CN111438368B - Gold-platinum bimetallic nanoenzyme prepared from silk fibroin solution and application thereof - Google Patents
Gold-platinum bimetallic nanoenzyme prepared from silk fibroin solution and application thereof Download PDFInfo
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Abstract
The invention relates to the field of synthesis of nano materials, in particular to a method for preparing gold-platinum bimetallic nanoenzyme by utilizing a silk fibroin solution in a green and mild way and application of the gold-platinum bimetallic nanoenzyme in catalysis, medicine, sensing and other aspects, which specifically comprises the following steps: (1) in-situ crystallization is carried out in the regenerated silk fibroin aqueous solution to generate the gold-platinum bimetallic nano enzyme. (2) The characteristics show that the surface of the prepared bimetallic nanoenzyme simultaneously forms a protein crown formed by silk fibroin, so that the composite nanomaterial has excellent biocompatibility and certain drug-loading capacity. (3) Experiments show that the composite nano enzyme can simulate the activities of oxidase, glucose oxidase and peroxidase in vivo and in vitro. Meanwhile, the nano-particles have certain photothermal response and can be used for photoacoustic imaging in biological tissues. Has great application potential in the biomedical field, the catalytic sensing field and other social and economic fields.
Description
Technical Field
The invention relates to the field of synthesis of nano materials, in particular to a method for preparing gold-platinum bimetallic nano enzyme by utilizing a silk fibroin solution in a green and mild manner and application of the gold-platinum bimetallic nano enzyme in catalysis, medicines, sensing and other aspects.
Background
As enzymes, biologically active substances having catalytic action, such as proteins and nucleic acids, have been thought to be the first. In 1926, biochemist James B. Sumner doctor discovered the first enzyme (urease) and confirmed it to be a protein molecule. Since then, proteins have been identified as a material constituent of all enzymes until nuclease class was discovered in 1982. However, these natural enzymes consisting of bioactive substances are present in very low amounts in the living body, are difficult to obtain in large quantities and are therefore expensive. Furthermore, natural enzymes have poor stability, and protease is inactivated by changes in pH or temperature. To overcome these limitations, research and development of stable and low-cost synthetic artificial mimic enzymes are receiving increasing attention. Nanoenzymes are an important class of these. The emergence of the nano-enzyme changes the traditional concept that people think that the inorganic nano-material is a biological inert substance in the past, reveals the inherent biological effect and new characteristics of the nano-material, enriches the research of the mimic enzyme, expands the research from organic compounds to inorganic nano-materials and expands the application range of the nano-materials. In view of the fact that the nano-enzyme has high catalytic activity of natural enzyme and characteristics of simulating enzyme stability and economy, research on the nano-enzyme rapidly rises since the report of HRP enzyme activity nano-enzyme in 2007, and the research related range is gradually wide, and the research includes different fields of material science, physics, chemistry, biology, medicine, environment and the like.
A distinguishing feature of biomineralization from general mineralization is the interaction at the interface by organic macromolecules and inorganic ions. The crystallization and growth of inorganic mineral phase are controlled from molecular level, so that the biomineral has special hierarchical structure and assembly mode. Recent studies have shown that the control of biomineralization by organisms is a complex, multi-layered process, in which the arrangement of biomacromolecules and their long-lasting action with inorganic mineral phases are two major aspects of the biomineralization process. The mineralization process in organisms is generally considered to be divided into four stages.
(1) Pre-organization of organic matter: insoluble organic matter in organisms forms an organized micro-reaction environment before mineral deposition, and the environment determines the nucleation position of inorganic matters and the function of mineral formation. This stage is a prerequisite for biomineralization to proceed.
(2) Interface molecule recognition: under the control of the formed organic macromolecular assemblies, inorganic substances nucleate at organic-inorganic interfaces in solution through electrostatic force action, chelation, hydrogen bonding, van der waals force and the like. Molecular recognition is a process with specific functions that control the nucleation, growth, and aggregation of crystals.
(3) Growth and modulation: in the process of inorganic mineral phase growth, the form, size, orientation and structure of the crystal are regulated and controlled by organism organic matters, and the subunit is obtained through preliminary assembly. This stage imparts unique structure and morphology to biomineralization through chemical vector regulation.
(4) And (3) epitaxial growth: with the participation of cells, subunits are assembled to form a multi-stage structure of biogenic minerals. This stage is the main reason for the difference between natural biomineralization materials and artificial materials. But also the final modification stage of complex hyperfine structures in cell activities.
Biomineralization is a complex dynamic process and is comprehensively regulated and controlled by biological organic matters, a crystal growth mechanism and the external environment. The establishment of a biomimetic mineralization model and the deep research of a related mechanism provide a theoretical basis for synthesizing inorganic materials in organic components, and further utilizing the mechanical property research of biogenic minerals to prepare the biomimetic materials with high fracture toughness and high strength.
Disclosure of Invention
The multifunctional bimetallic nano-enzyme is synthesized by taking regenerated silk fibroin solution, chloroauric acid and chloroplatinic acid as raw materials, has the multienzyme activities of peroxidase-like enzyme, oxidase-like enzyme and glucose oxidase-like enzyme, and the effects of consuming glutathione, loading drugs and performing photoacoustic imaging, and has the advantages of simple preparation process and particularly mild reaction conditions.
The technical scheme of the invention is as follows:
1. the method for preparing the gold-platinum bimetallic nanoenzyme by utilizing the silk fibroin solution is characterized by comprising the following steps:
(1) separating and purifying silk fibroin from silkworm cocoon to prepare a stable regenerated silk fibroin solution;
(2) adding HAuCl into the regenerated silk fibroin solution obtained in the step (1) under rapid magnetic stirring 4 And chloroplatinic acid H 2 PtCl 6 Immediately adjusting the pH value of the system to 3 by using a nitric acid solution, slowly crystallizing for 12 hours under uniform stirring, and obtaining a purple nano particle aqueous solution after the reaction is finished; putting the nano particle solution into a dialysis bag for dialysis for 12 hours to remove unreacted ions, and obtaining the composite bimetallic nano enzyme which is remained in the dialysis bag.
Further, the regenerated silk fibroin solution in the step (1) is extracted by putting the silkworm cocoons into a boiling 0.5% sodium carbonate solution according to the proportion of 40 grams per liter, and boiling for 30 minutes to remove sericin; the remaining silk fibroin was then dissolved at 90 degrees celsius and contained calcium chloride: ethanol: the molar ratio of water is 1: 2: 8, keeping the temperature of 90 ℃ in a water bath for two hours to fully dissolve the solution; and carrying out suction filtration and dialysis on the dissolved silk fibroin solution, and purifying to obtain a clear regenerated silk fibroin solution.
The concentration of the regenerated silk fibroin solution in the step (2) is 4 mg-mL -1 The concentration of the chloroauric acid and the chloroplatinic acid is 6 mM, and the volume ratio of the regenerated silk fibroin solution to the chloroauric acid and the chloroplatinic acid is 2:1: 1; the concentration of nitric acid used for adjusting the pH value is 1.5 wt%, and the molecular weight cut-off of the dialysis bag is 3500 kDa.
The gold-platinum bimetallic nanoenzyme has oxidase activity for catalyzing oxygen to generate superoxide anions, peroxidase activity for catalyzing hydrogen peroxide to generate hydroxyl radicals, glucose oxidase activity for oxidizing glucose to generate gluconic acid and hydrogen peroxide, and enzyme activity for consuming glutathione, and is used for performing photoacoustic imaging contrast under a near-infrared thermal imager and a photoacoustic imaging system; and the imaging contrast of the nano enzyme is enhanced by utilizing the drug loading capacity of the fibroin crown on the surface of the bimetal nano enzyme and carrying a near infrared dye Cy7.5.
2. The invention has the main advantages that:
the gold-platinum bimetallic nanoenzyme with good dispersibility and uniform appearance is prepared in a green and mild way in a regenerated silk fibroin solution by utilizing a crystallization method inspired by biomineralization. The artificial nano enzyme has strong catalytic activity, and can spontaneously catalyze oxygen, glucose and over-expressed hydrogen peroxide in a tumor region to generate active oxygen without stimulation of external light, electricity, heat and other conditions. The method can be used for enhancing oxidative stress in a tumor region so as to play a role in tumor treatment, and can also be used in the fields of catalysis, sensing and detection. The fibroin protein corona on the surface of the nano enzyme can play a role of a drug carrier and efficiently load various drugs or fluorescent molecules. In addition, the nano enzyme has the common inductive coupling photothermal conversion capability of inorganic non-metallic materials, and can be used as a photothermal adjuvant and a photoacoustic imaging contrast agent. Has wide application prospect in a plurality of fields such as catalysis, medicine, sensing and the like.
Drawings
In order to make the purpose, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings:
FIG. 1 is a schematic diagram of the synthesis of a gold-platinum bimetallic nanoenzyme in example 1 of the present invention.
Fig. 2 is a photograph of the regenerated silk fibroin solution and the nanoenzyme solution in example 1 of the present invention.
FIG. 3 is a transmission electron microscope image of the gold-platinum bimetallic nanoenzyme in example 1 of the present invention.
FIG. 4 is the scanning electron microscope image of thermal field emission of the gold-platinum bimetallic nanoenzyme in example 1 of the present invention.
FIG. 5 is a dynamic light scattering hydrated particle size distribution diagram of the gold-platinum bi-metal nanoenzyme in example 1 of the present invention.
FIG. 6 is the EDS energy spectrum of the gold-platinum bi-metal nanoenzyme in example 1 of the present invention.
FIG. 7 is the ESR energy spectrum of the activity of the gold-platinum bi-metal nanoenzyme in example 1 of the present invention.
FIG. 8 is a photo-acoustic image of the gold-platinum bimetallic nanoenzyme in the tumor-bearing mouse in example 1 of the present invention.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1 preparation of gold-platinum bimetallic nanoenzyme by using silk fibroin solution and application thereof
The synthetic flow chart of the gold-platinum bimetallic nanoenzyme is shown in figure 1, and comprises the following preparation steps:
(1) extracting silk fibroin: the silkworm cocoon is cut open along the symmetry axis by scissors, the silkworm pupa is taken out, and then the silkworm cocoon is cut into one eighth of small pieces. 80 g of silkworm cocoon pieces are added to 2L of 0.5% sodium carbonate solution and boiled for 30 minutes in a boiling state. The sodium carbonate solution was then decanted and the silk fibroin was repeatedly rinsed with secondary water. And (3) placing the washed silk fibroin in a blast drying oven at 35 ℃ for drying for 12 hours.
(2) Re-solubilization of silk fibroin: the molar ratio of the raw materials is 1: 2: and 8, preparing 50 g of calcium chloride, ethanol and water solution, adding 2 g of the silk fibroin prepared in the step (1) into the solution, and heating the solution in a water bath kettle at the temperature of 90 ℃ for 2 hours to fully dissolve the protein. Then, the residue was removed by suction filtration and dialyzed in a 3500 kDa dialysis bag for three days.
(3) Preparing the bimetallic nano enzyme: adding 5 ml of chloroauric acid (6 mmol) and 5 ml of chloroplatinic acid (6 mmol) into 10 ml of regenerated silk fibroin solution with the concentration of 4 mg per ml under rapid magnetic stirring, immediately adjusting the pH value of the system to 3 by using a nitric acid solution, slowly crystallizing for 12 hours under uniform stirring, and obtaining a purple nano particle aqueous solution after the reaction is finished. Putting the nano particle solution into a 3500 kDa dialysis bag, dialyzing for 12 hours to remove unreacted ions, and obtaining the composite bimetallic nano enzyme which is remained in the dialysis bag.
Fig. 2 shows the regenerated silk fibroin solution and the nanoenzyme solution, and fig. 3 is a transmission electron microscope image of the nanoenzyme, which shows that the average particle size of the nanoparticles is about 100 nm. The scanning electron microscope image of the nanoenzyme in fig. 4 and the hydrated particle size distribution diagram in fig. 5 show that the nanoenzyme has very uniform particle size and good dispersibility. The EDS spectrogram of figure 6 determines the chemical components and the content of the nanoenzyme, the ESR spectrogram of figure 7 verifies the enzyme catalytic activity of the nanoenzyme, and the tumor-bearing mouse photothermographic image of figure 8 shows the capability of the nanoenzyme as a photoacoustic imaging contrast agent.
Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.
Claims (3)
1. The preparation method for preparing the gold-platinum bimetallic nanoenzyme by utilizing the silk fibroin solution is characterized by comprising the following steps of:
(1) separating and purifying silk fibroin from silkworm cocoon to prepare a stable regenerated silk fibroin solution;
(2) adding HAuCl into the regenerated silk fibroin solution obtained in the step (1) under rapid magnetic stirring 4 And chloroplatinic acid H 2 PtCl 6 Immediately adjusting the pH value of the system to 3 by using a nitric acid solution, slowly crystallizing for 12 hours under uniform stirring, and obtaining a purple nano particle aqueous solution after the reaction is finished; putting the nano particle solution into a dialysis bag for dialysis for 12 hours to remove unreacted ions, and obtaining the compound bimetallic nano enzyme which is remained in the dialysis bag; the concentration of the regenerated silk fibroin solution in the step (2) is 4 mg-mL -1 The concentration of chloroauric acid and chloroplatinic acid is 6 mM, the volume ratio of the regenerated silk fibroin solution to the chloroauric acid to the chloroplatinic acid is 2:1:1, the concentration of nitric acid used for adjusting the pH value is 1.5 wt%, and the molecular weight cut-off of the dialysis bag is 3500 kDa.
2. The method for preparing the gold platinum bimetal nanoenzyme by using the silk fibroin solution according to claim 1, wherein the extraction of the regenerated silk fibroin solution in the step (1) is to boil bombyx mori cocoons in a boiling 0.5% sodium carbonate solution for 30 minutes according to a ratio of 40 g/l to remove sericin; the remaining silk fibroin was then dissolved at 90 degrees celsius and contained calcium chloride: ethanol: the molar ratio of water is 1: 2: 8, keeping the temperature of 90 ℃ in a water bath for two hours to fully dissolve the solution; and carrying out suction filtration and dialysis on the dissolved silk fibroin solution, and purifying to obtain a clear regenerated silk fibroin solution.
3. The method for preparing gold-platinum bimetallic nanoenzyme by using silk fibroin solution according to claim 1, characterized in that the gold-platinum bimetallic nanoenzyme has oxidase activity for catalyzing oxygen to generate superoxide anion, peroxidase activity for catalyzing hydrogen peroxide to generate hydroxyl radical, glucose oxidase activity for oxidizing glucose to generate gluconic acid and hydrogen peroxide, and enzyme activity for consuming glutathione, and is used for photoacoustic imaging radiography under a near-infrared thermal imager and a photoacoustic imaging system; and the imaging contrast of the nano enzyme is enhanced by utilizing the drug loading capacity of the fibroin crown on the surface of the bimetal nano enzyme and carrying a near infrared dye Cy7.5.
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