Preparation method and application of fluorescent copper nanocluster based on fulvic acid

Abstract

The invention relates to the technical field of metal nanoclusters, in particular to a fulvic acid-based fluorescent copper nanocluster preparation method and application thereof, and the fulvic acid-based fluorescent copper nanocluster preparation method comprises the following steps: S101, adding a 1 mM fulvic acid solution and a 9.6 mM copper sulfate solution into a reaction container, fixing the volume, then adding a 0.8 M ascorbic acid solution, and stirring; and S102, an oxygen consumption agent is flatly laid in a flat plate with holes, the flat plate is clamped into the reaction container, the flat plate is immersed into the solution without touching the bottom, stirring is conducted for 4-5 hours at the temperature of 50-60 DEG C, and the fluorescent copper nanocluster is prepared. The prepared blue fluorescent copper nano-cluster is uniform in size and high in stability, and the material shows good application potential in SPFX high-sensitivity detection and temperature detection.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of metal nanoclusters, and particularly relates to a preparation method of fluorescent copper nanoclusters based on fulvic acid and application thereof. BACKGROUND

[0002] In recent years, metal nanoclusters (MNCs) have shown broad application prospects in the fields of sensing, drug delivery, biological imaging, catalysis, and nanodevices due to their unique electrical, physical, and optical properties. Compared with quantum dots and organic dyes, MNCs have advantages such as high light stability, good biocompatibility, and large Stokes shift. The structure of MNCs is composed of ligands with end-capping protection and metal atoms, and the size is close to the Fermi wavelength of electrons, which endows MNCs with molecular-like properties, including discrete electronic states and size-dependent transitions. Copper, as a resource-rich, low-cost, and excellent-conductive metal, has more application potential in nanocluster research than noble metals such as gold and silver. In recent years, many researchers have developed various preparation methods for copper nanoclusters (Cu NCs). Commonly used methods for preparing Cu NCs include chemical reduction, ligand-assisted, photochemistry, and electrochemistry. Among them, chemical reduction is one of the most commonly used methods, which reduces copper ions under the action of reducing agents and templates or ligands to form nanoclusters, and is simple to operate and easy to control particle size and fluorescence performance. However, some chemical reduction methods are sensitive to reaction conditions, which can cause uneven distribution of products or unstable fluorescence performance. When using large molecules such as DNA, amino acids, or proteins as templates, the particle size of the obtained Cu NCs is usually large, which is prone to aggregation, and there are certain limitations in biocompatibility and structural stability. In addition, one-pot method has attracted much attention due to its simple operation, mild conditions, and simultaneous realization of reduction, ligand coating, and fluorescence regulation. By mixing copper salt, reducing agent, and small molecule template in a single reaction system, Cu NCs with uniform size and good dispersion can be generated. However, there are still some deficiencies, such as the presence of all reaction components in the same system, which may produce side reactions or ligand competition, thereby affecting the fluorescence quantum yield and stability. In addition, one-pot method is highly sensitive to reaction conditions such as temperature, pH value, and redox environment. If there is too much oxygen in the system, zero-valent copper will be oxidized to divalent copper, which will significantly reduce the fluorescence performance and structural stability of Cu NCs.

[0003] Sparfloxacin (SPFX) is a third-generation quinolone antibacterial drug that inhibits bacterial DNA synthesis to exert bactericidal effect, and is mainly used for the treatment of respiratory, urinary, intestinal, and skin soft tissue infections. Common analysis methods for SPFX include high-performance liquid chromatography and capillary electrophoresis high-frequency conductivity method, but these methods are complex and time-consuming, and there is an urgent need to develop a simple and highly sensitive detection method.

[0004] Temperature is a key parameter for regulating environment and biological processes, and intracellular temperature sensing plays a key role in many fields such as electronics and clinical diagnosis, and accurate sensing of intracellular temperature is helpful for the development of cell biology and biomedical science.

[0005] In order to overcome the above-mentioned deficiencies, the present application firstly adopts a small-molecule organic fulvic acid (FA) as a template to prepare copper nanoclusters with ultra-small size, uniformity and high stability, and the copper nanoclusters show good application potential in the high-sensitivity detection of SPFX and temperature detection. SUMMARY

[0006] In view of the problems existing in the prior art, the present application provides a preparation method of fluorescent copper nanoclusters based on fulvic acid and applications thereof.

[0007] The technical scheme for solving the above technical problems is as follows: a preparation method of fluorescent copper nanoclusters based on fulvic acid, comprising the following steps: S101, adding 1 mM fulvic acid solution and 9.6 mM copper sulfate solution into a reaction container, and then adding 0.8 M ascorbic acid solution and stirring; S102, laying an oxygen consumption agent on a hole plate, and clamping the hole plate into the reaction container, immersing the hole plate in the solution without touching the bottom, and stirring at 50-60 DEG C for 4-5 hours to obtain the fluorescent copper nanoclusters; The preparation method of the oxygen consumption agent comprises the following steps: S201, adding laccase and 10 mM tannic acid solution into 1.5% (w / v) sodium alginate solution and stirring for 5-10 min to obtain a sodium alginate mixed solution; S202, dropping the sodium alginate mixed solution into 100 mM calcium chloride solution, and standing for solidification, and washing to obtain sodium alginate microbeads; S203, loading the sodium alginate microbeads into a dialysis bag with a molecular weight cut-off of 3.5 kDa, and supplementing ultrapure water into the bag to make the total volume in the bag 5-7 mL, excluding air bubbles, and sealing by double-end ligation to obtain the oxygen consumption agent.

[0008] Further, the enzyme activity of the laccase is 10-30 U / mL.

[0009] Further, the volume ratio of the sodium alginate solution to the tannic acid solution in step S201 is (2-4):1.

[0010] Further, the standing for solidification in step S202 is 15-30 min.

[0011] Further, the washing condition in step S202 is washing twice with deionized water.

[0012] A fluorescent copper nanocluster based on fulvic acid, wherein the fluorescent copper nanocluster is prepared according to the above preparation method.

[0013] This invention provides a method for preparing fluorescent copper nanoclusters based on fulvic acid, resulting in fluorescent copper nanoclusters.

[0014] This invention provides an application of fulvic acid-based fluorescent copper nanoclusters in the detection of sparfloxacin.

[0015] This invention provides an application of fulvic acid-based fluorescent copper nanoclusters in temperature detection.

[0016] The beneficial effects of this invention are as follows: This invention provides a method for preparing blue fluorescent copper nanoclusters (FA-Cu NCs) using fulvic acid as a template via a one-pot method. The prepared FA-Cu NCs have the advantages of uniform size and good stability. These FA-Cu NCs can be used as fluorescent nanoprobes for sensitive and selective detection of SPFX. This material can also be used as a switch-type temperature sensor, exhibiting thermosensitivity and good reversibility over a wide temperature range (25-80℃), and can be used in smart nanothermometers.

[0017] In this invention, ascorbic acid reduces Cu 2+ The process is prone to self-oxidation, generating excess reactive oxygen species, such as hydrogen peroxide and free oxygen. If these are not removed in time, they will gradually accumulate in the solution, leading to Cu oxidation. 0 The nucleus is subjected to secondary oxidation, resulting in reduced yield and decreased fluorescence stability. To suppress this type of oxidation side reaction, an oxygen-consuming agent is added during the preparation process to mitigate this situation. The oxygen-consuming agent is a composite microcapsule structure composed of sodium alginate-laccase-tannic acid, placed as an independent module in the dialysis bag. It does not participate in the preparation process of FA-Cu NCs, but indirectly regulates the reaction environment through redox buffering and gas diffusion control. During the reaction, laccase catalyzes the slow oxidation of tannic acid, consuming excess hydrogen peroxide or free oxygen in the solution and preventing the formation of Cu. 0 It is oxidized again to Cu²⁺. At the same time, the porous network formed by the microcapsule structure has good gas and small molecule permeability, which can adsorb some dissolved oxygen and achieve slow release, so as to maintain the reaction system under micro-oxygen conditions and promote the formation of FA-Cu NCs. Attached Figure Description

[0018] Figure 1 This is a flow chart of the preparation process of the oxygen-consuming agent described in this invention; Figure 2 This is a flow chart of the preparation process of FA-Cu NCs described in this invention; Figure 3The structural characterization results of FA-Cu NCs prepared in Example 1 of this invention are shown below, where (a) is the optimal excitation, emission, and absorption spectrum; (b) is the ultraviolet absorption spectrum of different components; (c) is the system comparison diagram; (d) is the fluorescence spectrum of FA-Cu NCs at different excitation wavelengths; (e) is the TEM image; (f) is the infrared absorption spectrum of FA and FA-Cu NCs; (g) and (h) are the XPS survey spectrum and Cu 2p region of FA-Cu NCs; (i), (j), (k), and (l) are the responses of FA-Cu NCs to pH, ultraviolet light, NaCl, and organic solvents, respectively. Figure 4 The results of the detection of sparfloxacin and temperature experiments using FA-Cu NCs prepared in Example 1 of this invention are shown below. (a) is the fluorescence spectrum of FA-Cu NCs responding to different concentrations of SPFX; (b) shows the relationship between F / F0 and the final SPFX concentration in the range of 0-500 µM, where F / F0 and the final SPFX concentration show a linear relationship in the range of 0-40 µM; (c) and (d) are the fluorescence intensities of FA-Cu NCs in metal salt and macromolecular solutions, respectively; (e) shows the fluorescence spectrum of FA-Cu NCs and the UV-Vis spectrum of SPFX; (f) shows the UV-Vis spectra of FA-Cu NCs, SPFX, and the FA-Cu NCs / SPFX system; (g) shows the infrared spectra of FA, FA-Cu NCs, SPFX, and the FA-Cu NCs / SPFX system; (h) shows the time-resolved fluorescence decay curves of FA-Cu NCs probes without SPFX (red) and containing 30 µM SPFX (blue); (i) and (j) are the fluorescence intensities of FA-Cu NCs in the range of 0-500 µM and 0-40 µM SPFX, respectively. Fluorescence spectra of NCs solutions during heating and cooling processes; (k) shows the linear correlation between relative fluorescence intensity and temperature during heating and cooling; (l) shows the fluorescence intensity obtained during five cycles of heating and cooling in the range of 15℃-80℃. Detailed Implementation

[0019] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the reagents used in this invention are all conventional reagents in the art and can be obtained commercially. Unless otherwise specified, the experimental methods used in this invention are all conventional experimental methods in the art.

[0020] See attached document Figure 1 -Appendix Figure 4 The present invention provides: Example 1 1. Preparation of oxygen-consuming agents S201. Dissolve sodium alginate in deionized water to prepare a 1.5% (w / v) sodium alginate solution. Take 10 mL of the solution and add an appropriate amount of laccase to the beaker so that the final enzyme activity of laccase in the system is 20 U / mL. Then add 5 mL of 10 mM tannic acid solution and stir at 80 rpm for 8 min to obtain a sodium alginate mixture. S202. Prepare a 100 mM calcium chloride solution and measure 50 mL into a beaker. Use a syringe to slowly drip the sodium alginate mixture into the calcium chloride solution drop by drop. After dripping, solidify for 20 min. Then wash twice with deionized water to remove residual ions on the surface to obtain sodium alginate microbeads. S203. Boil a dialysis bag with a molecular cutoff of 3.5 kDa in boiling water for 10 minutes, then rinse it with distilled water. After that, put sodium alginate microbeads into the rinsed dialysis bag and add pure water to the bag so that the total volume inside the bag is 5 mL. Remove air bubbles and tie and seal both ends of the dialysis bag to obtain the oxygen-consuming agent.

[0021] 2. Preparation of fluorescent copper nanoclusters using fulvic acid as a template S101. Add 0.2 mL of 1 mM fulvic acid solution and 0.4 mL of 9.6 mM copper sulfate solution to the reaction vessel, bring the solution to a final volume of 50 mL with pure water, and stir at 80 rpm for 5 min at room temperature. Then add 0.4 mL of 0.8 M ascorbic acid solution and stir at 300 rpm for 2 min at room temperature. S102. The oxygen-consuming agent is spread evenly on a perforated plate, and the plate is clipped into the reaction vessel, immersed in the solution but not touching the bottom. The mixture is stirred continuously at 80 rpm for 4 h at 60 °C to obtain FA-Cu NCs, which are fluorescent copper nanoclusters with fulvic acid as a template.

[0022] Example 2 1. Preparation of oxygen-consuming agents S201. Dissolve sodium alginate in deionized water to prepare a 1.5% (w / v) sodium alginate solution. Take 15 mL of the solution and add an appropriate amount of laccase to the beaker so that the final enzyme activity of laccase in the system is 10 U / mL. Then add 5 mL of 10 mM tannic acid solution and stir at 80 rpm for 5 min to obtain a sodium alginate mixture. S202. Prepare a 100 mM calcium chloride solution and measure 50 mL into a beaker. Use a syringe to slowly drip the sodium alginate mixture into the calcium chloride solution drop by drop. After dripping, solidify for 15 min. Then wash twice with deionized water to remove residual ions on the surface and obtain sodium alginate microbeads. S203. Boil a dialysis bag with a molecular cutoff of 3.5 kDa in boiling water for 10 minutes, then rinse it with distilled water. After that, put sodium alginate microbeads into the rinsed dialysis bag and add pure water to the bag so that the total volume inside the bag is 6 mL. Remove air bubbles and tie and seal both ends of the dialysis bag to obtain the oxygen-consuming agent.

[0023] 2. Preparation of fluorescent copper nanoclusters using fulvic acid as a template S101. Add 0.2 mL of 1 mM fulvic acid solution and 0.4 mL of 9.6 mM copper sulfate solution to the reaction vessel, bring the solution to a final volume of 50 mL with pure water, and stir at 80 rpm for 5 min at room temperature. Then add 0.4 mL of 0.8 M ascorbic acid solution and stir at 300 rpm for 2 min at room temperature. S102. Spread the oxygen-consuming agent evenly in a perforated plate, and attach the plate to the reaction vessel. Immerse the plate in the solution but do not touch the bottom. Stir continuously at 80 rpm for 5 h at 50℃ to obtain FA-Cu NCs, which are fluorescent copper nanoclusters with fulvic acid as a template.

[0024] Example 3 1. Preparation of oxygen-consuming agents S201. Dissolve sodium alginate in deionized water to prepare a 1.5% (w / v) sodium alginate solution. Take 20 mL of the solution and put it in a beaker. Add an appropriate amount of laccase to the beaker so that the final enzyme activity of laccase in the system is 30 U / mL. Then add 5 mL of 10 mM tannic acid solution and stir at 80 rpm for 10 min to obtain a sodium alginate mixture. S202. Prepare a 100 mM calcium chloride solution and measure 50 mL into a beaker. Use a syringe to slowly drip the sodium alginate mixture into the calcium chloride solution drop by drop. After dripping, solidify for 30 min. Then wash twice with deionized water to remove residual ions on the surface to obtain sodium alginate microbeads. S203. Boil a dialysis bag with a molecular cutoff of 3.5 kDa in boiling water for 10 minutes, then rinse it with distilled water. After that, put sodium alginate microbeads into the rinsed dialysis bag and add pure water to the bag so that the total volume inside the bag is 7 mL. Remove air bubbles and tie and seal both ends of the dialysis bag to obtain the oxygen-consuming agent.

[0025] 2. Preparation of fluorescent copper nanoclusters using fulvic acid as a template S101. Add 0.2 mL of 1 mM fulvic acid solution and 0.4 mL of 9.6 mM copper sulfate solution to the reaction vessel, bring the solution to a final volume of 50 mL with pure water, and stir at 80 rpm for 5 min at room temperature. Then add 0.4 mL of 0.8 M ascorbic acid solution and stir at 300 rpm for 2 min at room temperature. S102. Spread the oxygen-consuming agent evenly in a perforated plate, and attach the plate to the reaction vessel. Immerse the plate in the solution but do not touch the bottom. Stir continuously at 80 rpm for 4.5 h at 55 °C to obtain FA-Cu NCs, which are fluorescent copper nanoclusters with fulvic acid as a template.

[0026] 3. Result Characterization The fluorescent copper nanoclusters prepared in Example 1 were characterized as shown in the attached figure. Figure 3 As shown in (a) and 3(c), the synthesized FA-Cu NCs are pale yellow under sunlight and exhibit strong blue fluorescence under 365 nm UV light. When the excitation wavelength is 365 nm, FA-Cu NCs show a maximum emission peak at 418 nm, with fluorescence intensity significantly higher than the other control groups. This indicates that the fluorescence originates from the synthesized FA-Cu NCs.

[0027] The ultraviolet absorption spectrum results are attached. Figure 3 As shown in (b), compared with FA, AA, and CuSO4, FA-Cu NCs exhibit a new strong absorption peak near 300 nm, but no characteristic absorption peak of large-sized copper nanoparticles formed by surface plasmon resonance was observed in the 560-600 nm range, indicating that the FA-Cu NCs prepared in this invention form ultra-small copper nanoclusters. Furthermore, as shown in the attached... Figure 3 As shown in (d), no obvious excitation dependence was observed when the excitation wavelength was in the range of 315-415 nm, which is related to the uniform diameter of the synthesized copper nanoclusters.

[0028] The prepared FA-Cu NCs were TEM scanned, and the results are as follows: Figure 3 As shown in (e), the prepared FA-Cu NCs are spherical with a relatively uniform size distribution, and their diameters are distributed between 2 and 18 nm. This result indicates that the prepared FA-Cu NCs form relatively uniform nanoclusters.

[0029] Fourier transform infrared spectroscopy was performed on the prepared FA-Cu NCs and FA to investigate their surface groups. The results are as follows: Figure 3 As shown in (f), FA-Cu NCs and FA have similar peak positions and major functional groups, indicating that FA was successfully attached to copper nanoclusters; at 1624.43 cm⁻¹ -1and 1112.89 cm -1 The transmission peak at 3364.63 cm⁻¹ indicates the presence of peaks corresponding to the C=O and CO extension vibrations in FA-Cu NCs. A new strong peak appears at 1768.50 cm⁻¹, suggesting that C=O and copper elements bond together, causing a peak shift in the bonded C=O peak. -1 The peak at that point is very broad, corresponding to the extension of the hydroxyl group.

[0030] The chemical valence state of copper in the prepared FA-Cu NCs was analyzed using high-resolution XPS spectroscopy, and the results are shown in the attached figure. Figure 3 As shown in (g) and 3(h). The spectrum of the copper 2p region shows that the two typical peaks at 932.6 and 952.5 eV correspond to Cu 2p3 / 2 and Cu 2p1 / 2 of Cu(0), respectively, indicating that the copper element was completely reduced. No satellite peaks appeared near 942 eV, indicating that there was no Cu(II) element in the prepared FA-Cu NCs.

[0031] 4. Stability Study of FA-Cu NCs Stability studies of FA-Cu NCs were conducted under different conditions, as shown in the attached figure. Figure 3 As shown in (i), 3(j), and 3(k), the results indicate that the prepared FA-Cu NCs can tolerate a wide pH range (2-11); after irradiation under 365 nm UV light for 60 minutes, the fluorescence intensity of FA-Cu NCs remained essentially unchanged; and the fluorescence intensity remained stable in sodium chloride solutions with concentrations as high as 0.8 M.

[0032] Further investigation was conducted on the fluorescence of FA-Cu NCs in different organic solvents, and the results are attached. Figure 3 As shown in Figure 1, FA-Cu NCs maintained stable fluorescence intensity in water (H₂O), lactic acid (LAC), N,N-dimethylformamide (DMF), acetic acid (HAC), tetrahydrofuran (THF), and formaldehyde (HCHO); however, the fluorescence intensity decreased in dimethyl sulfoxide (DMSO), ethylene glycol (EG), and toluene (Tol); and benzaldehyde (PhCHO) caused quenching of FA-Cu NCs. These results confirm that FA-Cu NCs possess superior overall stability and can be applied in sensing, bioimaging, and other fields.

[0033] 5. Sensitivity and selectivity of FA-Cu NCs for SPFX detection Take 0.2 mL of the FA-Cu NCs solution prepared in Example 1, and add 1.6 mL of pure water and 0.2 mL of SPFX solutions of different concentrations (0-5 mM) sequentially. After mixing the solutions, let them stand for 1 h and measure and collect the fluorescence spectra. To determine the selectivity of FA-CuNCs as an SPFX sensor, under the same conditions, add 500 µM SPFX and interfering agent solutions to the FA-Cu NCs solution, respectively, so that the final concentration of SPFX solution in the system is 0-500 µM.

[0034] The results show that FA-Cu NCs can be rapidly quenched by SPFX at room temperature. (See attached image.) Figure 4 As shown in (a) and 4(b), the fluorescence intensity of FA-Cu NCs at different concentrations of SPFX (0 ~ 500 µM) was monitored at an excitation wavelength of 365 nm. The results showed a good linear relationship between the final SPFX concentration in the range of 0-40 µM and the fluorescence intensity of FA-Cu NCs (F / F0 = -0.00121C + 0.99917, R...). 2 =0.99357), and the LOD is as low as 0.17µM when the signal-to-noise ratio is 3.

[0035] Further studies were conducted on the selectivity of FA-Cu NCs for other substances, and the results are attached. Figure 4 As shown in (c) and 4(d), the results indicate that the fluorescence of FA-Cu NCs is significantly quenched upon the addition of SPFX, while the effects of other substances are negligible. Even when these substances are present simultaneously, the fluorescence quenching remains stable, indicating that the obtained fluorescent probe has excellent selectivity for SPFX. These results demonstrate that FA-Cu NCs can be used as a rapid, highly selective, and highly sensitive SPFX quenching sensor.

[0036] 6. Quenching mechanism of FA-Cu NCs The UV-Vis absorption and fluorescence spectra of the FA-Cu NCs / SPFX system were measured, and the results are presented in the appendix. Figure 4 As shown in (e) and 4(f), the absorption spectrum of SPFX severely overlaps with the excitation spectrum of FA-Cu NCs, indicating that the quenching phenomenon is an internal filtration effect. The internal filtration effect typically occurs between the absorber and the fluorophore, caused by absorption in the excitation and emission bands of the fluorophore. The presence of SPFX did not cause a new absorption peak in FA-Cu NCs, ruling out the possibility of dynamic quenching, because in dynamic quenching, the analyte and the ground state of the fluorophore would form a new complex. To determine the type of quenching, fluorescence lifetime tests were also performed. (See attached...) Figure 4(h) shows that the fluorescence lifetime of the probe remains almost unchanged after the addition of SPFX. According to the double exponential fitting method, the average lifetimes of FA-Cu NCs without SPFX and with SPFX are calculated to be 1.3943 ns and 1.4192 ns, respectively [Equation: I(t) = B1exp(-t / τ1) + B2exp(-t / τ2); τ ave = (B1τ1) 2 + B2τ2 2 [) / (B1τ1+B1τ1)], ruling out the possibility of dynamic quenching and fluorescence resonance energy transfer. Meanwhile, as shown in the attached... Figure 4 As shown in (g), no significant changes were observed in the infrared spectrum of FA-Cu NCs compared to the FA-Cu NCs / SPFX system, indicating that no new substances were formed. These results suggest that the quenching of FA-Cu NCs by SPFX is a static quenching based on the internal filtration effect.

[0037] 7. Suitability test of FA-Cu NCs for detecting SPFX in biological samples First, serum samples were centrifuged at 10000 r / min for 10 min, and the supernatant was collected. To 0.2 mL of the supernatant, 1.4 mL of PBS buffer (10 mmol / L, pH=7.4), 0.2 mL of FA-Cu NCs solution, and 0.2 mL of SPFX solutions of different concentrations (10, 50, and 100 µM) were added sequentially. After thorough mixing, the mixture was allowed to stand for 1 h, and fluorescence spectra were measured and collected. The detection limit and recovery rate were calculated based on the working curve. The final concentrations of SPFX solution in the system were 1, 5, and 10 µM.

[0038] The experimental results are shown in Table 1. The detection amount and the spiking amount were very close, and the recovery rate of SPFX was in the range of 95.7%-101.35%, which was satisfactory. The results indicate that the fluorescent nanosensor based on FA-Cu NCs has great potential in drug detection.

[0039] Table 1. Quantitative detection of SPFX in serum samples

[0040] 8. Determination of the temperature response and temperature dependence of fluorescence intensity of FA-Cu NCs The fluorescence intensity response and temperature dependence of FA-Cu NCs were measured, and the results are attached. Figure 4As shown in (i), 4(j), 4(k), and 4(l), the fluorescence intensity of FA-Cu NCs gradually decreases as the temperature increases from 25 °C to 80 °C; when the temperature cools from 80 °C to 25 °C, FA-Cu NCs exhibit a significant reversible reaction. During heating and cooling, the relative fluorescence intensity (F / F0) shows a good linear relationship with temperature, with the heating process being F / F0 = -0.01183T + 1.23285, R... 2 =0.97079, the cooling process is F / F0=-0.01259T+1.24828, R 2 =0.97450; it can be easily subjected to five cycles of heating and cooling between 15-80 °C. Due to its good salt resistance, UV radiation resistance and temperature responsiveness, FA-Cu NCs can be used as a "switch" type fluorescent nanothermometer.

[0041] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing fluorescent copper nanoclusters based on fulvic acid, characterized in that, Includes the following steps: S101. Add 1 mM fulvic acid solution and 9.6 mM copper sulfate solution to the reaction vessel, make up to volume, then add 0.8 M ascorbic acid solution and stir. S102. Spread the oxygen-consuming agent evenly in a perforated plate, and clip the plate into the reaction vessel. Immerse the plate in the solution but do not touch the bottom. Stir at 50-60℃ for 4-5 hours to obtain the fluorescent copper nanoclusters. The method for preparing the oxygen-consuming agent includes the following steps: S201. Add laccase and 10 mM tannic acid solution to a 1.5% (w / v) sodium alginate solution and stir for 5-10 min to obtain a sodium alginate mixture. S202. Add the sodium alginate mixed solution dropwise into a 100 mM calcium chloride solution, let it stand to solidify, wash, and obtain sodium alginate microbeads. S203. Sodium alginate microbeads are packed into a dialysis bag with a molecular cutoff of 3.5 kDa, and ultrapure water is added to the bag to make the total volume of the bag 5-7 mL. Air bubbles are removed, and the bags are sealed at both ends to obtain the oxygen-consuming agent.

2. The method for preparing fluorescent copper nanoclusters based on fulvic acid according to claim 1, characterized in that, The enzyme activity of the laccase is 10-30 U / mL.

3. The method for preparing fluorescent copper nanoclusters based on fulvic acid according to claim 1, characterized in that, In step S201, the volume ratio of sodium alginate solution to tannic acid solution is (2-4):

1.

4. The method for preparing fluorescent copper nanoclusters based on fulvic acid according to claim 1, characterized in that, In step S202, the solidification is allowed to proceed for 15-30 minutes.

5. The method for preparing fluorescent copper nanoclusters based on fulvic acid according to claim 1, characterized in that, In step S202, the washing conditions are to wash twice with deionized water.

6. A fluorescent copper nanocluster based on fulvic acid, characterized in that, The fluorescent copper nanoclusters were prepared according to the preparation method described in any one of claims 1-5.

7. The application of fulvic acid-based fluorescent copper nanoclusters in the detection of sparfloxacin according to claim 6.

8. The application of fulvic acid-based fluorescent copper nanoclusters in temperature detection according to claim 6.

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