NL2028410A

NL2028410A - Antibacterial nanozyme and preparation method thereof

Info

Publication number: NL2028410A
Application number: NL2028410A
Authority: NL
Inventors: Zhou Hong; Liu Jing
Original assignee: Qingdao Univ Of Science And Technology
Priority date: 2020-06-11
Filing date: 2021-06-08
Publication date: 2021-08-30
Also published as: CN111632141A; NL2028410B1; WO2021248674A1

Abstract

The present disclosure relates to an antibacterial nanozyme and a preparation method thereof, and belongs to the technical field of antibacterial materials. The present disclosure includes the following steps: step l, heating and stirring an aqueous chloroauric acid solution, adding an aqueous sodium citrate solution thereto, boiling, and cooling to obtain a gold seed solution, step 2, mixing an aqueous ammonia solution, hemin, hydrazine hydrate, and a sonicated aqueous graphene oxide solution, heating and reacting, centrifuging, cleaning, and drying to obtain a graphene-hemin composite, and step 3, mixing an aqueous hydroxylamine hydrochloride solution, the gold seed solution obtained in step 1, the graphene-hemin composite obtained in step 2 and the aqueous chloroauric acid solution, and stirring until the solution turns blue-green, after stirring again, stopping stirring, and letting the solution stand to obtain a nanozyme. Photothermal properties generated by the nanozyme of the present disclosure under irradiation of an 808 nm near-infrared laser and reactive oxygen species produced on the surface of the nanozyme enable efficient antibiosis.

Description

ANTIBACTERIAL NANOZYME AND PREPARATION METHOD THEREOF TECHNICAL FIELD

[01] The present disclosure relates to the technical field of antibacterial materials, and in particular to an antibacterial nanozyme and a preparation method thereof.

BACKGROUND ART

[02] Bacterial contamination is one of the most common food and environmental pollution. Food contaminated by bacteria may bring about a plurality of diseases. Currently, commonly used disinfectants include oxidants, heavy metal salts, organic compounds, and the like. Chemotherapeutics, including antimetabolites or antibiotics, can act on a certain link in the metabolism of pathogenic microorganisms to inhibit growth thereof or cause death. However, chronic use thereof is likely to make bacteria resistant to drugs, which seriously affects the post-sterilization effect.

SUMMARY

[03] An objective of the present disclosure is to provide an antibacterial nanozyme and a preparation method thereof. Photothermal properties generated by the nanozyme of the present disclosure under irradiation of an 808 nm near-infrared laser and reactive oxygen species produced on the surface of the nanozyme enable efficient antibiosis.

[04] The present disclosure provides a method for preparing an antibacterial nanozyme, including the following steps:

[05] step 1, heating and stirring an aqueous chloroauric acid solution, adding an aqueous sodium citrate solution thereto, boiling for 20-30 min, and cooling to obtain a gold seed solution;

[06] step 2, mixing an aqueous ammonia solution, hemin, hydrazine hydrate, and a sonicated aqueous graphene oxide solution, heating to 60-70°C and reacting for 3-4 h, centrifuging, cleaning, and drying to obtain a graphene-hemin composite; and

[07] step 3, mixing an aqueous hydroxylamine hydrochloride solution, the gold seed solution obtained in step 1, the graphene-hemin composite obtained in step 2 and the aqueous chloroauric acid solution with a pH value of 11-12, and stirring until the solution turns blue-green; after stirring for 3 -5 min, stopping stirring, and letting the solution stand for 8-12 h to obtain a nanozyme;

[08] where there is no limitation to the time sequence of the steps 1 and 2.

[09] Preferably, in step 1, a mass percent concentration of chloroauric acid in the aqueous chloroauric acid solution may be 0.01%-0.02% by weight, a mass percent concentration of sodium citrate in the aqueous solution may be 1%-2% by weight, and the aqueous chloroauric acid solution and the aqueous sodium citrate solution may have a volume ratio of 50:(0.75-1.5).

[10] Preferably, a mass concentration of graphene oxide in the aqueous graphene oxide solution in step 2 may be 0.2-0.5 mg/mL.

[11] Preferably, the graphene oxide may be 200 nm to 1 um in particle size after the sonication in step 2.

[12] Preferably, in step 2, the volume of the aqueous ammonia solution, the mass of the hemin, the volume of the hydrazine hydrate, and the volume of the sonicated aqueous graphene oxide solution preferably may have a volume ratio of 60 pL: 10 mg: 10 pL: 40 mL.

[13] Preferably, the centrifugation in step 2 may be conducted at 11,000 rpm for 30 min.

[14] Preferably, in step 3, the volume of the aqueous hydroxylamine hydrochloride solution, the volume of the gold seed solution, the mass of the graphene-hemin composite, and the volume of the aqueous chloroauric acid solution with a pH value of 11-12 may have a volume ratio of 300 uL: 12 mL: 0.02 g: 20 mL; a molar concentration of hydroxylamine hydrochloride in the aqueous hydroxylamine hydrochloride solution may be 0.02 M; a mass percent concentration of chloroauric acid in the aqueous chloroauric acid solution may be 0.01% by weight.

[15] The present disclosure further provides an antibacterial nanozyme obtained by the preparation method according to the above technical solution.

[16] The disclosure further provides use of the antibacterial nanozyme obtained by the preparation method according to the above technical solution or antibacterial nanozyme according to the above technical solution in antibiosis.

[17] Preferably, in the use, an 808 nm near-infrared laser may be irradiated to the antibacterial nanozyme.

[18] The present disclosure provides a method for preparing an antibacterial nanozyme. The preparation method of the present disclosure synthesizes an antibacterial nanozyme composite based on graphene and flower-shaped gold nanoflower by reducing the chloroauric acid solution in situ on the surface of reduced graphene oxide.

The nanozyme synthesized by the preparation method of the present disclosure is rapidly heated up under an 808 nm laser irradiation, while producing reactive oxygen species that may produce a better antibacterial effect. Compared with conventional chemical antibacterial reagents, the nanozyme may have no toxic and side effects on the environment, and maintain excellent an antibacterial effect while avoiding bacterial drug resistance.

BRIEF DESCRIPTION OF THE DRAWINGS

[19] FIG. 11s a transmission electron microscopic (TEM) image of characterization of the nanozyme provided by the present disclosure;

[20] FIG. 2 illustrates the antibacterial effect of the nanozyme provided by the present disclosure.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[21] The present disclosure provides a method for preparing an antibacterial nanozyme, including the following steps:

[22] step 1, heating and stirring an aqueous chloroauric acid solution, adding an aqueous sodium citrate solution thereto, boiling for 20-30 min, and cooling to obtain a gold seed solution;

[23] step 2, mixing an aqueous ammonia solution, hemin, hydrazine hydrate, and a sonicated aqueous graphene oxide solution, heating to 60-70°C and reacting for 3-4 h, centrifuging, cleaning, and drying to obtain a graphene-hemin composite; and

[24] step 3, mixing an aqueous hydroxylamine hydrochloride solution, the gold seed solution obtained in step 1, the graphene-hemin composite obtained in step 2 and the aqueous chloroauric acid solution with a pH value of 11-12, and stirring until the solution turns blue-green; after 3-5 min, stopping stirring, and letting the solution stand for 8-12 h to obtain a nanozyme;

[25] where there is no limitation to the time sequence of the steps 1 and 2.

[26] The present disclosure heats and stirs an aqueous chloroauric acid solution, adds an aqueous sodium citrate solution thereto, boils for 20-30 min, and cools to obtain a gold seed solution. In the present disclosure, a mass percent concentration of chloroauric acid in the aqueous chloroauric acid solution may preferably be 0.01%-0.02% by weight,

a mass percent concentration of sodium citrate in the aqueous solution may preferably be 1%-2% by weight, and the aqueous chloroauric acid solution and the aqueous sodium citrate solution may preferably have a volume ratio of 50:(0.75-1.5). More specifically, in the present disclosure, it may be preferable to prepare, heat, and stir 50 mL of chloroauric acid solution with a concentration of 0.01% by weight, and quickly add 750 uL of aqueous sodium citrate solution with a concentration of 1% by weight thereto. In the present disclosure, it may be preferable to naturally cool, and preferably cool to room temperature. The gold seed solution obtained by the present disclosure may preferably be stored in a refrigerator at 4°C for later use. Sources of the chloroauric acid and sodium citrate are not particularly limited in the present disclosure, as long as conventional commercially available products of chloroauric acid and sodium citrate well known to those skilled in the art may be used.

[27] The present disclosure mixes an aqueous ammonia solution, hemin, hydrazine hydrate, and a sonicated aqueous graphene oxide solution, and heats to 60-70°C and reacts for 3-4 h, and more preferably at 60°C for 4 h, followed by centrifuging, cleaning, and drying to obtain a graphene-hemin composite. The source of the graphene oxide is not particularly limited in the present disclosure, and preferably, a 2 mg/mL graphene oxide dispersion purchased from Nanjing Xianfeng Nanomaterials Technology Co., Ltd. may be used. In the present disclosure, it may be preferable to add 5 mL of the graphene oxide dispersion to 35 mL of water, and sonicate the solution to obtain a sonicated aqueous graphene oxide solution. In the present disclosure, the sonication may preferably be conducted for 30 min. In the present disclosure, the graphene oxide may preferably be 200 nm to 1 um, and more preferably 500 nm, in particle size after the sonication. In the present disclosure, a mass concentration of graphene oxide in the aqueous graphene oxide solution may preferably be 0.2-0.5 mg/mL, and more preferably

0.25 mg/mL. In the present disclosure, the volume of the aqueous ammonia solution, the mass of the hemin, the volume of the hydrazine hydrate, and the volume of the sonicated aqueous graphene oxide solution may preferably have a volume ratio of 60 uL: 10 mg: 10 pL: 40 mL. Specifically, in the present disclosure, it may be preferable to add 60 pL of aqueous ammonia solution, 10 mg of hemin, and 10 pL of hydrazine hydrate to 40 mL of sonicated aqueous graphene oxide solution. In the present disclosure, the centrifugation may preferably be conducted at 11,000 rpm for 30 min. Sources of the aqueous ammonia solution, the hemin, and the hydrazine hydrate are not particularly limited in the present disclosure, as long as conventional commercially available products well known to those skilled in the art may be used, and the hemin may preferably be purchased from Sigma Co., Ltd. In the present disclosure, the cleaning may preferably be conducted with ultrapure water twice.

5 [28] After the gold seed solution and the graphene-hemin composite are obtained, the present disclosure mixes an aqueous hydroxylamine hydrochloride solution, the gold seed solution, the graphene-hemin composite, and the aqueous chloroauric acid solution with a pH value of 11-12, stirs until the solution turns blue-green, after stirring for another 3-5 min, stops stirring, and lets stand for 8-12 h to obtain a nanozyme. In the present disclosure, flower-shaped gold nanoflower particles are synthesized in sifu on the surface of the graphene oxide to construct a nanozyme composite. In the present disclosure, the volume of the aqueous hydroxylamine hydrochloride solution, the volume of the gold seed solution, the mass of the graphene-hemin composite, and the volume of the aqueous chloroauric acid solution with a pH value of 11-12 may preferably have a volume ratio of 300 uL: 12 mL: 0.02 g: 20 mL; a molar concentration of hydroxylamine hydrochloride in the aqueous hydroxylamine hydrochloride solution may be 0.02 M; a mass percent concentration of chloroauric acid in the aqueous chloroauric acid solution may be 0.01% by weight. In the present disclosure, the aqueous chloroauric acid solution may be more preferably at pH 11.5. Specifically, in the present disclosure, it may be preferable to adjust 50 mL of aqueous chloroauric acid solution (with a mass percent concentration of 0.01% by weight) to pH 11.5 with 1 M sodium hydroxide, and add 12 mL of gold seed solution, 300 pL of 0.02 M hydroxylamine hydrochloride, and 0.02 g of graphene-hemin composite. The source of the hydroxylamine hydrochloride is not particularly limited in the present disclosure, as long as commercially available products of hydroxylamine hydrochloride well known to those skilled in the art may be used.

[29] The present disclosure further provides an antibacterial nanozyme obtained by the preparation method according to the above technical solution. The nanozyme provided by the present disclosure is rapidly heated up under an 808 nm laser irradiation, while producing reactive oxygen species that may produce a better antibacterial effect. That is, the present disclosure utilizes strong photothermal and catalytic properties of the nanozyme to achieve a safe and efficient antibacterial effect.

[30] The disclosure further provides use of the antibacterial nanozyme obtained by the preparation method according to the above technical solution or antibacterial nanozyme according to the above technical solution in antibiosis. For example, the antibacterial nanozyme is used for sterilization and antibacterial treatment of drug- resistant Escherichia coli.

[BI] Inthe use provided by the present disclosure, an 808 nm near-infrared laser may need to be irradiated to the antibacterial nanozyme.

[32] The antibacterial nanozyme and the preparation method thereof provided by the present disclosure will be further described in detail below in conjunction with specific examples. The technical solutions of the present disclosure include, but are not limited to, the following examples.

[33] Example 1

[34] Preparation method of nanozyme

[35] First, a gold seed solution was synthesized; all glass containers were cleaned and dried. 50 mL of aqueous chloroauric acid solution with a mass percent concentration of 0.01% by weight was prepared, heated and stirred; 750 pL of aqueous sodium citrate solution with a mass percent concentration of 1% by weight was quickly added thereto; the mixed solution was kept boiling for 20 min, and heating was stopped; the mixed solution was cooled to room temperature naturally and stored in refrigerator at 4°C for later use.

[36] A graphene-hemin composite was synthesized; 5 mL of graphene oxide dispersion was added to 35 mL of water and sonicated for 30 min. 60 uL of aqueous ammonia solution, 10 mg of hemin and 10 pL of hydrazine hydrate was added and held at 60°C for 4 h. Subsequently, the solution was centrifuged (at 11,000 rpm for 30 min), during which the solution was washed twice with ultrapure water. The solution prepared was dried and stored in a refrigerator at 4°C for later use.

[37] Finally, a hemin-graphene-gold nanoflower composite was synthesized; 50 mL of chloroauric acid (0.01%) was adjusted to pH 11.5 with 1 M sodium hydroxide, mixed with 12 mL of well-prepared gold seed solution, 300 pL of 0.02 M hydroxylamine hydrochloride, and 0.02 g of well-prepared graphene-hemin composite, and stirred constantly. The solution turned blue-green. After 3 min, the stirring was stopped and let stand overnight to prepare a nanozyme solution, as shown in FIG. 1 (a TEM image of characterization the nanozyme synthesized, from which very flower-shaped gold nanoparticles (around 50 nm in particle size) are grown on the surface of graphene in situ, and the particle size is relatively uniform).

[38] Example 2

[39] Antibacterial property of the nanozyme against drug-resistant £. coli (Ampr E. coli) under laser irradiation

[40] 100 pg/mL nanozyme solution was co-incubated with 1.0 = 105 CFU mL* bacteria for 20 min; after 100-fold dilution, 100 pL of bacterial suspension was spread on an LB medium in a Petri dish, and incubated at 37°C for 18 h. An 808 nm laser was turned on, and the current was adjusted to 0.5 A; the Petri dish was placed and irradiated for 10 min under a laser probe, and the bacterial survival rate was recorded; at the same time, a bacterial Petri dish without nanozyme solution (bacterial concentration was 1.0 x 10° CFU mL!) was used as a control group, and the same experimental operation was adopted.

[41] The experimental results were as follows: after 808 nm laser irradiation for 10 min, the bacterial survival rate of the bacterial Petri dish without nanozyme solution was 99%, and that of the bacterial Petri dish with 100 ug mL"! nanozyme solution was 28%. It is indicated that the nanozyme has an excellent bactericidal effect on drug-resistant Z. coli (Ampr E. coli), as shown in FIG. 2 (illustrating an antibacterial effect of the nanozyme), where A illustrates the bacterial Petri dish without nanozyme solution after 808 nm laser irradiation for 10 min; B illustrates the bacterial Petri dish with 100 pg mL" !nanozyme solution after 808 nm laser irradiation for 10 min.

[42] Example 3

[43] Antibacterial properties of different concentrations of the nanozyme against drug-resistant E. coli (Ampr E. coli) under laser irradiation. The experiment was divided into five groups; 0 pg/mL (group 1), 50 pg/mL (group 2), 80 ug/mL (group 3), 100 pg/mL (group 4), and 120 pg/mL (group 5) nanozyme solutions were co-incubated with

1.0 x 10° CFU mL"! bacteria for 20 min, respectively; after 100-fold dilution, 100 uL of bacterial suspension was spread on an LB medium in a Petri dish, and incubated at 37°C for 18 h. An 808 nm laser was turned on, and the current was adjusted to 0.5 A; each Petri dish was placed and irradiated for 10 min under a laser probe, and the bacterial survival rate was recorded. The bacterial survival rate was 97.5% in group 1, 76.2% in group 2, 55.2% in group 3, 28.6% in group 4, and 18.8% in group 5. It is indicated that the bactericidal effect of the nanozyme on drug-resistant F. coli (Ampr E. coli) is related to the nanozyme concentration, and that greater nanozyme concentration exhibits a better bactericidal effect within a certain range.

[44] The above descriptions are merely preferred implementations of the present disclosure. It should be noted that a person of ordinary skill in the art may further make several improvements and modifications without departing from the principle of the present disclosure, but such improvements and modifications should be deemed as falling within the protection scope of the present disclosure.

Claims

-9- Conclusions L Method for preparing an antibacterial nanozyme, comprising the following steps: step 1, heating and stirring an aqueous chloroauric acid solution, adding an aqueous sodium citrate solution thereto, boiling for 20-30 minutes, and cooling to obtain a golden seed solution; step 2, mixing an aqueous ammonia solution, hemin, hydrazine hydrate and a sonicated aqueous graphene oxide solution, heating to 60 - 70°C and reacting for 3 - 4 hours, centrifuging, cleaning and drying to obtain a graphene hemin composite; and step 3, mixing an aqueous hydroxylamine hydrochloride solution, the gold seed solution obtained in step 1, the graphene hemine composite obtained in step 2, and the aqueous chloroauric acid solution having a pH of 11-12, and stirring until the solution turns blue-green; then stirring for 3-5 min, stopping stirring, and letting the solution stand for 8-12 h to obtain a nanozyme; wherein there is no limitation on the time sequence of steps 1 and 2.

The preparation method according to claim 1, wherein in step 1, a mass percent concentration of chloroauric acid in the aqueous chloroauric acid solution is 0.01 wt% - 0.02 wt%, a mass percent concentration of sodium citrate in the aqueous solution is 1 wt% - 2 wt. -%, and wherein the aqueous chloroauric acid solution and the aqueous sodium citrate solution have a volume ratio of 50:(0.75 - 1.5).

The preparation method according to claim 1, wherein a mass concentration of graphene oxide in the aqueous graphene oxide solution in step 2 is 0.2-0.5 mg/mL.

The preparation method according to claim 1, wherein the graphene oxide is 200 nm - 1 µm in particle size after the sonication in step 2.

The preparation method according to claim 1, wherein in step 2, the volume of the

- 10 - aqueous ammonia solution, the mass of the hemin, the volume of the hydrazine hydrate and the volume of the sonicated aqueous graphene oxide solution preferably have a volume ratio of 60 µL: 10 mg: 10 µL: 40 mL.

The preparation method according to claim 1, wherein the centrifugation in step 2 is performed at 11,000 rpm for 30 min.

The preparation method according to claim 1, wherein in step 3, the volume of the aqueous hydroxylamine hydrochloride solution, the volume of the gold hall solution, the mass of the graphene hemin composite and the volume of the aqueous chloroauric acid solution having a pH of 11 - 12 is a volume ratio of 300 µL: 12 mL: 0.02 g: 20 mL; wherein a molar concentration of hydroxylamine hydrochloride in the aqueous hydroxylamine hydrochloride solution is 0.02 M; wherein a mass percent concentration of chloroauric acid in the aqueous chloroauric acid solution is 0.01% by weight.

An antibacterial nanozyme obtained by the preparation method according to any one of claims 1 to 7.

Use of the antibacterial nanozyme obtained by the preparation method according to any one of claims 1 to 7 or the antibacterial nanozyme according to claim 8 in antibiotics.

Use according to claim 9, wherein, in use, an 808 nm near infrared laser beam is beamed to the antibacterial nanozyme.