CN109202098B

CN109202098B - Preparation method of keratin composite silver nanocluster

Info

Publication number: CN109202098B
Application number: CN201811063612.XA
Authority: CN
Inventors: 王金杰; 周永泉
Original assignee: Shanghai University of Engineering Science
Current assignee: Shanghai University of Engineering Science
Priority date: 2018-09-12
Filing date: 2018-09-12
Publication date: 2022-04-01
Anticipated expiration: 2038-09-12
Also published as: CN109202098A

Abstract

The present invention relates to a preparation method of keratin composite silver nano-cluster, which adopts the following steps: (1) mixing keratin solution and silver nitrate solution and adjusting the pH value; (2) adding sodium borohydride solution and allowing to stand for reaction for 30 minutes; (3) The reaction is terminated by a purification method to prepare fluorescent silver nanoclusters. Compared with the prior art, the present invention has the advantages of easily available and cheap raw materials, simple system, short synthesis time and good reproducibility. The prepared material has good biocompatibility and strong and stable fluorescence properties.

Description

Preparation method of keratin composite silver nanocluster

Technical Field

The invention belongs to the field of preparation of fluorescent nanoclusters, and particularly relates to a preparation method of a keratin composite silver nanocluster.

Background

Silver nanoclusters have gained much attention in recent years in the fields of catalysis, chemical sensors, electronics, and bioimaging due to their unique physical, electrical, and optical properties. Silver nanoclusters have a lower production cost than gold nanoclusters. The silver nanoclusters with high quantum yield, near-infrared emission wavelength and high biocompatibility have great application prospects in the field of biological imaging. The biomineralization method is a green and environment-friendly synthetic method which is started in the last decade, and the reaction principle is that groups such as amine, carboxyl, thiol group and the like in a protein or nucleic acid sequence are used as a stabilizer and a reducing agent in a synthetic reaction to replace the traditional chemical reagent. At present, nucleic acid sequences and bovine serum albumin are two commonly used biological raw materials, but the raw materials have the problem of higher cost, and the bovine serum albumin also needs to be pretreated in the synthesis process and has more complex steps; in addition, the existing method reduces the reaction speed by using a low-concentration reducing agent in the reaction so as to be convenient for terminating the reaction by adopting a method which needs long-time purification, such as dialysis and the like, and the time required by the whole reaction is more than 24 hours.

Keratin is ubiquitous in human and animal hair, and can be extracted from animal hair in laboratory by Soxhlet extraction methodThe keratin is extracted, and the raw materials are easy to obtain and are low in cost; the keratin polypeptide has a complete chain segment structure and functional groups such as hydroxyl, carboxyl, amino, disulfide bond and the like which can form stronger acting force with metal cations; however, for silver clusters, the protein with large molecular weight is used as a template for synthesis, and the current literature is few, mainly because the content of sulfydryl in general protein is not enough, and in addition, NaBH is used as a main reason₄The time for participating in the reaction system is difficult to control.

Chinese patent CN108031857A discloses a method for preparing red fluorescence-emitting gold nanoclusters, which comprises preparing a chloroauric acid solution and a keratin solution respectively, slowly adding the chloroauric acid solution into the keratin solution at room temperature, and then fully stirring to uniformly mix the solutions to obtain a light yellow solution; adjusting the pH value of the solution to 8-12; incubating the solution at 25-60 ℃ to obtain a brown gold nanocluster solution; and pouring the gold nanocluster solution into a dialysis bag with the molecular weight cutoff for dialysis, and then freezing and drying to obtain the gold nanocluster particles. The silver nanoclusters and the gold nanoclusters belong to noble metal nanoclusters, and the metal clusters with red fluorescence can be used as fluorescent probes. Compared with gold nanoclusters, a reducing agent is needed in the synthesis process of the silver nanoclusters, and the synthesis process is relatively complex; but the synthesis cost of the silver nanoclusters is lower.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a preparation method of keratin composite silver nanoclusters with good biocompatibility and strong and stable fluorescence properties.

The purpose of the invention can be realized by the following technical scheme:

a preparation method of keratin composite silver nanoclusters comprises the following steps:

(1) stirring and mixing the keratin solution and the silver nitrate solution for 5 minutes and continuing to react for 15 minutes after adjusting the pH value;

(2) adding a sodium borohydride solution, and standing for reaction for 30 minutes;

(3) and terminating the reaction by a purification method to prepare the fluorescent silver nanocluster.

In the step (1):

the reaction temperature is controlled to be 0-40 deg.C, and 25 deg.C is preferably selected from 0 deg.C, 25 deg.C and 40 deg.C.

The mass ratio of the silver nitrate to the keratin is 1:6.59-1:39.54, and the preferred ratio is 1: 13.18.

Adding 0.1-10mol/L sodium hydroxide solution, preferably 1mol/L sodium hydroxide to adjust pH value to 12.4.

The keratin is extracted and purified from the feather by the following steps: thoroughly washing the feathers with water, degreasing the feathers with acetone, immersing the degreased and crushed feathers into a mixed solution containing 8mol/L urea, 0.2mol/L sodium dodecyl sulfate and 0.5mol/L sodium metabisulfite, filtering keratin degradation liquid to remove insoluble substances after heating treatment, putting the keratin degradation liquid into a dialysis bag with the cut-off size of 8000-14000Da for dialysis, and finally freeze-drying the keratin solution into powder.

In the step (2), the molar ratio of silver nitrate to sodium borohydride is 1:0.1-1:1, and the preferable ratio is 1: 1.

And (3) performing column purification by using a chromatographic column with Sephadex G25 gel as a filler, and judging the position of the fluorescent silver nanocluster in the chromatographic column by using an ultraviolet lamp, wherein the process takes about 10 minutes.

The size of the prepared silver nanocluster is 2.5nm, strong fluorescence emission is achieved at 710nm, and the fluorescence quantum yield is 1.7% (with rhodamine B as a contrast). The prepared material has good biocompatibility and strong and stable fluorescence.

Compared with the prior art, the invention has low cost and good biocompatibility; the invention adopts a gel column-passing method to quickly remove the reducing agent micromolecules in the reaction system and accurately regulate and control the termination time of the reaction, thereby using high-concentration NaBH in the reaction₄The reaction speed is accelerated, the synthesis time of the method only needs 1 hour, the operation is simple, and the repeatability is good; based on abundant sulfydryl in keratin, the silver nanocluster synthesized by the method has near-infrared emission wavelength, higher quantum yield, stable fluorescence intensity in aqueous solution and huge application in biological imagingAnd (4) foreground.

The method for synthesizing the silver nanoclusters by using the keratin as the template has the advantages of good biocompatibility, excellent fluorescence performance, good stability, simplicity, rapidness and lower cost by virtue of the unique advantage of abundant disulfide bonds of the keratin and the characteristic that the gel column chromatography can quickly terminate the reaction.

Drawings

Fig. 1 is a schematic diagram of a transmission electron microscope and a particle size distribution of silver nanoclusters synthesized in example 1 of the present invention.

Fig. 2 is an absorption spectrum of the silver nanoclusters synthesized in example 1 of the present invention.

FIG. 3 is a graph showing the fluorescence stability of silver nanoclusters synthesized in example 1 of the present invention.

Detailed Description

The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the spirit of the invention. All falling within the scope of the present invention.

Raw materials of reagents

The solvent used in the reaction is ultrapure water; sodium hydroxide, sodium borohydride (96%), nitric acid, phosphoric acid (95%), hydrogen peroxide (30%) were purchased from national pharmaceutical group chemicals, ltd; silver nitrate (99.8%) was purchased from shanghai Lingfeng Chemicals, ltd; keratin is extracted from feather by itself.

The fluorescence spectra were recorded by a fluorescence spectrometer (Edinburgh, England FS-5); the UV-vis absorption spectrum was measured with a UV-2450 spectrophotometer (Shimadzu, Japan); transmission Electron Microscopy (TEM) was performed under a JEOL JEM-2100 ultra-high resolution transmission electron microscope.

Preparation of the solutions used:

(1) preparation of keratin:

the keratin is automatically extracted and purified from the feathers by a laboratory, and the specific purification steps are as follows: the feathers were thoroughly washed with water and degreased with acetone. 10g of degreased and crushed feather is immersed into 100mL of solution containing 8mol/L urea, 0.2mol/L sodium dodecyl sulfate and 0.5mol/L sodium metabisulfite. The above solution was heated to 70 ℃ for 1 hour. Then filtering the keratin degradation solution to remove insoluble substances, and then putting the keratin degradation solution in a dialysis bag with the cut-off size of 8000-14000Da for dialysis. Finally, the keratin solution is freeze-dried into powder for later use.

(2) Preparation of the solutions used:

0.0849g of silver nitrate solid is weighed to prepare 5mL of silver nitrate solution (0.1mol/L), and keratin is prepared into 5 parts of ultrapure water with the concentration of 5-30 mg/mL; weighing 0.4000g of sodium hydroxide solid to prepare 1mL of sodium hydroxide (10mol/L) solution, and diluting the solution by two parts of 1mL of sodium hydroxide (1mol/L) and 1mL of sodium hydroxide (0.1 mol/L); 0.1892g of sodium borohydride solid is weighed and prepared into 5mL of sodium borohydride solution (1mol/L) by using 1mol/L of sodium hydroxide solution, and then the sodium borohydride solution is diluted into 0.1mol/L sodium borohydride solution and 0.01mol/L sodium borohydride solution respectively by using 0.1mol/L sodium hydroxide solution and 0.01mol/L sodium borohydride solution.

Example 1

Fully reacting 5mL of keratin solution (10mg/mL) with 0.2mL of silver nitrate solution (0.1mol/L) for 5 minutes at room temperature under the stirring state, adding 0.15mL of sodium hydroxide solution (1mol/L), stirring for reacting for 15 minutes, adding 0.23mL of sodium borohydride solution (0.1mol/L) into the solution, uniformly mixing, standing for 30 minutes, stopping the reaction by a Sephadex-G25 gel chromatography column purification method to obtain a stable product silver nano cluster, preparing 1mg/mL of silver nano cluster, exciting by exciting light of 400nm, and measuring that the fluorescence at 710nm is strongest.

Example 2

At room temperature and in a stirring state, 5mL of keratin solution (5mg/mL, 7.5mg/mL, 15mg/mL and 30mg/mL) and 0.2mL of silver nitrate solution (0.1mol/L) are fully reacted for 5 minutes, 0.15mL of sodium hydroxide solution (1mol/L) is added, stirring is carried out for 15 minutes, 0.23mL of sodium borohydride solution (0.1mol/L) is added into the solution, the mixture is uniformly mixed and then stands for 30 minutes, the reaction is stopped by a Sephadex-G25 gel chromatography column purification method, and the product silver nanocluster is obtained, 1mg/mL of silver nanocluster is prepared and is excited by exciting light with 400nm, and the measured light intensity is respectively 0.12, 0.65, 0.70 and 0.49 times of the product in the example 1.

Example 3

Fully reacting 5mL of keratin solution (10mg/mL) with 0.2mL of silver nitrate solution (0.1mol/L) for 5 minutes at room temperature under the stirring state, adding 0.15mL of sodium hydroxide solution (0.1mol/L and 10mol/L) to stir for 15 minutes, adding 0.23mL of sodium borohydride solution (0.1mol/L) to the solution, uniformly mixing, standing for 30 minutes, stopping the reaction by a Sephadex-G25 gel chromatography column purification method to obtain a stable product silver nano cluster, preparing 1mg/mL of silver nano cluster, exciting by 400nm exciting light, and measuring the light intensity to be 0.00 and 0.19 times of that of the product in the example 1.

Example 4

Fully reacting 5mL of keratin solution (10mg/mL) with 0.2mL of silver nitrate solution (0.1mol/L) for 5 minutes at room temperature under the stirring state, adding 0.15mL of sodium hydroxide solution (1mol/L), stirring for reacting for 15 minutes, adding 0.23mL of sodium borohydride solution (0.01mol/L) into the solution, uniformly mixing, standing for 30 minutes, stopping the reaction by a Sephadex-G25 gel chromatography column purification method to obtain a stable product silver nano cluster, preparing 1mg/mL of silver nano cluster, exciting by exciting light of 400nm, and measuring the light intensity to be 0.70 times of that of the product of example 1.

Example 5

Under the condition of stirring at 0 ℃ and 40 ℃, 5mL of keratin solution (10mg/mL) and 0.2mL of silver nitrate solution (0.1mol/L) are fully reacted for 5 minutes, 0.15mL of sodium hydroxide solution (1mol/L) is added, stirring is carried out for 15 minutes, 0.23mL of sodium borohydride solution (0.1mol/L) is added into the solution, the mixture is uniformly mixed and stands for 30 minutes, the reaction is stopped by a Sephadex-G25 gel chromatographic column purification method, and then a stable product silver nano cluster is obtained, 1mg/mL of silver nano cluster is prepared, excitation light with 400nm is used, and the measured light intensity is respectively 0.36 times and 0.23 times of that of the product in the example 1.

Example 6

Under the condition of room temperature and stirring, 5mL of keratin solution (10mg/mL) and 0.2mL of silver nitrate solution (0.1mol/L) are fully reacted for 5 minutes, 0.15mL of sodium hydroxide solution (1mol/L) is added, stirring is carried out for 15 minutes, 0.23mL of sodium borohydride solution (0.1mol/L) is added into the solution, the mixture is uniformly mixed and stands for 30 minutes, the reaction is stopped by a purification method (ultrapure water and BR buffer solution with pH equal to 6), and stable product silver nanoclusters are obtained, 1mg/mL of silver nanoclusters are prepared, and the stable product silver nanoclusters are excited by exciting light with the wavelength of 400nm, and the measured light intensity is respectively 0.50 times and 0.67 times of that of the product of the example 1.

FIG. 1 is a schematic transmission electron microscope and a particle size distribution (2.53. + -. 0.54nm) of the silver nanoclusters synthesized in example 1.

FIG. 2 is an absorption spectrum of the silver nanoclusters synthesized in example 1, wherein line 1 is an ultraviolet absorption spectrum of a keratin solution of 1 mg/mL; line 2 is the ultraviolet absorption spectrum of the silver nanocluster at 1 mg/mL; line 3 is the fluorescence absorption spectrum of 1mg/mL silver nanoclusters. As a result, no large-size particle size existed in the silver nanocluster, and the maximum fluorescence emission peak position was 710nm at an excitation light of 400 nm.

Fig. 3 is a graph representing the fluorescence stability of the silver nanoclusters synthesized in example 1. Wherein, the line 1 and the line 2 are fluorescence intensities at the end of synthesis and after three months of storage, respectively. The fluorescence intensity of the silver nanocluster can be maintained at more than 80% after three months, which shows the stability of the silver nanocluster synthesized by the method.

The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes and modifications may be made by one skilled in the art within the scope of the appended claims without departing from the spirit of the invention.

Claims

1. a preparation method of keratin composite silver nano-cluster, is characterized in that, the method adopts the following steps:

(1) mix keratin solution and silver nitrate solution and adjust pH value; The mass ratio of described silver nitrate and described keratin is 1: (6.59-39.54);

(2) Add sodium borohydride solution and let stand for reaction for 30 min; the molar ratio of silver nitrate to sodium borohydride is 1: (0.1-1);

(3) The reaction is terminated by a purification method to prepare fluorescent silver nanoclusters;

Among them, the purification method adopts a chromatography column with Sephadex G25 gel as a filler to carry out column purification, and uses an ultraviolet lamp to determine the position of the fluorescent silver nanoclusters in the chromatography column.

2 . The method for preparing keratin composite silver nanoclusters according to claim 1 , wherein the reaction temperature is controlled to be 0-40° C. in step (1). 3 .

3 . The method for preparing keratin composite silver nanoclusters according to claim 1 , wherein in step (1), sodium hydroxide solution is added to adjust the pH value. 4 .

4. the preparation method of a kind of keratin composite silver nanocluster according to claim 3, is characterized in that, the concentration of described sodium hydroxide solution is 0.1-10mol/L.

5 . The method for preparing a keratin composite silver nanocluster according to claim 1 , wherein the pH value is adjusted to 12.4 in step (1). 6 .

6 . The method for preparing a keratin composite silver nanocluster according to claim 1 , wherein in step (1), keratin is extracted and purified from feathers, and the following steps are adopted: thoroughly wash the feathers with water, and use Acetone is used for degreasing operation, and the degreasing and pulverized feathers are immersed in a mixed solution of urea, sodium lauryl sulfate and sodium metabisulfite. Dialysis was performed in Da's dialysis bag, and finally the keratin solution was lyophilized to a powder.