Arg-1AuNCs via a one-pot synthesis method. Then, a double-gold nanocluster nanosatellite fluorescent probe is constructed by self-assembly using hydrogen bonding between nitrogen atoms on the surface of green fluorescent ATT AuNCs and guanidino groups on the surface of orange-yellow fluorescent GSH / GSH-Arg-1AuNCs. The ligand-derived fluorescent probe exhibits emission characteristics of both ATT AuNCs and GSH / GSH-Arg-1AuNCs. The surface ligand layer of the probe is rigidified, and the dual-ligand shell endows the AuNCs with excellent switchable emission properties. The double-gold nanoclusters exhibit a fluorescence emission peak at 554 nm. The pH of the probe is controlled using sodium hydroxide and dilute hydrochloric acid, where chloride and sodium ions have no effect. The probe displays a rich fluorescence color change from green to orange-yellow at different pH values. The theoretical pH range for the precipitation titration of calcium carbonate is 11-9. The fluorescence color of the dual-gold nanoclusters / nanosatellites exhibits significant changes before and after the stoichiometric point of this precipitation titration reaction. These fluorescence color changes can be used to determine the titration endpoint, and the fluorescence data can be used to monitor the formation process of calcium carbonate precipitation. In this invention, the probe was adjusted to pH 6 to pH 13 for four cycles. The fluorescence data showed a small range of change, demonstrating that the fluorescent probe has good reversible stability. Attached Figure Description

[0015] Figure 1 The fluorescence spectrum, UV-Vis absorption spectrum, and infrared spectrum of ATT AuNCs are shown.

[0016] Figure 2 The fluorescence spectrum, UV-Vis absorption spectrum, and infrared spectrum of GSH / GSH-Arg-1AuNCs are shown.

[0017] Figure 3 Fluorescence spectra of double gold nanoclusters constructed from GSH / GSH-Arg-1 AuNCs synthesized with ATT AuNCs and different ligands GSH and GSH-Arg-1 in ratios (1.5:8.5, 1:9, 0.5:9.5).

[0018] Figure 4 The fluorescence spectrum, UV-Vis absorption spectrum, and infrared spectrum of the double gold nanocluster fluorescent probe are shown.

[0019] Figure 5 This study investigates the pH-responsiveness of a double gold nanocluster fluorescent probe.

[0020] Figure 6 To investigate the properties of fluorescent probes for double gold nanoclusters.

[0021] Figure 7This demonstrates the application of dual-gold nanocluster fluorescent probes in precipitation titration reactions.

[0022] Figure 8 The fitted curve is used to indicate the precipitation process by the fluorescent probe of the double gold nanocluster. Detailed Implementation

[0023] To illustrate the present invention more clearly, the following embodiments are provided, but the scope of protection of the present invention is not limited to the following embodiments.

[0024] Example 1

[0025] Synthesis of ATT AuNCs:

[0026] Add 15 mL of 10 mg / mL HAuCl4 solution to 15 mL of ATT (80 mM) solution containing 0.2 M NaOH, and place the mixture in the dark and stir continuously at 25 °C for 1 h. The synthesized ATT AuNCs were purified by ultrafiltration (50 kDa), and the precipitate was dispersed in 30 mL of ultrapure water and stored in the dark at 4 °C before use.

[0027] Synthesis of GSH / GSH-Arg-1AuNCs:

[0028] 500 μL of a 20 mM HAuCl4 solution, 127.5 μL of a 100 mM GSH solution, and 22.5 μL of a 100 mM GSH-Arg-1 solution were mixed at room temperature, and 4.35 mL of ultrapure water was added. All solutions were prepared fresh before use. The mixture was gently stirred at 70 °C for 24 h to obtain orange-yellow fluorescent GSH / GSH-Arg-1 AuNCs. Furthermore, the synthesized gold nanoclusters were purified by first filtering the solution through a 0.22 μm filter to remove larger impurities, then precipitating with ethanol and centrifuging repeatedly until the precipitate was dispersed in 5 mL of ultrapure water. The orange-yellow luminescent GSH / GSH-Arg-1 AuNCs dispersion could be stored at 4 °C for 6 months.

[0029] Synthesis of fluorescent probes for dual-gold nanoclusters and nanosatellites:

[0030] The purified ATT AuNCs aqueous dispersion and the GSH / GSH-Arg-1AuNCs aqueous dispersion were mixed at a molar ratio of 1:5. The pH was adjusted to 7 with hydrochloric acid and sodium hydroxide solution, and the mixture was reacted at 37°C for 20 min to obtain a pale yellow ATT AuNCs / GSH / GSH-Arg-1AuNCs solution. The prepared gold nanoclusters were first filtered through a 0.22 μm filter to remove larger impurities, then ethanol was added to precipitate the nanoclusters, and the mixture was centrifuged repeatedly until the precipitate was dispersed in ultrapure water. The orange-yellow luminescent ATT AuNCs / GSH / GSH-Arg-1AuNCs dispersion could be stored at 4°C for 6 months.

[0031] Study on the fluorescence properties of the prepared ATT AuNCs:

[0032] 15 mL of 10 mg / mL HAuCl4 solution was added to 15 mL of ATT (80 mM) solution containing 0.2 M NaOH. The reaction was carried out at pH 10 for one hour to obtain a gold nanocluster solution. The fluorescence excitation and emission spectra were then measured using a fluorescence spectrophotometer. The results are as follows: Figure 1 As shown in Figure a. The double gold nanoclusters were tested using UV-Vis spectroscopy in the 200-600 nm range, and the results are as follows. Figure 1 As shown in b, its absorption spectrum shows no surface plasmon resonance (SPR) peak at 520 nm, confirming the absence of aggregates or large particles. Fourier transform infrared spectroscopy (FT-IR) was used for testing, as shown... Figure 1 As shown in c, the pure ATT is at 3144cm. -1 The peak at 2567 cm⁻¹ represents the stretching vibration of -NH₃. -1 The peak at the thiol level represents the characteristic infrared peak of -SH, but the thiol peak disappeared after the synthesis of ATT-AuNCs. The results indicate that Au-S bonds were formed between the ATT ligand and AuNCs, confirming the presence of ATT on the AuNCs surface.

[0033] Study on the optical properties of the prepared GSH / GSH-Arg-1AuNCs:

[0034] 500 μL of a 20 mM HAuCl4 solution, 127.5 μL of a 100 mM GSH solution, and 22.5 μL of a 100 mM GSH-Arg-1 solution were mixed at room temperature, and 4.35 mL of ultrapure water was added. The reaction was allowed to proceed for 24 hours. The optical properties of the GSH / GSH / Arg-1 AuNCs were characterized using fluorescence spectroscopy and other methods. Figure 2As shown in a and 2b, the optimal excitation peak for the fluorescence of GSH / GSH / Arg-1AuNCs is at 410 nm, and the maximum emission peak is at 570 nm. The absorption peak in the UV-vis spectrum shows a shoulder peak at approximately 400 nm, consistent with previously reported absorption peaks. Based on the FT-IR spectrum (… Figure 2 c), showing a value of 2620cm -1 The disappearance of the -SH stretching vibration characteristic peak centered on the GSH indicates the successful modification of GSH / GSH-Arg-1.

[0035] Study on the optical properties of the prepared double gold nanocluster nanosatellite fluorescent probe:

[0036] First, the concentration of ATT AuNCs was fixed at a 40-fold dilution. Then, GSH / GSH-Arg-1AuNCs ligands were synthesized in a one-pot method at GSH to GSH-Arg-1 molar ratios of 1.5:8.5, 1:9, and 0.5:9.5. The two gold nanoclusters were then incubated at room temperature for 15 minutes. Figure 3 As shown, the optimal molar ratio of GSH to GSH-Arg-1 is 1.5:8.5, as determined by fluorescence spectroscopy. In the UV-Vis spectrum, as... Figure 4 As shown in b, two shoulder peaks are observed at 415 nm and 490 nm. The absorption spectrum of the gold nanocluster nanosatellites shows no surface plasmon resonance (SPR) peak at 520 nm, confirming the absence of aggregates or large particles. Fourier transform infrared spectroscopy (FTIR) characterization was used to test the characteristic functional groups of ATT AuNCs / GSH / GSH-Arg-1 AuNCs. Compared with ATT AuNCs and GSH / GSH-Arg-1 AuNCs, the dual gold nanocluster nanosatellites exhibited superior absorption at 3200-3500 nm. -1 Redshift occurred in the nearby broadband area ( Figure 4 c) This demonstrates the enhanced hydrogen bonding, proving the successful synthesis of the fluorescent probe.

[0037] pH-responsiveness study of dual-gold nanocluster fluorescent probes:

[0038] The fluorescence intensity table of ATT AuNCs / GSH / GSH-Arg-1AuNCs exhibits sensitive pH responsiveness. For example... Figure 5As shown, the fluorescence spectra of ATT AuNCs / GSH / GSH-Arg-1 AuNCs at different pH values ​​(6.04-12.89) were measured. When the pH of the solution increased from 6.04 to 12.52, the fluorescence of the double gold nanoclusters increased with increasing pH, reaching its highest intensity at 12.52. Further increases in pH to 12.89 resulted in a decreasing fluorescence intensity. During the pH increase, the optimal fluorescence emission peak of the double gold nanoclusters shifted from 570 nm to 523 nm, indicating that the double gold nanoclusters exhibited a GSH / GSH-Arg-1AuNCs fluorescence peak at low pH values, while exhibiting an ATT AuNCs fluorescence peak as the solution became more alkaline.

[0039] Properties of dual-gold nanocluster fluorescent probes:

[0040] To investigate the changes in fluorescence color of double-gold nanoclusters and nanosatellites under different pH stimuli, images of double-gold nanoclusters and nanosatellites and ATT AuNCs solutions at different pH values ​​were taken under sunlight and ultraviolet light irradiation. Figure 6 As shown, ATT AuNCs at different pH values ​​are colorless under sunlight, but their fluorescence color is green under ultraviolet light, and the fluorescence color becomes brighter with increasing pH. The solution of the double gold nanoclusters / nanosatellites is colorless under sunlight. When the pH value increases from 6 to 10, the fluorescence color of the double gold nanoclusters / nanosatellites under 365nm ultraviolet light is orange-yellow. When the pH value increases to 11.5, its fluorescence color changes to yellow-green. Further increasing the pH value from 12 to 13, the fluorescence color changes to green. In summary, compared with ATT AuNCs probes, the double gold nanoclusters probe exhibits more diverse fluorescence color changes over a wide pH range. Further investigation into the stability of the gold nanoclusters / nanosatellites fluorescent probe was conducted, with four consecutive cycles of measurement. The double gold nanoclusters probe is completely reversible. According to the four cycles, the response and recovery times of the sample solution are rapid, and the different fluorescence colors of the solution vary between orange-yellow (pH=6) and green (pH=12.5). It is evident that the gold nanocluster nanosatellite fluorescent probe exhibits a highly reversible response to pH. These results demonstrate that the gold nanocluster nanosatellite fluorescent probe possesses stable fluorescence properties and can serve as a suitable fluorescent probe for monitoring changes in the pH environment.

[0041] Application Example 1

[0042] Application of dual-gold nanocluster fluorescent probes in precipitation titration reactions:

[0043] To investigate the application of the dual-gold nanocluster fluorescent probe in precipitation titration reactions, 200 μL of the dual-gold nanocluster fluorescent probe was added to 1 mL of 0.1 M Na₂CO₃ solution, and different volumes of 0.1 M CaCl₂ solution were gradually added to react. The reaction was photographed under ultraviolet light. Figure 7 As shown in the figure, without the addition of CaCl2 solution, the solution fluorescence color is green, mainly due to the luminescence of ATT AuNCs. When 10 μL of CaCl2 solution is added, the fluorescence color changes to yellow-green. Adding another 50 μL of CaCl2 solution changes the fluorescence color from yellow-green to yellow. When 800 μL of CaCl2 solution is added, the fluorescence color completely turns orange-yellow. Adding a final 1000 μL of CaCl2 solution completes the reaction between Na2CO3 and CaCl2, resulting in a final orange-yellow fluorescence color. Therefore, this fluorescent probe can visually monitor the precipitation titration reaction process of sodium carbonate and calcium chloride through changes in fluorescence color.

[0044] Application Example 2

[0045] Fitting curve of the precipitation formation process indicated by the fluorescent probe of the double gold nanocluster:

[0046] To investigate the process of precipitate formation indicated by fluorescent probes, such as Figure 8 As shown, in the first stage, the volume of calcium chloride solution added increases from 0 to 100 μL. No precipitate forms in the solution, indicating a supersaturated state. As the ion concentration increases, the conductivity increases, showing a positive linear correlation between conductivity and the volume of calcium chloride added. This stage represents the nucleation stage of crystal-forming ions. When the volume of calcium chloride solution added reaches 150 μL, the solution becomes turbid, indicating crystal nucleation and a decrease in conductivity. With continued addition, precipitate continues to form in the reaction system, building upon the existing calcium carbonate crystals. Fitting the conductivity data reveals a good linear relationship between solution conductivity and the volume of calcium chloride. When the volume of calcium chloride solution added reaches 800 μL, precipitate continues to form in the reaction system, indicating crystal growth and precipitation. Continued addition of calcium chloride solution completes the precipitation reaction, increasing the electrolyte concentration and consequently the conductivity. Simultaneously, linear fitting was performed on the fluorescence data during the reaction process, and the changes in conductivity matched the four stages of fluorescence changes, thus proving that the formation process of calcium carbonate precipitate can be indicated by a dual-gold nanocluster nanosatellite fluorescent probe.

Claims

1. A method for preparing a fluorescent probe for a dual-gold nanocluster nanosatellite, characterized in that, The specific operation of the preparation method is as follows: 6-aza-2-thiothymidine gold nanocluster aqueous dispersion and gold nanocluster aqueous dispersion co-modified with glutathione and arginine-modified glutathione are mixed and the pH is adjusted to 6.8-7.

2. The mixture is reacted at 30-40℃ for 15-25 min, filtered through a 0.22 μm filter membrane, and then ethanol or isopropanol is added to precipitate and centrifuged. The precipitate is dispersed in ultrapure water to obtain the dual gold nanocluster nanosatellite fluorescent probe. The synthesis method of the 6-aza-2-thiothymidine gold nanoclusters is as follows: ATT solution with a pH of 10.5-11.5 and HAuCl4 solution are mixed, stirred and reacted in the dark, ultrafiltered, and the precipitate is dispersed in ultrapure water to obtain the product. The method for synthesizing the gold nanoclusters co-modified with glutathione and arginine-modified glutathione is as follows: HAuCl4 solution, GSH solution and GSH-Arg-1 solution are mixed and stirred at 68-72 °C for 20-24 h. The mixture is then filtered through a 0.22 μm filter membrane, and ethanol or isopropanol is added to precipitate the precipitate. After centrifugation, the precipitate is dispersed in ultrapure water to obtain the final product.

2. The preparation method according to claim 1, characterized in that, The molar ratio of 6-aza-2-thiothymidine gold nanoclusters to gold nanoclusters co-modified with glutathione and arginine-modified glutathione is 1:3-5.

3. The application of the double gold nanocluster nanosatellite fluorescent probe prepared according to claim 1 or 2 as a pH indicator.

4. The application of the double gold nanocluster nanosatellite fluorescent probe prepared according to claim 1 or 2 in indicating the calcium carbonate precipitation reaction.

5. The application according to claim 4, characterized in that, The method for indicating calcium carbonate precipitation reaction by the dual-gold nanocluster nanosatellite fluorescent probe is as follows: the dual-gold nanocluster nanosatellite fluorescent probe is added to a solution containing calcium ions or a solution containing carbonate ions, and the titration endpoint is determined by color change under ultraviolet light during the titration process.

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