CN108949145B - Urease-gold nanocluster fluorescent material and preparation method thereof - Google Patents

Urease-gold nanocluster fluorescent material and preparation method thereof Download PDF

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CN108949145B
CN108949145B CN201811064151.8A CN201811064151A CN108949145B CN 108949145 B CN108949145 B CN 108949145B CN 201811064151 A CN201811064151 A CN 201811064151A CN 108949145 B CN108949145 B CN 108949145B
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urease
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gold nanocluster
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nanocluster fluorescent
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CN108949145A (en
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陈伟
邓豪华
彭花萍
陈睿婷
何少斌
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Fujian Medical University
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Abstract

The invention discloses a urease-gold nanocluster fluorescent material and a preparation method thereof. The method takes urease as a biological template to synthesize the gold nanocluster fluorescent material in situ, and the urease is used as a stabilizer and a reducing agent to control the formation of the gold nanoclusters. The invention is a preparation method of a novel gold nanocluster fluorescent material, and has the advantages of simple and environment-friendly preparation and no need of a reducing agent. The prepared urease-gold nanocluster has the characteristics of strong red fluorescence (the maximum emission wavelength is 630 nm), long fluorescence life (1.74 mu s), high stability (time, salt and serum stability), larger Stokes shift (-140 nm) and the like.

Description

Urease-gold nanocluster fluorescent material and preparation method thereof
Technical Field
The invention relates to a urease-gold nanocluster fluorescent material and a preparation method thereof, and belongs to the technical field of nanometer.
Background
In recent years, fluorescent metal nanoclusters have been attracting attention as a novel fluorescent nanomaterial. The metal nanoclusters are molecular aggregates consisting of several to hundreds of metal atoms under the protection of certain molecular layers. Due to the unique physical, electrical and optical properties, the metal nanoclusters show wide application prospects in the fields of monomolecular photoelectricity, catalysis, biological imaging, sensors and the like. Among all metal nanocluster materials, gold nanoclusters (AuNCs) are the most studied metal nanocluster material at present because of their advantages of stable chemical properties, good biocompatibility, and the like.
The action of organisms to form mineral structures by uptake of metal ions is called biomineralization, a natural process that is an important mechanism for the body to evade the harmful effects of toxic metal ions. Inspired by this process, several research efforts have shown that nanomaterials can be synthesized by the uptake and alignment of inorganic materials by organisms or biological macromolecules. The protein has good size regulation and control capability, and the nano material can be easily prepared through the residue structure in the molecule. Therefore, the combination of proteins and metal precursors has been developed as an important branch of metal nanocluster synthesis. In 2009, Xie et al successfully prepared AuNCs containing 25 gold atoms by simulating biomineralization by using the template effect of Bovine Serum Albumin (BSA) for the first time. Under alkaline conditions, aromatic amino acid residues in the protein can reduce chloroauric acid to form a gold core, and opening of disulfide bonds can provide a good stabilizing effect for the gold core. To date, in addition to BSA, researchers have found that other proteins, such as human serum albumin, transferrin, trypsin, pepsin, lysozyme, horseradish peroxidase, and insulin, can be prepared to AuNCs. Compared with other AuNCs preparation methods, the protein biological template method has the advantages of simple preparation method, mild reaction conditions, no need of adding any exogenous reducing agent, high stability of the prepared AuNCs, good biocompatibility and large application prospect in the research of the fields of medicine, sensors and the like, and the maximum emission wavelength of the AuNCs is usually positioned in a near infrared region.
The invention takes urease as a biological template to synthesize the gold nanocluster fluorescent material in situ under the condition of no addition of any other reducing agent. Urease acts as a stabilizer and reducing agent to control the formation of gold nanoclusters.
Disclosure of Invention
The invention aims to provide a urease-gold nanocluster fluorescent material and a method for synthesizing the gold nanocluster fluorescent material in situ by taking urease as a biological template under the condition that no other reducing agent is added.
In order to achieve the purpose, the invention adopts the following technical scheme:
the preparation method of the urease-gold nanocluster fluorescent material is characterized by comprising the following reaction steps of: sequentially adding a sodium hydroxide solution and a chloroauric acid solution with the concentration of 0.2-0.2 mol/L into a urease solution with the concentration of 0-50 mg/mL, uniformly mixing, placing in a 37 ℃ water bath for constant-temperature reaction for 0-12 hours, after the reaction is finished, dialyzing and purifying the reaction liquid by using a dialysis bag with the molecular weight cutoff of 7000 to obtain a urease-gold nanocluster fluorescent material aqueous solution, freeze-drying the urease-gold nanocluster fluorescent material aqueous solution to obtain urease-gold nanocluster fluorescent material powder, wherein urease is used as a stabilizer and a reducing agent to control the formation of gold nanoclusters, and the volume ratio of the sodium hydroxide solution to the chloroauric acid solution to the urease solution is 1: 4: 4, the total volume of the three components is 45 mL.
The concentration and volume of the used sodium hydroxide solution are 1 mol/L and 5 mL, the concentration and volume of the chloroauric acid solution are 10 mmoL/L and 20 mL, the concentration and volume of the urease solution are 50 mg/mL and 20 mL, the reaction time is 12 hours, and the reaction temperature is 37 ℃.
The urease-gold nanocluster fluorescent material prepared by the method is characterized in that the urease-gold nanocluster fluorescent material aqueous solution is light brown, a characteristic absorption peak of protein appears at 280 nm of an ultraviolet-visible spectrum, strong red fluorescence is generated under the irradiation of an ultraviolet lamp, the maximum excitation wavelength and the emission wavelength are 490 nm and 630 nm respectively, the quantum yield is 17%, and the fluorescence life is 1.74 mu s.
After the urease-gold nanocluster fluorescent material aqueous solution is stored for one month in a dark place at 4 ℃, the relative fluorescence intensity is kept above 85%.
After the urease-gold nanocluster fluorescent material aqueous solution is placed in 0.2-2 mol/L sodium chloride solution for incubation for 30 minutes, the relative fluorescence intensity is kept above 90%.
After the urease-gold nanocluster fluorescent material aqueous solution is placed in 10% human serum to be incubated for 30 minutes, the relative fluorescence intensity is kept above 90%.
The method does not add any other reducing agent, and urease is used as a stabilizing agent and the reducing agent to control the formation of the gold nanoclusters.
The preparation of the urease-gold nanocluster fluorescent material comprises the following steps: all glassware used in the following process is soaked in aqua regia, thoroughly washed with double distilled water and dried. The preparation method of the urease-gold nanocluster fluorescent material comprises the following steps: 5 mL of NaOH solution with the concentration of 1 moL/L and 20 mL of chloroauric acid with the concentration of 10 mmoL/L are sequentially added into 20 mL of freshly prepared urease solution with the concentration of 50 mg/mL, mixed evenly and placed in a constant-temperature water bath at 37 ℃ for incubation for 8 hours. And after the reaction is finished, dialyzing and purifying the reaction solution for 48 hours by using a dialysis bag with the molecular weight of 7000 to obtain the urease-gold nanocluster fluorescent material aqueous solution.
The invention has the advantages that:
(1) the invention takes urease as a biological template to synthesize the gold nanocluster fluorescent material in situ under the condition of no addition of any other reducing agent. The preparation method is green and environment-friendly, and has simple, convenient and quick operation and good reproducibility.
(2) The urease-gold nanocluster prepared by the invention has the characteristics of strong red fluorescence (the maximum emission wavelength is 630 nm), long fluorescence life (1.74 mu s), high stability (time, salt and serum stability), larger Stokes shift (140 nm) and the like.
Drawings
FIG. 1 is a comparison graph of appearance of urease-gold nanocluster fluorescent nanomaterial under visible light (A) and ultraviolet light (B), respectively.
FIG. 2 is a diagram of ultraviolet-visible absorption spectra of urease-gold nanocluster fluorescent nanomaterials.
FIG. 3 is a fluorescence spectrum of urease-gold nanocluster fluorescent nanomaterial.
FIG. 4 is a graph showing the effect of urease concentration on the fluorescence intensity of urease-gold nanoclusters.
FIG. 5 is a graph showing the effect of sodium hydroxide concentration on the fluorescence intensity of urease-gold nanoclusters.
FIG. 6 is a graph showing the effect of reaction time on fluorescence intensity of gold urease-nanoclusters.
FIG. 7 is a transmission electron microscope image of urease-gold nanocluster fluorescent nanomaterial.
FIG. 8 is an energy scattering X-ray energy spectrum of urease-gold nanocluster fluorescent nanomaterial.
FIG. 9 is a fluorescence lifetime diagram of urease-gold nanocluster fluorescent nanomaterial.
FIG. 10 is an X-ray photoelectron spectrum of urease-gold nanocluster fluorescent nanomaterial. In the figure: a is a 4f plot of gold and B is a 2p plot of sulfur.
Fig. 11 is a time stability graph of urease-gold nanoclusters.
FIG. 12 is a graph of salt stability of urease-gold nanoclusters.
Fig. 13 is a serum stability graph of urease-gold nanoclusters.
Detailed Description
Example 1:
5 mL of NaOH solution with the concentration of 1 moL/L and 20 mL of chloroauric acid with the concentration of 10 mmoL/L are sequentially added into 20 mL of freshly prepared urease solution with the concentration of 50 mg/mL, mixed evenly and placed in a constant-temperature water bath at 37 ℃ for incubation for 8 hours. And after the reaction is finished, dialyzing and purifying the reaction solution for 48 hours by using a dialysis bag with the molecular weight of 7000 to obtain the urease-gold nanocluster fluorescent material aqueous solution. The obtained urease-gold nanocluster solution is light brown under visible light (see A in figure 1), strong red fluorescence is generated under the irradiation of an ultraviolet lamp (see B in figure 1), a characteristic absorption peak of protein appears at 280 nm in an ultraviolet-visible spectrum (see figure 2), the maximum excitation wavelength and the emission wavelength are 490 nm and 630 nm respectively (see figure 3), and the quantum yield is 17%.
Example 2:
5 mL of NaOH solution with the concentration of 1 moL L/L and 20 mL of chloroauric acid with the concentration of 10 mmoL/L are sequentially added into 20 mL of freshly prepared urease solution with the concentration of 0-80 mg/mL (0 of 0-80 mg/mL is more than 0 mg/mL, and can be 0.01mg/mL, namely 20 mL of 0.01 mg/mL-80 mg/mL), mixed uniformly and placed in a constant-temperature water bath tank at 37 ℃ for incubation for 8 hours. And after the reaction is finished, dialyzing and purifying the reaction solution for 48 hours by using a dialysis bag with the molecular weight of 7000 to obtain the urease-gold nanocluster fluorescent material aqueous solution. As shown in FIG. 4, the fluorescence intensity value (F) of the solution at 630 nm630) The maximum is reached at a urease solution concentration of 50 mg/mL.
Example 3:
sequentially adding 5 mL of NaOH solution with the concentration of 0.2-2 moL/L and 20 mL of chloroauric acid with the concentration of 10 mmoL/L into 20 mL of freshly prepared urease solution with the concentration of 50 mg/mL, uniformly mixing, and then placing in a constant-temperature water bath at 37 ℃ for incubation for 8 hours. After the reaction is finished, the reaction liquid is dialyzed by a dialysis bag with the molecular weight of 7000Dialyzing and purifying for 48 hours to obtain the urease-gold nanocluster fluorescent material aqueous solution. As shown in FIG. 5, the fluorescence intensity value (F) of the solution at 630 nm630) An equilibrium was reached at a sodium hydroxide solution concentration of 1 moL/L.
Example 4:
sequentially adding 5 mL of NaOH solution with the concentration of 1 moL/L and 20 mL of chloroauric acid with the concentration of 10 mmoL/L into 20 mL of freshly prepared urease solution with the concentration of 50 mg/mL, uniformly mixing, and then placing in a 37 ℃ constant-temperature water bath for incubation for 0-12 hours (0 in 0-12 hours is more than 0 hour, and can be 30 seconds, namely 30 seconds-12 hours). And after the reaction is finished, dialyzing and purifying the reaction solution for 48 hours by using a dialysis bag with the molecular weight of 7000 to obtain the urease-gold nanocluster fluorescent material aqueous solution. As shown in FIG. 6, the fluorescence intensity value (F) of the solution at 630 nm630) The maximum is reached at a reaction time of 8 hours.
Example 5:
5 mL of NaOH solution with the concentration of 1 moL/L and 20 mL of chloroauric acid with the concentration of 10 mmoL/L are sequentially added into 20 mL of freshly prepared urease solution with the concentration of 50 mg/mL, mixed evenly and placed in a constant-temperature water bath at 37 ℃ for incubation for 8 hours. And after the reaction is finished, dialyzing and purifying the reaction solution for 48 hours by using a dialysis bag with the molecular weight of 7000 to obtain the urease-gold nanocluster fluorescent material aqueous solution. The resulting solution was drop coated onto a copper mesh. Transmission electron microscopy analysis (see fig. 7) showed that the particle size of the urease-gold nanoclusters was about 2.4 nm. Energy scatter X-ray spectroscopy (see fig. 8) indicated that the product contained gold.
Example 6:
5 mL of NaOH solution with the concentration of 1 moL/L and 20 mL of chloroauric acid with the concentration of 10 mmoL/L are sequentially added into 20 mL of freshly prepared urease solution with the concentration of 50 mg/mL, mixed evenly and placed in a constant-temperature water bath at 37 ℃ for incubation for 8 hours. And after the reaction is finished, dialyzing and purifying the reaction solution for 48 hours by using a dialysis bag with the molecular weight of 7000 to obtain the urease-gold nanocluster fluorescent material aqueous solution. The obtained solution was subjected to fluorescence lifetime measurement, and the fluorescence lifetime value of the urease-gold nanocluster was measured to be 1.74. mu.s (see FIG. 9).
Example 7:
5 mL of NaOH solution with the concentration of 1 moL/L and 20 mL of chloroauric acid with the concentration of 10 mmoL/L are sequentially added into 20 mL of freshly prepared urease solution with the concentration of 50 mg/mL, mixed evenly and placed in a constant-temperature water bath at 37 ℃ for incubation for 8 hours. And after the reaction is finished, dialyzing and purifying the reaction solution for 48 hours by using a dialysis bag with the molecular weight of 7000 to obtain the urease-gold nanocluster fluorescent material aqueous solution. The resulting solution was freeze-dried to obtain a powder, and the obtained powder was subjected to X-ray photoelectron spectroscopy to show 4f peaks at 84.4 eV and 88.1 eV for cash and 2p peaks at 162.9 eV and 163.8 eV for sulfur (see FIG. 10, A is a 4f diagram for gold and B is a 2p diagram for sulfur).
Example 8:
5 mL of NaOH solution with the concentration of 1 moL/L and 20 mL of chloroauric acid with the concentration of 10 mmoL/L are sequentially added into 20 mL of freshly prepared urease solution with the concentration of 50 mg/mL, mixed evenly and placed in a constant-temperature water bath at 37 ℃ for incubation for 8 hours. And after the reaction is finished, dialyzing and purifying the reaction solution for 48 hours by using a dialysis bag with the molecular weight of 7000 to obtain the urease-gold nanocluster fluorescent material aqueous solution. After the obtained solution was stored in a dark place at 4 ℃ for one month, the relative fluorescence intensity of the urease-gold nanoclusters was 85.7% (fig. 11).
Example 9:
5 mL of NaOH solution with the concentration of 1 moL/L and 20 mL of chloroauric acid with the concentration of 10 mmoL/L are sequentially added into 20 mL of freshly prepared urease solution with the concentration of 50 mg/mL, mixed evenly and placed in a constant-temperature water bath at 37 ℃ for incubation for 8 hours. And after the reaction is finished, dialyzing and purifying the reaction solution for 48 hours by using a dialysis bag with the molecular weight of 7000 to obtain the urease-gold nanocluster fluorescent material aqueous solution. After the obtained solution is placed in 0.2-2 mol/L sodium chloride solution for incubation for 30 minutes, the relative fluorescence intensity of the urease-gold nanoclusters is kept above 90% (fig. 12).
Example 10:
5 mL of NaOH solution with the concentration of 1 moL/L and 20 mL of chloroauric acid with the concentration of 10 mmoL/L are sequentially added into 20 mL of freshly prepared urease solution with the concentration of 50 mg/mL, mixed evenly and placed in a constant-temperature water bath at 37 ℃ for incubation for 8 hours. And after the reaction is finished, dialyzing and purifying the reaction solution for 48 hours by using a dialysis bag with the molecular weight of 7000 to obtain the urease-gold nanocluster fluorescent material aqueous solution. After incubating the resulting solution in a 10% human serum solution for 30 minutes, the relative fluorescence intensity of the urease-gold nanoclusters was 90.0% (fig. 13).

Claims (6)

1. A preparation method of urease-gold nanocluster fluorescent material is characterized by comprising the following reaction steps: sequentially adding a sodium hydroxide solution and a chloroauric acid solution with the concentration of 0.2-2.0 mol/L into a urease solution with the concentration of 0-50 mg/mL, uniformly mixing, placing in a 37 ℃ water bath for constant-temperature reaction for 0-12 hours, after the reaction is finished, dialyzing and purifying the reaction liquid by using a dialysis bag with the molecular weight cutoff of 7000 to obtain a urease-gold nanocluster fluorescent material aqueous solution, freeze-drying the urease-gold nanocluster fluorescent material aqueous solution to obtain urease-gold nanocluster fluorescent material powder, wherein urease is used as a stabilizer and a reducing agent to control the formation of gold nanoclusters, and the volume ratio of the sodium hydroxide solution to the chloroauric acid solution to the urease solution is 1: 4: 4, the total volume of the three components is 45 mL.
2. The method for preparing the urease-gold nanocluster fluorescent material as claimed in claim 1, wherein the concentration and volume of the sodium hydroxide solution used are 1 mol/L and 5 mL, the concentration and volume of the chloroauric acid solution are 10 mmoL/L and 20 mL, the concentration and volume of the urease solution are 50 mg/mL and 20 mL, the reaction time is 8 hours, and the reaction temperature is 37 ℃.
3. A urease-gold nanocluster fluorescent material prepared by the method of any one of claims 1-2, which is characterized in that the urease-gold nanocluster fluorescent material aqueous solution is light brown, a characteristic absorption peak of protein appears at 280 nm in an ultraviolet-visible spectrum, strong red fluorescence is generated under the irradiation of an ultraviolet lamp, the maximum excitation wavelength and the emission wavelength are 490 nm and 630 nm respectively, the quantum yield is 17%, and the fluorescence lifetime is 1.74 mu s.
4. The urease-gold nanocluster fluorescent material as claimed in claim 3, wherein the relative fluorescence intensity is kept above 85% after the urease-gold nanocluster fluorescent material aqueous solution is placed in a dark place at 4 ℃ for one month.
5. The urease-gold nanocluster fluorescent material as claimed in claim 3, wherein the relative fluorescence intensity is kept above 90% after the urease-gold nanocluster fluorescent material aqueous solution is placed in 0.2-2 mol/L sodium chloride solution for incubation for 30 minutes.
6. The urease-gold nanocluster fluorescent material as claimed in claim 3, wherein the relative fluorescence intensity is kept above 90% after the urease-gold nanocluster fluorescent material aqueous solution is placed in 10% human serum and incubated for 30 minutes.
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