Preparation method of gold nanocluster fluorescent material as well as product and application of gold nanocluster fluorescent material

Abstract

The invention provides a preparation method of a gold nanocluster fluorescent material as well as a product and application of the gold nanocluster fluorescent material. The method comprises the following steps: step 1, preparing a carboxylated chitosan aqueous solution; step 2, fully mixing a NaOH solution and a HAuCl4 solution, adding the mixture into the carboxylated chitosan aqueous solution prepared in the step 1, then adding a DTT solution, and fully stirring and uniformly mixing; and step 3, incubating the mixed solution in the step 2 in a water bath at 5-50 DEG C for 8 hours until the color of the mixture gradually changes from faint yellow to colorless in the reaction process. The obtained gold nanocluster fluorescent material CC / DTT-AuNCs can be used as a fluorescent probe of a mitomycin C detection system, and can be used for detecting mitomycin C in plasma. The method is easy to operate, mild in reaction condition, environmentally friendly, capable of being applied to detection of MMC in plasma and high in practicability; the fluorescent material has good accuracy, selectivity and sensitivity in the aspect of MMC detection, and the detection limit is as low as 0.3 mu M.

Description

[Technical field]

[0001] The present invention relates to the field of nanomaterials, and in particular to a preparation method of a gold nanocluster fluorescent material, a product thereof and an application thereof. [Background technology]

[0002] Mitomycin C (MMC) is a broad-spectrum antitumor antibiotic isolated and extracted from the culture medium of Streptomyces, which has a significant therapeutic effect on various cancers. Its working principle is that it can depolymerize the DNA of cells and hinder the replication of DNA, thereby inhibiting the division of tumor cells. However, long-term use of MMC has serious adverse reactions such as intestinal reactions and bone marrow suppression. At present, the detection methods of MMC include HPLC, capillary electrophoresis amperometry, HPLC-mass spectrometry, flow injection, etc. However, most of these methods have the disadvantages of using toxic solvents, complex procedures, and long response time. Therefore, it is of certain research value to establish a simple, reliable, and green MMC detection method.

[0003] Fluorescence analysis has the advantages of simplicity and high efficiency and has been widely used in the detection of drugs and biomolecules. Gold nanoclusters (AuNCs) have attracted extensive attention in the field of chemical and biological sensing due to their advantages such as easy modification, low toxicity, and visible to near-infrared photoluminescence. In particular, the PET process of AuNCs is very sensitive to changes in the surrounding environment and can be simply controlled by the redox state and size of the metal core, the surface charge, and the length of the ligand shell. Therefore, PET-induced changes in AuNCs photoluminescence have been proven to be a promising approach to construct fluorescent sensors.

[0004] MMC is a derivative of benzoquinone and a typical electron acceptor. Therefore, efficient detection of MMC can be achieved by rationally constructing a PET fluorescence sensor. Based on the above background, this experiment intends to use the PET theory of AuNCs to screen suitable gold nanoclusters as fluorescent probes, study the PET process of MMC and the screened AuNCs, and rationally construct a fluorescence sensor based on AuNCs to achieve efficient detection of MMC and use it for the detection of MMC in plasma samples. [Summary of the invention]

[0005] The technical problem to be solved by the present invention is to provide a method for preparing a gold nanocluster fluorescent material, a product and an application thereof. The method is simple to operate, has mild reaction conditions, is green and environmentally friendly, and can be applied to the detection of MMC in plasma, with high practicality. The fluorescent material has good accuracy, selectivity and sensitivity in detecting MMC, with a detection limit as low as 0.3 μM.

[0006] The present invention is achieved in that:

[0007] A method for preparing a gold nanocluster fluorescent material, the method comprising the following steps:

[0008] Step 1, preparing a carboxylated chitosan aqueous solution;

[0009] Step 2, fully mix the NaOH solution and the HAuCl4 solution, add them to the carboxylated chitosan aqueous solution prepared in step 1, then add the DTT solution, and stir and mix thoroughly;

[0010] Step 3: Place the mixture of step 2 in a 5-50°C water bath and incubate for 8 hours. During the reaction, the color of the mixture gradually changes from light yellow to colorless.

[0011] Furthermore, the gold nanoclusters obtained in step 3 were dialyzed in deionized water for 24 hours using a dialysis bag with a molecular weight cutoff of 3500 to obtain purified CC / DTT-AuNCs; the purified gold nanoclusters CC / DTT-AuNCs were then sealed and stored in a 4°C refrigerator away from light.

[0012] Furthermore, the concentration of the carboxylated chitosan aqueous solution is 6.76 mg / mL; the concentration of the HAuCl4 solution is 20 mg / mL; and the concentration of the DTT solution is 0.1M.

[0013] Furthermore, the pH value of the mixed solution in step 2 is adjusted to 6-11 by using NaOH solution.

[0014] Furthermore, a method for preparing a gold nanocluster fluorescent material obtains the gold nanocluster fluorescent material CC / DTT-AuNCs.

[0015] Furthermore, the gold nanocluster fluorescent material CC / DTT-AuNCs is used as a fluorescent probe in the mitomycin C detection system.

[0016] Furthermore, the gold nanocluster fluorescent material CC / DTT-AuNCs is used for the detection of mitomycin C in plasma.

[0017] Furthermore, the detection limit of mitomycin C of the gold nanocluster fluorescent material CC / DTT-AuNCs fluorescent probe is as low as 0.3 μM.

[0018] The present invention has the following advantages:

[0019] The present invention uses CC / DTT-AuNCs as a fluorescent probe for the MMC detection system, and MMC can significantly inhibit the fluorescence of CC / DTT-AuNCs through the PET mechanism. On this basis, a new MMC fluorescence detection method based on the PET behavior of CC / DTT-AuNCs was successfully constructed. The new fluorescence detection method has good accuracy, selectivity and sensitivity in detecting MMC, with a detection limit as low as 0.3μM, which is lower than the existing MMC detection limit; and the method is simple to operate, mild reaction conditions, does not require the addition of additional solvents, is green and environmentally friendly, and can be applied to the detection of MMC in plasma, with high practicality.

Brief Description of the Drawings

[0020] The present invention will be further described below in conjunction with embodiments with reference to the accompanying drawings.

[0021] Figure 1 Schematic diagram of the preparation principle of CC / DTT-AuNCs of the present invention.

[0022] Figure 2 Schematic diagram of the characterization results of CC / DTT-AuNCs according to an embodiment of the present invention.

[0023] Figure 3 Schematic diagram of the mechanism of MMC quenching CC / DTT-AuNCs fluorescence according to an embodiment of the present invention.

[0024] Figure 4 This is a schematic diagram of the effects of different concentrations of MMC on the fluorescence quenching of CC / DTT-AuNCs according to an embodiment of the present invention.

[0025] Figure 5 Schematic diagram of the anti-interference experimental results of CC / DTT-AuNCs according to an embodiment of the present invention. [Specific implementation method]

[0026] The following will be combined with the attached Figure 1-5 The technical scheme of the present invention is clearly and completely described in detail with specific embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are all conventional products that can be purchased commercially.

[0027] Example

[0028] 1. Solution Preparation

[0029] All glass containers, quartz containers, and stirring bars used were soaked in aqua regia (the ratio of concentrated hydrochloric acid to nitric acid was 3:1) for 10 min before use and rinsed with deionized water.

[0030] Chloroauric acid solution: Dissolve the chloroauric acid solid required for the experiment in deionized water to prepare a chloroauric acid solution with a concentration of 20 mg / mL, and store it in a refrigerator, sealed and protected from light. According to the needs of the experiment, dilute it with deionized water to different concentrations of chloroauric acid solution for later use.

[0031] Dithiothreitol solution: Accurately weigh 0.23 g of DTT solid, add 15 mL of deionized water to dissolve, and prepare a 0.1 M DTT solution.

[0032] Carboxylated chitosan solution: Accurately weigh 50 mg of CC solid, transfer to a conical flask and add 7.4 mL of deionized water and stir until completely dissolved to prepare a CC solution of the desired concentration.

[0033] MMC solution: Accurately weigh a certain amount of mitomycin C solid and prepare it with deionized water to the concentration required for the experiment.

[0034] NaOH solution: Accurately weigh 0.16 g of NaOH solid, dissolve it in deionized water and dilute to 10 mL to prepare a 0.4 M NaOH solution.

[0035] PB buffer: Weigh a certain amount of Na2HPO4 and NaH2PO4 solids respectively, prepare a solution with a concentration of 20mM with deionized water, and adjust the pH value of the solution to the required pH.

[0036] All other reagents used were of analytical grade and all solutions were prepared with deionized water.

[0037] 2. Preparation and purification of CC / DTT-AuNCs

[0038] Add 50 mg of carboxylated chitosan and 7.4 mL of deionized water to a conical flask, stir until completely dissolved to obtain a CC solution. Thoroughly mix 1 mL of 0.4 M NaOH solution and 1.6 mL of 20 mg / mL HAuCl4 solution, add to the above CC solution, then add 10 mL of 0.1 M DTT solution, stir thoroughly to mix, and incubate in a 37 °C water bath for 8 hours. During the reaction, the color of the mixture gradually changes from light yellow to colorless. The reacted gold nanoclusters were dialyzed in deionized water for 24 hours using a dialysis bag with a molecular weight cutoff of 3500 to obtain purified CC / DTT-AuNCs. The gold nanoclusters were sealed in a 4 °C refrigerator and stored away from light. The preparation principle of the CC / DTT-AuNCs is as follows Figure 1 shown.

[0039] 3.MMC Determination

[0040] The MMC detection was performed by the following steps: 0.1 mL CC / DTT-AuNCs solution was added to 1.9 mL standard MMC solution of different concentrations (in 20 mM phosphate buffer, pH 6.0), and the emission spectrum of the mixture was directly monitored at an excitation wavelength of 290 nm.

[0041] 4. Screening of AuNCs

[0042] The Rehm-Weller equation was used to calculate the driving force of electron transfer of MMC-CC / DTT-AuNCs. The Rehm-Weller equation is as follows:

[0043]

[0044] ΔG is the driving force for PET, E ox (D) is the oxidation potential of CC / DTT-AuNCs, E red (A) is the reduction potential of MMC, E excit is the excited state energy of CC / DTT-AuNCs, which can be obtained from the spectral characteristics of CC / DTT-AuNCs. is the Coulomb interaction energy, which can be ignored in water systems. The calculation results show that the electron transfer driving force of MMC-CC / DTT-AuNCs is -1.24eV. ​​The value of the electron transfer driving force is very close to the reorganization energy of the CC / DTT-AuNCs system (1.03eV), indicating that MMC can significantly quench the fluorescence of CC / DTT-AuNCs through the PET effect.

[0045] 5. Characterization of CC / DTT-AuNCs

[0046] CC / DTT-AuNCs were prepared and characterized. CC / DTT-AuNCs showed two weak single electron transition absorption peaks in its UV-visible absorption spectrum, rather than the typical plasma peaks observed in gold nanoparticles. Figure 2 , Figure 2 A is the 3D luminescence spectrum of the prepared CC / DTT-AuNCs solution, and B is the TEM image of CC / DTT-AuNCs. As can be seen from the figure, the 3DPL spectrum analysis of AuNCs shows that the maximum excitation wavelength and emission wavelength are located at 290nm and 640nm, respectively (as shown in Figure 2). Figure 2 A). Transmission electron microscopy (TEM) images show that CC / DTT-AuNCs particles are approximately spherical and uniformly dispersed, with an average particle size of <3.0 nm (as shown in Figure 2B). This is consistent with the characteristics of CC / DTT-AuNCs, indicating that CC / DTT-AuNCs have been successfully prepared.

[0047] 6. Study on the mechanism of MMC quenching CC / DTT-AuNCs fluorescence

[0048] Figure 3 Schematic diagram of the mechanism of MMC quenching CC / DTT-AuNCs fluorescence, including (A) the emission spectrum of CC / DTT-AuNCs (purple line) and the emission spectrum of CC / DTT-AuNCs after 100μM MMC (red line), inset: photos of CC / DTT-AuNCs before (left) and after (right) adding 100μM MMC; (B) the normalized emission spectrum (blue line) and normalized excitation spectrum (red line) of CC / DTT-AuNCs and the normalized absorption spectrum (green line) of MMC; (C) the effect of solvent on MMC-induced CC / DTT-AuNCs fluorescence quenching, where F and F0 are the steady-state fluorescence intensities of CC / DTT-AuNCs in the presence and absence of MMC, respectively. (D) Inner filter effect test: the actual measurement curve (blue line) and calibration curve (red line) of CC / DTT-AuNCs fluorescence inhibition efficiency (E%) after adding different concentrations of MMC.

[0049] like Figure 3 As shown in Figure A, after mixing MMC with CC / DTT-AuNCs, the results are consistent with theoretical predictions. MMC can significantly inhibit the fluorescence intensity of CC / DTT-AuNCs. Then, it is ruled out that MMC quenches the fluorescence of CC / DTT-AuNCs through FRET and IFE mechanisms. First, the occurrence of FRET generally requires that the emission spectrum of the fluorophore and the absorption spectrum of the quencher have a certain effective overlap, such as Figure 3 As shown in Figure B, since there is no cross-overlap between the absorption spectrum of MMC and the emission spectrum of CC / DTT-AuNCs, FRET can be ruled out as the cause of the fluorescence quenching of CC / DTT-AuNCs. In addition, due to the strong solvent-dependent quenching (e.g. Figure 3 C), it is reasonable to further exclude that FRET is not the mechanism of fluorescence quenching of CC / DTT-AuNCs caused by MMC, because the appearance of FRET will not produce an electron transfer state that is sensitive to solvent polarity. Figure 3 In B, it can also be found that the ultraviolet absorption spectrum of MMC partially overlaps with the fluorescence excitation spectrum of CC / DTT-AuNCs, which may lead to the occurrence of IFE, thereby causing the fluorescence quenching of CC / DTT-AuNCs. Therefore, it is necessary to further verify whether IFE inhibits the fluorescence intensity of CC / DTT-AuNCs. The following formula can be used to exclude the influence of IFE. The formula is as follows:

[0050]

[0051] In the above formula, CF is the correction factor, F obsd is the fluorescence intensity of MMC-CC / DTT-AuNCs, F cor is the corrected fluorescence intensity of MMC-CC / DTT-AuNCs, A ex is the absorbance measured at the maximum excitation wavelength (290 nm), A em is the absorbance measured at the maximum emission wavelength (640nm). s is the thickness of the electron beam (0.1cm), g is the distance between the electron beam and the edge of the cuvette (0.4cm), and d is the width of the cuvette (1cm). The calibration results are shown in Figure 3 D and Table 1, after calculation, it was found that the corrected fluorescence quenching efficiency was almost the same at different concentrations of MMC, which means that IFE is not the reason why MMC causes the fluorescence quenching of CC / DTT-AuNCs. Based on the above discussion, MMC quenches the fluorescence of CC / DTT-AuNCs through the PET mechanism.

[0052] Table 1 Effect of IFE on the fluorescence intensity of CC / DTT-AuNCs

[0053]

[0054] 4. Construction of MMC detection method

[0055] Under the optimal reaction conditions (pH 6.0 and 30 °C), in order to ensure the high sensitivity of this method for MMC detection, the effect of different concentrations of MMC on the fluorescence quenching of CC / DTT-AuNCs was further studied. Figure 4 As shown in the figure, (A) Fluorescence emission spectra of CC / DTT-AuNCs in the presence of different concentrations of MMC. From top to bottom, the concentrations of MMC are 0, 1.0, 2.5, 5.0, 7.5, 10.0, 15.0, 20.0, 25.0, 50.0, 75.0, 100.0 μM; (B) Linear relationship between ΔF and MMC concentration. Figure 4 As shown in Figure A, with the increase of MMC concentration, the fluorescence intensity of CC / DTT-AuNCs was gradually suppressed. Figure 4 As shown in B, when the MMC concentration was in the range of 1-25 μM, ΔF = F0-F = 10.63[MMC] + 32.60, R 2 =1.00. After calculation, the minimum detection limit of MMC is 0.3μM (the detection limit is based on 3σ / S, where σ and S are the standard deviation of the blank signal and the slope of the working curve, respectively). This detection limit is lower than or close to the detection limit of MMC reported in previous literature, and the detection method is much simpler than before, and the detection time is greatly shortened.

[0056] In order to verify the selectivity of the detection method for MMC, common interfering substances in plasma such as metal ions and proteins (using bovine serum albumin as a model) and combined therapeutic drugs (5-fluorouracil, ascorbic acid, reduced glutathione, etc.) were selected for experiments. The results are as follows: Figure 5 As shown, the X-axis numbers in the figure correspond to blank, MMC, Fe 3+ , Fe 2+ Mg 2+ , Ca 2+ , K + 、Na + 、HCO3 - 、Zn 2+ , urea, glucose, GSH, BSA, ascorbic acid, DL-histidine, L-cysteine, N-acetyl-L-cysteine, 5-fluorouracil. The MMC concentration used in Figure (A) is 10μM. Except for the bovine serum albumin concentration of 40μg / mL, the concentrations of other interfering substances are all 10μM. As can be seen from the figure, only MMC has a significant inhibitory effect on the fluorescence of CC / DTT-AuNCs, while the effects of other interfering substances on the fluorescence of CC / DTT-AuNCs are negligible. The interference verification experiment proves that the detection method has good specificity for MMC and the detection method is safe and reliable.

[0057] 5. Detection of MMC in human serum samples

[0058] Since MMC has severe bone marrow toxicity, rapid and accurate detection of MMC blood concentration is of great significance in clinical treatment. The experiment used human serum samples as the research object to verify the feasibility of this method for MMC detection in actual samples. 200μL serum and 1.5mL PB buffer with pH7.4 and concentration of 20mM were shaken and mixed, 100μL CC / DTT-AuNCs solution was added and shaken and mixed again, and finally 200μL MMC solution of different concentrations was added and shaken and mixed. Under the condition of excitation wavelength of 290nm, the fluorescence spectrum of the obtained solution was measured. From the results shown in Table 2, it can be seen that the recovery rate of the three spiked samples is between 98.0-104.0%, and the relative standard deviation value is 1.12-1.71% (n=3). The data show that this method is stable and reliable and can be used for the detection of MMC content in actual clinical samples.

[0059] Table 2 MMC spike test in human serum

[0060]

[0061] In summary, the present invention uses CC / DTT-AuNCs as a fluorescent probe for the MMC detection system, and MMC can significantly inhibit the fluorescence of CC / DTT-AuNCs through the PET mechanism. On this basis, a new method for MMC fluorescence detection based on the PET behavior of CC / DTT-AuNCs was successfully constructed. This new fluorescence detection method has good accuracy, selectivity and sensitivity in detecting MMC, with a detection limit as low as 0.3μM, which is lower than the existing MMC detection limit; and the method is simple to operate, mild reaction conditions, no additional solvents need to be added, green and environmentally friendly, and can be applied to the detection of MMC in plasma, with high practicality.

[0062] Although the specific implementation modes of the present invention are described above, those skilled in the art should understand that the specific implementation modes described are only illustrative and are not intended to limit the scope of the present invention. Equivalent modifications and changes made by those skilled in the art in accordance with the spirit of the present invention should be included in the scope of protection of the claims of the present invention.

Claims

1. A method for preparing a gold nanocluster fluorescent material, characterized in that: The method steps are as follows: Step 1, preparing a carboxylated chitosan aqueous solution; Step 2, fully mix the NaOH solution and the HAuCl4 solution, add them to the carboxylated chitosan aqueous solution prepared in step 1, then add the DTT solution, and stir and mix thoroughly; Step 3: Place the mixture of step 2 in a 5-50°C water bath and incubate for 8 hours. During the reaction, the color of the mixture gradually changes from light yellow to colorless.

2. The method for preparing a gold nanocluster fluorescent material according to claim 1, characterized in that: The gold nanoclusters obtained in step 3 were dialyzed in deionized water for 24 hours using a dialysis bag with a molecular weight cutoff of 3500 to obtain purified CC / DTT-AuNCs; the purified gold nanoclusters CC / DTT-AuNCs were then sealed and stored in a 4°C refrigerator away from light.

3. The method for preparing a gold nanocluster fluorescent material according to claim 1, characterized in that: The concentration of the carboxylated chitosan aqueous solution is 6.76 mg / mL; the concentration of the HAuCl4 solution is 20 mg / mL; and the concentration of the DTT solution is 0.1M.

4. The method for preparing a gold nanocluster fluorescent material according to claim 1, characterized in that: The pH value of the mixed solution in step 2 was adjusted to 6-11 with NaOH solution.

5. A gold nanocluster fluorescent material CC / DTT-AuNCs prepared according to the method for preparing a gold nanocluster fluorescent material according to any one of claims 1 to 4.

6. Use of the gold nanocluster fluorescent material according to claim 5 in the detection of mitomycin C, characterized in that: The gold nanocluster fluorescent material CC / DTT-AuNCs is used as a fluorescent probe in the mitomycin C detection system.

7. The use of the gold nanocluster fluorescent material in the detection of mitomycin C according to claim 6, characterized in that: The gold nanocluster fluorescent material CC / DTT-AuNCs is used for detecting mitomycin C in plasma.

8. The use of the gold nanocluster fluorescent material in the detection of mitomycin C according to claim 6, characterized in that: The detection limit of mitomycin C of the gold nanocluster fluorescent material CC / DTT-AuNCs fluorescent probe is as low as 0.3 μM.

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