CN105597159B

CN105597159B - Antibacterial product based on gold nanoparticles and preparation method and application thereof

Info

Publication number: CN105597159B
Application number: CN201610028789.0A
Authority: CN
Inventors: 蒋兴宇; 郑问枢
Original assignee: Beijing Institute of Nanoenergy and Nanosystems
Current assignee: Beijing Institute of Nanoenergy and Nanosystems
Priority date: 2016-01-15
Filing date: 2016-01-15
Publication date: 2020-04-07
Anticipated expiration: 2036-01-15
Also published as: CN105597159A

Abstract

The invention provides an antibacterial product based on gold nanoparticles, which comprises an antibacterial layer and a base material, wherein the antibacterial layer comprises DAPT modified gold nanoparticles, the surface of the base material is provided with negative charges, the antibacterial layer is combined on the surface of the base material through electrostatic action, and the negative charges on the surface of the base material are obtained by treating the base material in a plasma cleaning device.

Description

Antibacterial product based on gold nanoparticles and preparation method and application thereof

Technical Field

The invention relates to an antibacterial product, in particular to an antibacterial product based on gold nanoparticles, and a preparation method and application thereof.

Background

Bacterial infection has been a serious disease threatening the health of human beings since ancient times, and in the 40's of the 20 th century, antibiotics were invented to combat bacterial infection, thereby greatly reducing medical accidents caused by bacterial infection. However, in recent years, with the abuse of antibiotics, some novel multidrug-resistant bacteria have gradually appeared. These bacteria are not killed by the antibiotics currently commercialized, and are called superbacteria. With the advent of superbacteria, bacterial infections again represent a significant threat to human health.

Hospitals naturally produce many superbacteria as one of the most frequently used sites for antibiotics. Recently, many patients die from nosocomial infections in various hospitals worldwide every year, and these nosocomial infections are mostly caused by infections of medical instruments. For nosocomial infections caused by these superbacteria, the most effective method is currently to form an antimicrobial layer on the medical devices so that no bacteria are present on the medical devices, thereby reducing the risk of nosocomial infections caused by the medical devices.

At present, there are many methods for preparing an antibacterial layer on the surface of a solid, and traditional antibiotics can be used as a coating to modify the surface of the solid, but the method can greatly increase the risk of generating new superbacteria. High molecular materials are also widely applied to the antibacterial layer on the solid surface, but the materials generally have the defects of high preparation cost and complex preparation process, and the materials are often high in viscosity, so that the physical properties of the material surface are influenced.

In recent years, due to the vigorous development of nanotechnology, many novel nanomaterials are used for the preparation of antibacterial layers, including some copper nanoparticles, mercury nanoparticles, etc., but these materials are too toxic, limiting their widespread use.

Silver nanoparticles are the only approved nanomaterial by the U.S. drug administration (FDA) for surface modification of medical devices. Many commercial silver nanoparticle-decorated materials are commercially available, including silver-coated catheters and the like. However, in recent years there has been a constant worry about the following problems: silver nanoparticles are also toxic. Silver nanoparticles can continuously release silver ions, causing silver poisoning. Some recent studies have also shown that silver nanoparticles have strong blood coagulation side effects. This is not conducive to the widespread use of silver nanoparticle modified medical devices, such as syringes. In addition, some silver nanoparticle-containing films, coatings, composites, etc. have been disclosed in the prior art, but they do not fundamentally overcome the above toxicity problem of silver nanoparticles, and some suffer from the corresponding disadvantages and limitations due to the use of polymeric materials.

Meanwhile, how to stably attach the nanoparticles having the antibacterial effect to the solid surface is also a problem that needs further research and consideration. Methods currently available include direct coating of the nanopaste, chemical vapor deposition, chemical modification of the substrate, and the like. Among these methods, some nanoparticles have poor adhesion to solid surfaces (e.g., direct coating), some have severe manufacturing conditions (e.g., high temperature or negative pressure is required), and some require additional chemical reagents and reaction time (e.g., chemical modification), which are not advantageous for industrial scale production of antibacterial products having nanoparticles.

In view of the above, there is still a need to develop a surface coating and an antibacterial product with better biocompatibility, low cost and antibacterial activity, especially on multi-drug resistant antibacterial activity.

Disclosure of Invention

Therefore, the present invention is directed to overcome the above-mentioned drawbacks of the prior art, and provides an antibacterial article based on gold nanoparticles, which is convenient to prepare, has a wide application range and better biocompatibility, and also provides a preparation method and an application of the antibacterial article.

The invention provides an antibacterial product based on gold nanoparticles, which comprises an antibacterial layer and a base material, wherein the antibacterial layer comprises 4, 6-diamino-2-mercaptopyrimidine (DAPT) modified gold nanoparticles, the surface of the base material is negatively charged, and the antibacterial layer is combined on the surface of the base material through electrostatic action.

Preferably, the negative charge of the substrate surface is obtained by treating the substrate in a plasma cleaning device.

As mentioned before, in addition to silver nanoparticles, more and more nanomaterials are available for the preparation of the antibacterial layer, including copper nanoparticles, metal oxide nanoparticles. However, these nano materials have the same disadvantage as silver nanoparticles, namely, high toxicity. In addition, some carbon materials including fullerenes, graphene, etc. have also been found to have some antimicrobial activity. However, these particles tend to have the disadvantage of having a low antimicrobial activity. Some nano titanium oxide particles and gold nanorods are also considered to have the prospect of being used as an antibacterial layer, but these nano materials have antibacterial activity only under the auxiliary action of laser.

The gold nanoparticles modified by DAPT can kill multi-drug resistant bacteria in a solution, and the particles can change the potential of a cell membrane, further inhibit the synthesis of ATP of cells and finally kill bacteria. And the gold nanoparticles can not generate Reactive Oxygen Species (ROS) in the process of killing bacteria, so that the gold nanoparticles only cause the smallest possible damage to normal cells and can be ignored.

Theoretically, there are many methods for preparing an antibacterial layer by using the DAPT modified gold nanoparticles, but common coating preparation methods such as a solution synthesis method and an evaporation method are time-consuming and labor-consuming, or have certain requirements on the size and shape of a substrate material. The present inventors have tried many different schemes in order to simply and conveniently prepare a nano-coating that can be modified on the surface of various materials. For example, the present inventors tried to dry the nanoparticle solution directly on the surface of the substrate, but such a coating could not be stably adsorbed on the surface of the solid substrate, and thus the antibacterial activity could be easily lost. Co-spinning of nanoparticles and macromolecules to prepare an antibacterial layer by electrospinning has also been attempted, and we have found that the antibacterial activity of the co-spun nanoparticles is greatly reduced, probably because only the nanoparticles exposed to the surface have antibacterial activity, while many nanoparticles are wrapped inside the nanomaterial during co-spinning.

The inventors found that these DAPT-modified gold nanoparticles exhibit a positive surface potential in solution due to the presence of surface ligands, whereas the surface of the solid phase material (substrate) has a certain negative charge when subjected to surface plasma treatment. Therefore, the gold nanoparticles can perform electrostatic self-assembly with the surface of the treated solid phase material. This action causes the gold nanoparticles to be stably adsorbed on the surface of the solid material, thereby forming a surface coating layer having a certain antibacterial function, i.e., an antibacterial layer. Once bacteria are close to the antibacterial layer, the bacteria are attracted and killed by the coating, so that the antibacterial effect of the solid surface is realized. The above principle can be understood with reference to fig. 1.

The antibacterial product provided by the invention is characterized in that the molar ratio of DAPT to gold element on the gold nanoparticles is 0.1: 1-0.45: 1, preferably 0.45: 1. More preferably, the average particle size of the gold nanoparticles is 1-5 nm, and is preferably 3 nm. DAPT may be purchased from Sigma Aldrich and/or Arcos.

The antibacterial article according to the present invention, wherein the substrate is an organic material or an inorganic material; preferably, the substrate is selected from one or more of glass, silica gel, polyvinyl chloride Plastic (PVC), polystyrene plastic (PS plastic), polycarbonate plastic (PC plastic), polypropylene plastic (PP plastic), and Polydimethylsiloxane (PDMS); more preferably, the substrate is a biological or medical consumable. The biological consumables can include glass tubes, test tubes, centrifuge tubes, cell culture plates (such as 6-hole plates, 12-hole plates, 24-hole plates, 48-hole plates and 96-hole plates) and the like, and the medical consumables can include infusion sets, injectors, infusion pumps, catheters, drainage tubes, respiratory cannulas, stomach tubes, catheters, medical dressings and the like.

The invention also provides a preparation method of the gold nanoparticle-based antibacterial product, which comprises the following steps:

(1) mixing chloroauric acid, DAPT and a surfactant to obtain a mixed solution;

(2) adding a reducing agent into the mixed solution obtained in the step (1) for reduction to obtain a DAPT modified gold nanoparticle solution;

(3) placing the base material in a plasma cleaning device for treatment to obtain the base material with negative charges on the surface;

(4) and (3) immersing the substrate with the negative charges on the surface in the step (3) into the DAPT modified gold nanoparticle solution in the step (2), taking out, cleaning and drying to obtain the antibacterial product.

The preparation method comprises the step (1), wherein the molar ratio of the DAPT to the chloroauric acid in the mixed solution is 5: 1-1: 1, preferably 1: 1. DAPT may be purchased from Sigma Aldrich and/or Arcos.

According to the preparation method of the invention, the surfactant is tween, preferably tween-80. Preferably, the volume content of the surfactant in the mixed solution is 0.1%. More preferably, the reducing agent is sodium borohydride (NaBH)₄) Or sodium ascorbate. More preferably, the molar ratio of the reducing agent to the chloroauric acid is 5:1 to 1:1, and is preferably 3: 1.

The preparation method provided by the invention is characterized in that the reduction reaction time in the step (2) is 1-2 h, and the reaction temperature is 0 ℃. Preferably, the concentration of the DAPT-modified gold nanoparticle solution in step (2) is 0.6 mg/mL. More preferably, step (2) further comprises adding NaCl to the DAPT-modified gold nanoparticle solution. Still more preferably, the concentration of NaCl in the DAPT modified gold nanoparticle solution is 0.125-2M.

According to the preparation method, in the step (3), the time for treating the substrate by the plasma cleaning device is 1-10 min, preferably 4 min. The treatment time of the plasma cleaning device is 15-60 minutes, but in the preparation method of the invention, the plasma treatment time is only 1-10 minutes or preferably 4 minutes, so the required treatment time is shorter. Preferably, the power of the plasma cleaning device is 10-150W, preferably 60W, and the frequency of the plasma generator is 13.56 mHz. More preferably, the plasma cleaning device is an ultrasonic plasma cleaner.

The preparation method provided by the invention is characterized in that the immersion time in the step (4) is 3-15 h, preferably 12 h. Preferably, the washing in step (4) comprises three washes with Phosphate Buffered Saline (PBS) followed by a 30min ultrasonic wash.

The inventor finds that the ultraviolet absorption spectrum of the antibacterial product has an absorption peak at 520nm, and the peak intensity is in positive correlation with the surface density of the gold nanoparticles. The intensity of the absorption peak can be regulated and controlled by adjusting the concentration of the DAPT modified gold nanoparticle solution, the concentration of NaCl in the DAPT modified gold nanoparticle solution and/or the immersion time of the base material in the DAPT modified gold nanoparticle solution, so as to control the surface density of the gold nanoparticles on the base material. For example, it is found from the experiments of the present inventors (see example 2) that when the concentration of NaCl in a DAPT-modified gold nanoparticle solution is 0 to 2M, the absorption peak intensity is in a positive correlation with the concentration. The peak intensity was highest at a NaCl concentration of 2M. In addition, it can be known that the absorption peak intensity is in positive correlation with the concentration of the DAPT modified gold nanoparticle solution, and/or the absorption peak intensity is in positive correlation with the immersion time of the base material in the DAPT modified gold nanoparticle solution. The density of gold nanoparticles in the antibacterial layer is regulated, so that the antibacterial activity and biocompatibility of the antibacterial layer can be indirectly regulated.

The present inventors have also found that gold nanoparticles of about 13nm hardly form a stable antibacterial layer on a substrate. Gold nanoparticles of around 5nm can form a relatively stable coating on the surface of the substrate, but the stability is still not good, for example, after exposure in air for 3 days, the gold nanoparticles can fall off from the surface of the substrate, so that the antibacterial activity of the coating is lost. When the gold nanoparticles reduced to about 3nm are adopted, a more stable coating can be formed on the base material, and the coating still can keep good antibacterial activity after being exposed in air, water, PBS and ethanol for two weeks through tests. Although not wishing to limit the invention, it is speculated that this may be due to the unique conformation of DAPT on gold nanoparticles of this size, which is more favorable for the formation of a tight electrostatic adsorption, and also due to the larger specific surface area of the smaller gold nanoparticles, so that the same volume of gold nanoparticles may carry more DAPT molecules for electrostatic adsorption.

The invention also provides the use of the antibacterial product of the invention or prepared according to the method of the invention in the biological and/or medical field.

Compared with the prior art, the gold nanoparticle-based antibacterial product has the following beneficial effects:

1. the plasma cleaning technology can well treat metal, semiconductor, oxide and most high molecular materials such as polypropylene, polyester, polyimide, polrvinyl chloride, epoxy, even polytetrafluoroethylene and the like without being divided into substrate types, so that the antibacterial layer can be combined on the surfaces of substrates with different shapes and compositions, the application range of the antibacterial product is very wide, the operation is simple and convenient, the requirement on reaction conditions is not high, and the method is suitable for large-scale industrial production. In addition, the substrate is treated by using the plasma cleaning technology, so that the cleaning step of the substrate is saved, and the two purposes are achieved.

2. The antibacterial product has stable property, and the antibacterial layer can stably exist in vacuum condition, air, aqueous solution, organic solvent, PBS and other solutions for at least two weeks.

3. The antibacterial product of the invention shows good multidrug-resistant antibacterial activity, and comprises wild type and multidrug-resistant escherichia coli, pseudomonas aeruginosa, klebsiella pneumoniae and the like which are attracted and killed by the coating after being close to the surface of a solid phase. Therefore, the medical device can be widely applied to various medical devices, such as catheters, syringes, breathing machines and the like, is used for preventing the infection in a bacterial hospital caused by the medical devices, and has wide application prospect.

4. The antibacterial product of the invention has good biocompatibility. After the surfaces of the antibacterial layers are inoculated with cells, the cells have good cell survival rate, the antibacterial layers do not generate blood coagulation phenomenon, and simultaneously have certain anticoagulation effect, so that the toxicity and the defects of the silver-containing material are avoided.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

fig. 1 schematically shows a schematic diagram of the preparation and antibacterial process of an antibacterial article based on gold nanoparticles of the present invention;

fig. 2 shows the gold nanoparticle-based antimicrobial article prepared in example 1 and a comparison (glass tube (a), syringe (B), and catheter (C));

fig. 3 shows a 96-well plate (a) prepared in example 2, and adjustment of the absorption peak intensity (i.e., adjustment of the surface density of gold nanoparticles accordingly) by different concentrations of nacl (b), different concentrations of gold nanoparticle solution (C), and different immersion times (D);

FIG. 4 shows a comparison of gold element content before and after washing with PBS after immersion of the antibacterial 96-well plate of example 3 and the comparative 96-well plate at the highest modification concentration (0.6mg/mL of DAPT-modified gold nanoparticle solution) for 12 hours.

Detailed Description

The invention is further illustrated by the following specific examples, which, however, are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever.

This section generally describes the materials used in the testing of the present invention, as well as the testing methods. Although many materials and methods of operation are known in the art for the purpose of carrying out the invention, the invention is nevertheless described herein in as detail as possible. It will be apparent to those skilled in the art that the materials and methods of operation used in the present invention are well within the skill of the art, provided that they are not specifically illustrated.

Example 1

This example serves to illustrate the gold nanoparticle-based antimicrobial article of the present invention and its preparation.

The substrates selected in this example were glass tubing (made of glass and purchased from beijing koilong corporation), syringe (made of polypropylene and purchased from shanghai zhiyu medical devices, ltd.) and catheter (made of silica gel and purchased from guangzhou wili medical devices, ltd.).

A. The preparation method of the gold nanoparticle-based antibacterial glass tube comprises the following steps:

(1) chloroauric acid, DAPT, and a surfactant were mixed to obtain a mixed solution. Wherein the molar ratio of DAPT to chloroauric acid in the mixed solution is 1: 1. The surfactant is tween-80, and the volume content of the tween-80 in the mixed solution is 0.1 percent.

(2) And (2) adding a reducing agent into the mixed solution obtained in the step (1) for reduction to obtain a DAPT modified gold nanoparticle solution. Wherein the reducing agent is NaBH₄The molar ratio of the gold chloride to the gold chloride acid is 3: 1. The reduction reaction time is 1h, the reaction temperature is 0 ℃, the concentration of the obtained DAPT modified gold nanoparticle solution is 0.6mg/mL, the centrifuged gold nanoparticles are dialyzed for 24 hours by a dialysis bag of 8000-.

(3) And (3) placing the glass tube (substrate) in a plasma cleaning device for treatment to obtain the glass tube with the surface with negative charges. Wherein the used Plasma cleaning device is a Plasma cleaner (model PDC-MG, Chengdu constant technology development Co., Ltd.), and the time for cleaning the glass tube is 4 min. The power of the Plasma scrubber was set at 60W and the frequency of the Plasma generator was 13.56 mHz.

(4) And (3) immersing the glass tube with the surface with negative charges in the step (3) in the DAPT modified gold nanoparticle solution in the step (2) for 12h, taking out, washing with PBS for three times, then ultrasonically washing for 30min, and drying to obtain the gold nanoparticle-based antibacterial glass tube.

As shown in fig. 2(a), the glass tube located at the upper left portion is an antibacterial glass tube prepared, and the glass tube located at the lower right portion is an untreated glass tube of the same specification. The contrast shows that the antibacterial glass tube of the invention is changed into brownish black from colorless and transparent.

B. The preparation steps of the gold nanoparticle-based antibacterial syringe are as follows:

(1) chloroauric acid, DAPT, and a surfactant were mixed to obtain a mixed solution. Wherein the molar ratio of DAPT to chloroauric acid in the mixed solution is 5: 1. The surfactant is tween-80, and the volume content of the tween-80 in the mixed solution is 0.1 percent.

(2) And (2) adding a reducing agent into the mixed solution obtained in the step (1) for reduction to obtain a DAPT modified gold nanoparticle solution. Wherein the reducing agent is NaBH₄The molar ratio of the gold chloride to the gold chloride is 5: 1. The time of the reduction reaction is 2h, the reaction temperature is 0 ℃, and the concentration of the obtained DAPT modified gold nanoparticle solution is 0.6 mg/mL. Wherein the average particle diameter of the gold nanoparticles is 3nm, and the molar ratio of DAPT on the gold nanoparticles to gold element is 0.4:1

(3) The injector (substrate) was treated in a plasma cleaning apparatus to obtain an injector having a negatively charged surface. Wherein the used Plasma cleaning device is a Plasma cleaner (model PDC-MG, Chengdu constant technology development Co., Ltd.), and the time for cleaning the injector is 1 min. The power of the Plasma scrubber was set to 10W and the frequency of the Plasma generator was 13.56 mHz.

(4) And (3) immersing the injector with the negative charges on the surface in the step (3) in the DAPT modified gold nanoparticle solution in the step (2) for 3h, taking out, washing with PBS for three times, then washing with ultrasound for 30min, and drying to obtain the gold nanoparticle-based antibacterial injector.

As shown in fig. 2(B), the syringe located at the upper part was an antibacterial syringe prepared, and the syringe located at the lower part was an untreated syringe of the same specification. In contrast, the antibacterial syringe of the present invention changed from colorless transparency to brownish black.

C. The preparation method of the gold nanoparticle-based antibacterial catheter comprises the following steps:

(1) chloroauric acid, DAPT, and a surfactant were mixed to obtain a mixed solution. Wherein the molar ratio of DAPT to chloroauric acid in the mixed solution is 3: 1. The surfactant is tween-80, and the volume content of the tween-80 in the mixed solution is 0.1 percent.

(2) And (2) adding a reducing agent into the mixed solution obtained in the step (1) for reduction to obtain a DAPT modified gold nanoparticle solution. Wherein the reducing agent is sodium ascorbate, and the molar ratio of the sodium ascorbate to the chloroauric acid is 1: 1. The time of the reduction reaction is 1h, the reaction temperature is 0 ℃, and the concentration of the obtained DAPT modified gold nanoparticle solution is 0.6 mg/mL. Wherein the average particle diameter of the gold nanoparticles is 1-5 nm, and the molar ratio of DAPT on the gold nanoparticles to gold element is 0.1: 1.

(3) And (3) placing the catheter (substrate) in a plasma cleaning device for treatment to obtain the catheter with the surface with negative charges. Wherein the used Plasma cleaning device is a Plasma cleaner (model PDC-MG, Chengdu constant technology development Co., Ltd.), and the time for cleaning the catheter is 10 min. The power of the Plasma scrubber was set at 150W and the frequency of the Plasma generator was 13.56 mHz.

(4) And (3) immersing the catheter with the negative surface charge in the step (3) in the DAPT modified gold nanoparticle solution in the step (2) for 15h, taking out, washing with PBS for three times, then ultrasonically washing for 30min, and drying to obtain the gold nanoparticle-based antibacterial catheter.

As shown in fig. 2(C), the catheter on the left side was the prepared antibacterial catheter, and the catheter on the right side was the untreated catheter of the same specification. The contrast shows that the antibacterial catheter of the invention is changed into brownish black from colorless and transparent.

Example 2

The substrate selected for this example was a 96-well plate (polystyrene, available from Corning).

The preparation method of the antibacterial 96-well plate based on the gold nanoparticles comprises the following steps:

(2) And (2) adding a reducing agent into the mixed solution obtained in the step (1) for reduction to obtain a DAPT modified gold nanoparticle solution. Wherein the reducing agent is NaBH₄The molar ratio of the gold chloride to the gold chloride acid is 3: 1. The reduction reaction time is 1h, the reaction temperature is 0 ℃, the concentration of the obtained DAPT modified gold nanoparticle solution is 0.6mg/mL, the average particle size of the gold nanoparticles is 3nm, and the molar ratio of DAPT on the gold nanoparticles to gold element is 0.45: 1.

(3) And (3) placing the 96-well plate (substrate) in a plasma cleaning device for treatment to obtain the 96-well plate with the negatively charged surface. Wherein the used Plasma cleaning device is a Plasma cleaner (model PDC-MG, Chengdu constant technology development Co., Ltd.), and the time for cleaning the 96-well plate is 4 min. The power of the Plasma scrubber was set at 60W and the frequency of the Plasma generator was 13.56 mHz.

(4) And (3) immersing the 96-well plate with the surface with negative charges in the step (3) in the DAPT modified gold nanoparticle solution in the step (2) for 12h, taking out, washing with PBS for three times, then washing with ultrasound for 30min, and drying to obtain the gold nanoparticle-based antibacterial 96-well plate (as shown in fig. 3(A), the 96-well plate is changed into brown black from colorless and transparent).

The ultraviolet absorption spectra of the antibacterial 96-well plate and the antibacterial glass tube, the antibacterial syringe and the antibacterial catheter in example 1 all show an absorption peak at 520nm, which indicates that the ultraviolet absorption spectra of the antibacterial product of the invention all show a peak at 520nm, and the intensity of the absorption peak is in positive correlation with the surface density of the gold nanoparticles. To investigate this property, the present inventors conducted the following comparative experiment:

preparing 8 96-well plates by the same method as the above stepsAn antibacterial 96-well plate, differing only in that: the step (2) further comprises the step of adding NaCl into the DAPT modified gold nanoparticle solution to ensure that the concentration of NaCl in the DAPT modified gold nanoparticle solution is 0M and 2M respectively^-5M、2^-4M、2^-3M、2^-2M、2^-1M、2⁰M、2¹And M. The absorption at 520nm of these antibacterial 96-well plates was measured using an ultraviolet absorption spectrometer (model UV2450spectrophotometer, Shimadzu Corp.), as shown in FIG. 3 (B).

Taking 8 96-well plates, preparing an antibacterial 96-well plate according to the same method as the steps, wherein the difference is only that: the concentrations of the DAPT-modified gold nanoparticle solution in step (2) were 0 × 0.6mg/mL, 1/64 × 0.6mg/mL, 1/32 × 0.6mg/mL, 1/16 × 0.6mg/mL, 1/8 × 0.6mg/mL, 1/4 × 0.6mg/mL, 1/2 × 0.6mg/mL, and 1 × 0.6mg/mL, respectively. The absorption at 520nm of these antibacterial 96-well plates was measured using an ultraviolet absorption spectrometer (model UV2450spectrophotometer, Shimadzu Corp.), as shown in FIG. 3 (C).

Taking 6 96-well plates, preparing an antibacterial 96-well plate according to the same method as the steps, wherein the differences are only that: the immersion time in the step (4) is 0h, 3h, 6h, 9h, 12h and 15h respectively. The absorption at 520nm of these antibacterial 96-well plates was measured using an ultraviolet absorption spectrometer (model UV2450spectrophotometer, Shimadzu Corp.), as shown in FIG. 3 (D).

Therefore, the intensity of the absorption peak can be regulated and controlled by adjusting the concentration of NaCl in the DAPT modified gold nanoparticle solution, the concentration of the DAPT modified gold nanoparticle solution and/or the immersion time of the base material in the DAPT modified gold nanoparticle solution, so that the surface density of the gold nanoparticles on the base material can be controlled.

Example 3

This example serves to illustrate the physical properties of the antimicrobial layer of the antimicrobial article of the present invention.

Gold content (μ g/cm) of the antibacterial 96-well plate prepared in example 2 and the comparative 96-well plate was measured using an inductively coupled plasma emission spectrometer (ICP-OES) (instrument model Optima5300DV, available from Pekin-Elmer, USA)²)。

Wherein the comparative 96-well plate was prepared by the following steps: a common 96-well plate with the same specification as the antibacterial 96-well plate in example 2 was taken, directly immersed in the DAPT-modified gold nanoparticle solution in example 2 for 12 hours, taken out, washed three times with PBS, then ultrasonically washed for 30min, and dried.

The measurement results are shown in FIG. 4. In fig. 4, a column filled with a left-upper-right downward slant line indicates the gold element content when not washed with PBS after 12 hours of immersion. It can be seen that the gold elements are present on both the antibacterial 96-well plate and the comparative 96-well plate, but the gold element content on the antibacterial 96-well plate is significantly higher, while the gold element on the comparative 96-well plate is a small amount of gold nanoparticles non-specifically adsorbed onto the surface thereof. The columns filled with left-bottom to right-top oblique lines indicate the gold element content after washing with PBS. Clearly, there was essentially no change in gold content on the antimicrobial 96-well plates, whereas the gold content on the comparative 96-well plates had been washed away with PBS. It can be seen that the antibacterial layer of the antibacterial article of the present invention has good adhesion properties. Furthermore, the inventors have determined that the antibacterial layer can be stably present in a solution such as an aqueous solution, an organic solvent, or PBS for two or more weeks under vacuum conditions.

In addition, contact angles of the antibacterial 96-well plate and the ordinary 96-well plate were also measured by a contact angle measuring instrument (model DSA100, Kruss corporation). In the absence of gold nanoparticles, the contact angle of a common 96-well plate with the same specification is about 87 degrees, and the plate is relatively hydrophobic; the contact angle of the antibacterial 96-pore plate is about 50 degrees, and the plate is hydrophilic. Therefore, by carrying out gold nanoparticle coating modification on the base material, the hydrophilic and hydrophobic properties of the surface of the material can be controlled.

Example 4: antimicrobial Activity test

After counting the E.coli, 10 are counted⁵CFU of Escherichia coli (ATCC 11775, available from Peking Tiantan Hospital) and 10⁵CFU-resistant escherichia coli (obtained from beijing tiantan hospital) was inoculated onto the surfaces of the antibacterial glass tube (experimental group) and the ordinary glass tube (control group) of the same specification in example 1(a), respectively, and 24 hours later, the bacterial colonies were counted by plate coating method to obtain a bacterial clearance (number of control group colonies-number of experimental group colonies)/number of control group colonies100% to compare the antibacterial activity of the materials.

The antibacterial syringe and antibacterial catheter prepared in example 1, the antibacterial 96-well plate prepared in example 2, and the antibacterial Tips (Tips) prepared in the same manner as in example 1(a) were examined in the same manner. The results of the bacterial clearance test are shown in table 1 below.

TABLE 1 bacterial clearance test results

Test object (Experimental group)	Material	Clearance rate of bacteria
			Antibacterial glass tube	SiO₂	>99.99％
Antibacterial syringe	PP	>99.99％
			Antibacterial catheter	Silicone rubber/PVC	>99.99％
Antibacterial 96-well plate	PS/PC	>99.99％
			Antibacterial suction head	PP	>99.99％

The results show that the invention has good antibacterial effect on antibacterial products prepared from any materials. Generally, a bacteria clearance of > 95% is considered to have good bacteria clearance, whereas the bacteria clearance of the antibacterial product of the present invention is above 99.99%.

In addition, the inventor also tests the bacteria clearance rate of the antibacterial product of the invention on multidrug resistant pseudomonas aeruginosa, klebsiella pneumoniae and the like by the same method, and proves that the bacteria clearance rate is above 99.99 percent.

Example 5: cytotoxicity test

The antibacterial 96-well plate prepared in example 2 was used, and 5000 cells were inoculated into each well. After 48 hours of cell growth, CCK8 reagent (available from bi yun biotechnology) was added as an experimental group; taking a common 96-well plate with the same specification, adding cells, and taking the common 96-well plate as a blank group without adding a CCK8 reagent; taking a common 96-well plate with the same specification, adding cells and a CCK8 reagent as a negative control group; OD450 values in cell culture broth were tested. The OD450 value of the gold nanoparticle coating was negligible. Cell viability was 100% (experimental-blank)/(control-blank).

Three cells, human smooth muscle cells, human fibroblasts and human umbilical vein vascular endothelial cells, were selected for testing. The results show that the antimicrobial layer of the antimicrobial article of the present invention is not toxic to these cells. (the cell survival rate is 95-105 percent)

Example 6: hemolysis test

Hemolysis is one of the side effects of many traditional antibiotics. This example tests whether the antimicrobial layer of the antimicrobial article of the present invention is subject to hemolysis.

Fresh rabbit blood was diluted to 4% with PBS buffer, and the diluted blood (100uL) was incubated in 96-well plates with different surface densities of gold nanoparticles prepared in the comparative experiment of example 2, and blood incubated on the surface of a common syringe of the same specification was taken as a negative control, and blood to which 10uL of surfactant was added was taken as a positive control. Blood was incubated on the surface for 2h, then centrifuged and the supernatant was measured for visible light absorption at 576nm using a microplate reader (model Tecan infinite 200 multimodernicroplate readers, Tecan corporation). Percent hemolysis was 100% (experimental OD 576-negative control OD 576)/(positive control OD 576-negative control OD 576). The inventor finds that the percent of hemolysis of the antibacterial product with the gold nanoparticle antibacterial layer is less than 1 percent, and proves that the antibacterial layer can not generate the coagulation phenomenon while resisting bacteria.

Example 7: thrombin generation assay

Blood from healthy persons was taken for thrombin generation time testing. After blood was collected from healthy volunteers, ascorbic acid was added to obtain platelet-rich plasma by centrifugation, and at this time, the platelet-rich plasma and thrombin fluorogenic substrate were rapidly added to 96-well plates having different surface densities of gold nanoparticles prepared in the comparative experiment of example 2, followed by addition of CaCl₂Activating the coagulation mechanism of platelets. The fluorescence intensity of the cells was measured at different times. The Thrombin generation time and the Thrombin generation amount are calculated by the Thrombin scope software, and the result shows that the Thrombin generation time is gradually delayed and the Thrombin generation amount is continuously reduced along with the increase of the surface density of the gold nanoparticles.

Example 8: platelet adhesion test

Healthy human serum was placed on the surfaces of the antibacterial syringe prepared in example 1 and the common syringe of the same specification, respectively, and incubated at 37 ℃ for two hours. After incubation, the surface was washed three times with PBS. Then fixed with paraformaldehyde for 10 minutes. Then, dehydration treatment was performed with alcohols of different concentrations. Finally, the sample was observed under a scanning electron microscope after drying, and as a result, it was found that platelet adhesion was hardly observed on the surface of the antibacterial syringe of the present invention, while platelet adhesion was relatively significant on the surface of the conventional syringe.

Although the present invention has been described to a certain extent, it is apparent that appropriate changes in the respective conditions may be made without departing from the spirit and scope of the present invention. It is to be understood that the invention is not limited to the described embodiments, but is to be accorded the scope consistent with the claims, including equivalents of each element described.

Claims

1. An antibacterial product based on gold nanoparticles is characterized by comprising an antibacterial layer and a base material, wherein the antibacterial layer comprises gold nanoparticles modified by DAPT, the surface of the base material is provided with negative charges, the antibacterial layer is combined on the surface of the base material through electrostatic action, the average particle size of the gold nanoparticles is 3nm, and the molar ratio of DAPT to gold elements on the gold nanoparticles is 0.1: 1-0.45: 1;

wherein the antibacterial product is prepared by the following method:

(1) mixing chloroauric acid, DAPT and a surfactant to obtain a mixed solution;

(4) immersing the substrate with the negative charges on the surface in the step (3) into the DAPT modified gold nanoparticle solution in the step (2), taking out, cleaning and drying to obtain the antibacterial product;

and (2) adding NaCl into the DAPT modified gold nanoparticle solution, wherein the concentration of the NaCl in the DAPT modified gold nanoparticle solution is 0.125-2M.

2. The antimicrobial article of claim 1, wherein the negative charge on the surface of the substrate is obtained by treating the substrate in a plasma cleaning device.

3. The antimicrobial article of claim 1, wherein the molar ratio of DAPT to gold elements on the gold nanoparticles is 0.45: 1.

4. The antimicrobial article of any one of claims 1 to 3, wherein the substrate is an organic material or an inorganic material.

5. The antimicrobial article of claim 4, wherein the substrate is selected from one or more of glass, silicone, polyvinyl chloride plastic, polystyrene plastic, polycarbonate plastic, polypropylene plastic, and polydimethylsiloxane.

6. The antimicrobial article of claim 5, wherein the substrate is a consumable for a biological or medical application.

7. The method for preparing an antibacterial article based on gold nanoparticles according to any one of claims 1 to 6, characterized in that it comprises the following steps:

(1) mixing chloroauric acid, DAPT and a surfactant to obtain a mixed solution;

and (3) adding NaCl into the DAPT modified gold nanoparticle solution, wherein the concentration of the NaCl in the DAPT modified gold nanoparticle solution is 0.125-2M.

8. The method according to claim 7, wherein in the step (1), the molar ratio of DAPT to chloroauric acid in the mixed solution is 5:1 to 1: 1.

9. The method of claim 8, wherein the molar ratio of DAPT to chloroauric acid in the mixed solution is 1: 1.

10. The method according to any one of claims 7 to 9, wherein the surfactant is tween.

11. The method according to claim 10, wherein the surfactant is tween-80.

12. The production method according to claim 10, wherein the surfactant is contained in the mixed solution in an amount of 0.1% by volume.

13. The method of claim 10, wherein the reducing agent is sodium borohydride or sodium ascorbate.

14. The method according to claim 10, wherein the molar ratio of the reducing agent to the chloroauric acid is 5:1 to 1: 1.

15. The method according to claim 14, wherein the molar ratio of the reducing agent to the chloroauric acid is 3: 1.

16. The preparation method according to claim 7, wherein the reduction reaction time in the step (2) is 1-2 h, and the reaction temperature is 0 ℃.

17. The method of claim 16, wherein the concentration of the DAPT-modified gold nanoparticle solution in step (2) is 0.6 mg/mL.

18. The method according to claim 7, wherein in the step (3), the time for the plasma cleaning device to treat the substrate is 1-10 min.

19. The production method according to claim 18, wherein in the step (3), the time for which the plasma cleaning apparatus treats the substrate is 4 min.

20. The method of claim 19, wherein the power of the plasma cleaning device is 10 to 150W, and the frequency of the plasma generator is 13.56 mHz.

21. The method of claim 20, wherein the power of the plasma cleaning device is 60W.

22. The method of claim 18, wherein the plasma cleaning device is an ultrasonic plasma cleaner.

23. The method according to claim 7, wherein the immersion time in the step (4) is 3 to 15 hours.

24. The method of claim 23, wherein the immersion time in step (4) is 12 hours.

25. The method of claim 23, wherein the washing in step (4) comprises washing three times with a phosphate buffer solution, followed by ultrasonic washing for 30 min.

26. Use of an antimicrobial article according to any one of claims 1 to 6 or prepared according to the process of any one of claims 7 to 25 in the preparation of a biological and/or pharmaceutical product.