CN105345021B

CN105345021B - A kind of method using staphylococcus aureus biosynthesis with biocompatibility nanogold

Info

Publication number: CN105345021B
Application number: CN201510664346.6A
Authority: CN
Inventors: 邱小忠; 华文熙; 王乐禹
Original assignee: Southern Medical University
Current assignee: Southern Medical University
Priority date: 2015-10-14
Filing date: 2015-10-14
Publication date: 2018-02-16
Anticipated expiration: 2035-10-14
Also published as: CN105345021A

Abstract

The invention discloses a kind of method using staphylococcus aureus biosynthesis nanogold, this method is to mix staphylococcus aureus liquid and gold chloride, mixed liquor is reacted 4~6h；Reaction solution is centrifuged, supernatant is collected and continues 0.5~48h of reaction；Reaction solution is centrifuged again, collection, which precipitates, produces nanogold.The nanogold of biosynthesis of the present invention has biological capacitive, does not have toxicity to cell；It is gained nanogold uniform particle sizes, controllable, required size can be synthesized on request.Gained nanogold can keep stable for a long time and not reunite, and nanogold is kept stable without using interfacial agent.The present invention can avoid using the pollution caused by organic solvent and reduce the consumption of the energy because synthesis is carried out under room temperature, chamber pressure, and staphylococcus aureus acquisition is easy, economical, renewable.

Description

It is a kind of that there is biocompatibility nanogold using staphylococcus aureus biosynthesis Method

Technical field

The present invention relates to a kind of method using staphylococcus aureus biosynthesis with biocompatibility nanogold.

Background technology

Nanogold：Also known as " collaurum ", because being easily gathered into bulk in atmosphere, so typically nanogold is prepared molten In liquid system.Nanogold size generally makes solution be more than 100 nanometers with red, size and then make less than 100 nanometers in the solution Solution is in blueness or purple.Nanogold has special optical property, electrical properties, molecular recognition property and well biological Capacitive, nanogold is set to be applied to various different fields, such as electron microscope at present into widest nano material is studied , electronics, material science, biological detection, optical detection, drug delivery, catalytic reaction, disease treatment, electronic engineering and mould Hardened crystalline substance.

The synthetic method of nanogold has physics synthetic method, chemical synthesis and biological synthesis process at present.

(1) the physics synthetic method of nanogold

Vapor condensation method：Make the particle body such as material gasification or formation with the methods of vacuum evaporation, heating, high-frequency induction, so After be quenched.Its feature purity is high, crystalline structure is good, granularity is controllable, but technical equipment requires high.

Physical crushing method：The methods of being exploded by mechanical crushing, electric spark obtains nano-particle.Its feature is simple to operate, Cost is low, but product purity is low, and distribution of particles is uneven.

Mechanical attrition method：Using ball grinding method, appropriate condition is controlled to the nanoparticle of pure element, alloy or composite Son.Its feature is simple to operate, cost is low, but product purity is low, and distribution of particles is uneven.

Epitaxy stacking：Gold is decomposed into the Ultrafine nano Au particle of atomic structure with physical process for dispersing, Again with epitaxy Stack Technology, the gold particle of atomic state is stacked into reduction.Purity is high, can manufacture particle size from 0.5nm to 100nm。

(2) chemical synthesis process of nanogold

Chemical vapour deposition technique：Utilize the chemical reactive synthesis nano material of metal compound vapor.Its feature product is pure Degree is high, narrow particle size distribution.

The precipitation method：Precipitating reagent is added to after being reacted in salting liquid, precipitation is thermally treated resulting in nano material.The letter of its feature It is single easy, but purity is low, and particle radius is big.

Chemical reduction method is most widely used that in noble metal nano particles synthesis, will typically add and reduce in metal salt Agent, it is reduced to nano-particle, while to avoid metal nanoparticle from assembling, add interfacial agent or macromolecule mostly Polymer controls the growth of metal nanoparticle and aggegation as protective agent.Common interfacial agent has：Cetyl Base trimethylammonium (CTAB), lauryl sodium sulfate (SDS), two (2- ethylhexyls) Succinate sodium sulfonates (AOT), and macromolecule Polymer then often utilizes：Polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), perfluoromethylvinyl base The water soluble polymers such as ether (PMVE).

Green Chemistry advocates 12 principles：Including 1, avoid discarded object from producing, 2, raw material economics service efficiency it is high, 3, reduce Virose chemical synthesis, 4, design the chemicals of hypotoxicity, 5, solvent and adminicle safe to use, 6, high-energy source uses Rate, 7, the use of continue resource forever be raw material, 8 reduce the generations of non-essential derivatives, 9, using catalyst to improve yield, 10, set Count decomposable chemicals, 11, pollutant monitors in time and the control of manufacturing process, the chemicals of 12 careful selection manufacturing process Matter, to reduce the generation of unexpected storms.

The method that tradition prepares nanogold is mainly physics and chemical method, and these methods need strict condition, power consumption Height, the expensive reagents used.Conventional physical method has friction and pyrolysis, and production process not only needs to expend mass energy to tie up High temperature and high pressure is held, and speed is slow, and price is high；It is commonly used not only expensive in traditional wet chemical method but also has virose examination Agent has a large amount of unreacted reagent residuals in the solution as reducing agent and stabilizer, though cost is low, reaction is fast, Environment is polluted, application of the obtained nano-particle to fields such as food inspection, biomedicines has harmful effect.

(3) biosynthesis nanogold：

The green syt of nanogold is mainly synthesized using biomaterials such as microorganism and plants at present.Major defect Have：1st, the existing nanogold synthetic method consuming time is long, and this method can synthesize substantial amounts of nanogold in a short time.2nd, it is existing There is nanogold synthetic yield low, be primarily due to gold chloride and bacterial reaction overlong time, a large amount of nanogold is adhered to bacterium Surface causes to waste by centrifuging a large amount of nanogold as bacterium is centrifuged, and this nanogold synthetic method is to make bacterium in gold chloride Environment incretion reducing agent leaves away bacterium by the effect of centrifugation in mixed liquor, leaves supernatant and is further continued for reacting it and is produced Raw nanogold is using nanogold, therefore improves nanogold yield.3rd, the nanogold composition of biosynthesis now is not It is pure, and have larger toxicity to cell, this nanogold synthetic method enables nanogold to purify by the method for gradient centrifugation, makes The application of the nanogold of biosynthesis is achieved.

Staphylococcus aureus, also referred to as " S. aureus L-forms ", staphylococcus aureus have stronger vitality and adaptability, Distributed in nature is wide.Staphylococcus aureus nutritional requirement is not high, the well-grown on ordinary culture medium, aerobic or facultative detest Oxygen, 37 DEG C, the most suitable growth pH7.4 of optimum growth temperature, it can survive the several months under dry environment.But can it carry out the conjunction of nanogold Into still unknown at present.

The content of the invention

In order to solve above-mentioned problem, the invention provides a kind of more easy, quick, high-purity, bio-compatible The good nano gold biological synthetic method of property.

There is biocompatibility nanometer using staphylococcus aureus biosynthesis it is an object of the invention to provide one kind The method of gold.

The technical solution used in the present invention is：

A kind of method using staphylococcus aureus biosynthesis nanogold, comprise the following steps：

1) staphylococcus aureus liquid and gold chloride are mixed, mixed liquor is reacted 4~6h；

2) upper step reaction solution is centrifuged, collects supernatant and continue 0.5~48h of reaction；

3) upper step reaction solution is centrifuged, collection, which precipitates, produces nanogold.

Further, the concentration of Staphylococcus aureus is 1 × 10 in the step 1) mixed liquor⁸~1 × 10⁹CFU/mL。

Further, the concentration of gold chloride is 1 × 10 in the step 1) mixed liquor^-2~1 × 10²mmol/L。

Further, the temperature of step 1) the mixed liquor reaction is 15~37 DEG C.

Further, the rotating speed of the step 2) centrifugation is 3000~6000rpm.

Further, the temperature of the step 2) reaction is 20~80 DEG C.

Further, the rotating speed of the step 3) centrifugation is 10000~15000rpm.

Further, the time of the step 3) centrifugation is 20~60min.

Further, prepared nanogold has biocompatibility.

The beneficial effects of the invention are as follows：

1st, staphylococcus aureus obtains easy, economical, renewable.

2nd, because synthesis is carried out under room temperature, chamber pressure, therefore it can avoid using the pollution caused by organic solvent and subtract The consumption of few energy.

3rd, staphylococcus aureus can be lost activity by heating, surrounding environment will not be impacted.

4th, nanogold yield height obtained by biosynthesis nanogold will not cause to waste.

5th, biosynthesis nanogold can keep stabilization not reunite for a long time, nanogold is kept steady without using interfacial agent It is fixed.

6th, biosynthesis nanogold uniform particle sizes, controllable, can synthesize required size on request.

7th, the nanogold of biosynthesis has biocompatibility, does not have toxicity to cell.

Brief description of the drawings

Fig. 1 is nanogold prepared by embodiment 1 and its ultraviolet light visible absorbance curve, A are for centrifuged pellet Nanogold (centrifuge tube ttom of pipe part)；B figures are that (being can for solution after precipitated nanocrystals gold is resuspended with water by the inverted purpose of centrifuge tube With more convenient, more accurate observation nano-Au solution color and solubility)；C figures are nanogold ultraviolet light visible absorbance curve；

Fig. 2 is the scanning electron microscope (SEM) photograph of nanogold prepared by embodiment 1；

Fig. 3 is nanogold and its ultraviolet light visible absorbance curve prepared by embodiment 2；

Fig. 4 is nanogold and its ultraviolet light visible absorbance curve prepared by embodiment 3；

Fig. 5 is the nanogold of Escherichia coli synthesis；Left figure is that centrifuged pellet is nanogold (centrifuge tube bottom of the tube Point)；Right figure is the solution after precipitated nanocrystals gold is resuspended with water；

Fig. 6 is the nanogold of fecal bacteria synthesis；

Fig. 7 is the nanogold of enterococcus synthesis；

Fig. 8 is the influence of different nanogold cell growths；1. being blank control group, any nanogold is not added；2. it is Experimental group, add nanogold prepared by embodiment 1；3. be control group, add the step 3) of embodiment 1 in 4000rpm centrifugation after on Nanogold in clear liquid.

Embodiment

Preferably, the concentration of Staphylococcus aureus is 1 × 10 in the step 1) mixed liquor⁸~1 × 10⁹CFU/mL。

Preferably, the concentration of gold chloride is 1 × 10 in the step 1) mixed liquor^-2~1 × 10²mmol/L。

It is furthermore preferred that the concentration of gold chloride is 0.5~1.5mmol/L in the step 1) mixed liquor.

Preferably, the temperature of step 1) the mixed liquor reaction is 15~37 DEG C, more preferably 22~35 DEG C.

Preferably, the rotating speed of the step 2) centrifugation is 3000~6000rpm；More preferably 4000~5000rpm.

Preferably, the temperature of the step 2) reaction is 20~80 DEG C；More preferably 40~60 DEG C.

Preferably, step 2) the continuation reaction time is 3~10h.

Preferably, the rotating speed of the step 3) centrifugation is 10000~15000rpm；More preferably 12000~13000rpm.

Preferably, the time of the step 3) centrifugation is 20~60min；More preferably 30~40min.

Preferably, prepared nanogold has biocompatibility.

With reference to specific embodiment, the present invention is further illustrated, but is not limited thereto.

Embodiment 1

1) 1mmol/L gold chlorides (HAuCl is configured₄) the aqueous solution.

2) 25ml bacteria culture medias are added into appropriate staphylococcus aureus overnight incubation 3000rpm and bacterium are collected by centrifugation, 2ml sterilized waters are resuspended.

3) 1ml staphylococcus aureuses suspension (5 × 10 is taken⁸CFU/mL) with 2ml HAuCl₄Magnetic agitation, it is well mixed, Reaction 6 hours.

4) upper step reaction solution is centrifuged through 4000rpm and removes precipitated impurities, take supernatant to react 48h, 12000rpm in 20 DEG C It is nanogold to centrifuge 30min and collect precipitation.

Precipitated nanocrystals obtained by the present embodiment are golden as shown in the A figure centrifuge tube ttom of pipe part in Fig. 1, after it is resuspended with water (centrifuge tube inversion can be more convenient, more accurate observation nanogold color and solubility) as shown in the B figures in Fig. 1 for pink, Its ultraviolet light visible absorbance curve only has single absworption peak as shown in the C figures in Fig. 1 at 524nm, illustrates nanogold particle diameter Distribution is in 5~20nm, and purity is high.

Scanning electron microscope (SEM) photograph shown in Fig. 2 further illustrates nanogold particle diameter manufactured in the present embodiment in the range of 5~20nm, and Particle size is highly uniform.

The present embodiment biosynthesis nanogold (up to more than 6 months) can keep stabilization not reunite for a long time, without using interface Activating agent keeps the stability of nanogold.

The present embodiment reacts under normal temperature, normal pressure can synthesize that particle diameter is smaller, uniform nanogold, and purity is high, stable Property is good.

Embodiment 2

1) 1mmol/L gold chlorides (HAuCl is configured₄)。

2) 25ml bacteria culture medias add appropriate staphylococcus aureus overnight incubation 3000rpm and bacterium, 2ml are collected by centrifugation Sterilized water is resuspended.

4) products therefrom is collected, 4000rpm centrifugations remove precipitated impurities, take supernatant to react 0.5h in 80 DEG C of water-baths, It is nanogold that 12000rpm centrifugations 30min, which collects precipitation,.

It is pink (illustration in such as Fig. 3) after precipitated nanocrystals gold obtained by the present embodiment is resuspended with water, its ultraviolet light can See absorption curve as shown in figure 3, only there is single absworption peak at 524nm, illustrate nanogold particle size distribution range 5~ 20nm, and purity is high.

The present embodiment can shorten the reaction time to 30min by heating in the short period of time.

Embodiment 3

1) 10mmol/L gold chlorides (HAuCl is configured₄)。

3) 1ml staphylococcus aureuses suspension (5 × 10 is taken⁸CFU/mL) with 2ml HAuCl₄It is well mixed, react 6 hours.

4) products therefrom is collected, 4000rpm centrifugations remove precipitated impurities, take supernatant to react 0.5h in 80 DEG C of water-baths, It is nanogold that 12000rpm centrifugations 30min, which collects precipitation,

It is bluish violet (illustration in such as Fig. 4) after precipitated nanocrystals gold obtained by the present embodiment is resuspended with water, its ultraviolet light can See absorption curve as shown in figure 4, only there is single absworption peak at 540nm, illustrate nanogold particle size distribution range 30~ 80nm, and purity is high.

The present embodiment can synthesize the larger nanogold of particle diameter by improving the solubility of nanogold, illustrate the inventive method pair The particle diameter of gained nanogold has controllability, can synthesize the nanogold of purpose size as needed.

Comparative example 1

1) 1mmol/L gold chlorides (HAuCl is configured₄)。

2) 25ml bacteria culture medias add appropriate Escherichia coli overnight incubation 3000rpm and bacterium, 2ml sterilized waters are collected by centrifugation It is resuspended.

3) 1ml escherichia coli suspensions (5 × 10 are taken⁸CFU/mL) with 2ml HAuCl₄Magnetic agitation, it is well mixed, reaction 6 is small When.

4) products therefrom is collected, 4000rpm centrifugations remove precipitated impurities, take supernatant to react 30min, 12000rpm in 80 DEG C It is nanogold to centrifuge 30min and collect precipitation.

Precipitated nanocrystals gold is light after it is resuspended with water as shown in Fig. 5 left figure centrifuge tube ttom of pipe part obtained by this comparative example Pink is as shown in Fig. 5 right figures；There it can be seen that the amount of the generation nanogold of comparative example 1 is considerably less than the life of embodiment 1~3 Cheng Liang, illustrate in bacteria concentration and HAuCl₄In the case of dosage identical, the ability of staphylococcus aureus biosynthesis nanogold Considerably beyond Escherichia coli.

This comparative example result illustrates, although Escherichia coli can synthesize nanogold, compared with staphylococcus aureus, Escherichia coli synthesis nanogold yield is relatively low, and color is shallow compared with nanogold caused by staphylococcus aureus, gained nanometer after centrifugation Gold precipitation is also fewer.

Comparative example 2

1) 1mmol/L gold chlorides (HAuCl is configured₄)。

2) 25ml bacteria culture medias add appropriate fecal bacteria overnight incubation 3000rpm and bacterium, 2ml sterilized water weights are collected by centrifugation It is outstanding.

3) 1ml fecal bacterias suspension and 2ml HAuCl are taken₄Magnetic agitation, it is well mixed, reacts 6 hours.

4) products therefrom is collected, 4000rpm centrifugations remove precipitated impurities, take supernatant to be reacted 30 minutes in 80 DEG C, It is nanogold that 12000rpm, which is centrifuged and collected within 30 minutes precipitation,.

Precipitated nanocrystals gold is light after it is resuspended with water as shown in Fig. 6 left figure centrifuge tube ttom of pipe part obtained by this comparative example Pink is as shown in Fig. 6 right figures；There it can be seen that the amount that comparative example 2 generates nanogold is considerably less than in embodiment 1~3 Growing amount, illustrate in bacterial concentration and HAuCl₄In the case of dosage identical, staphylococcus aureus biosynthesis nanogold Ability is considerably beyond fecal bacteria.

This comparative example result illustrates, although fecal bacteria can synthesize nanogold, the excrement compared with staphylococcus aureus Coccus synthesis nanogold yield substantially reduces.

Comparative example 3

1) 1mmol/L gold chlorides (HAuCl is configured₄)。

2) 25ml bacteria culture medias add appropriate enterococcus overnight incubation 3000rpm and bacterium, 2ml sterilized water weights are collected by centrifugation It is outstanding.

3) 1ml enterococcus suspension and 2ml HAuCl are taken₄Magnetic agitation, it is well mixed, reacts 6 hours.

Precipitated nanocrystals gold is light after it is resuspended with water as shown in Fig. 7 left figure centrifuge tube ttom of pipe part obtained by this comparative example Pink is as shown in Fig. 7 right figures；There it can be seen that the amount of the generation nanogold of comparative example 3 is considerably less than the life of embodiment 1~3 Cheng Liang, illustrate in bacteria concentration and HAuCl₄In the case of dosage identical, the ability of staphylococcus aureus biosynthesis nanogold Considerably beyond enterococcus.

This comparative example result illustrates, although enterococcus can synthesize nanogold, the intestines compared with staphylococcus aureus Coccus synthesis nanogold yield substantially reduces.

The biocompatibility of the nanogold prepared below to the present invention detects.

Experimental group：

1) it is 60%~70% to cultivate U251 cells length to density；

2) nanogold (nanogold obtained after 12000rpm centrifugations) that prepared by Example 1, is handled by 20 μ g/ml concentration Cell 12 hours, detect cell growth status.

Control group：Except nanogold used is different with experimental group, other same experimental groups, this group nanogold used is Nanogold in the step 3) of embodiment 1 in 4000rpm centrifuged supernatants.

Blank control group：Nanogold is not added with except used, other operations are identical with experimental group.

Testing result is as shown in figure 8, there it can be seen that nanogold processing U251 cells prepared by embodiment 1, cell life It is long good, a large amount of nanogold are swallowed into the cell, are illustrated that the biocompatibility of nanogold prepared by the present invention is very good, are not influenceed thin The growth of born of the same parents, and be easily phagocytized by cells.And after the nanogold processing cell in 4000rpm centrifuged supernatants, have no cell Survival, cell also unopsonized nanogold, illustrates that the nanogold has high cell toxicity.Though the nanogold after 4000rpm centrifugations Large granular impurity so is eliminated, but still there is cytotoxicity.

Claims

A kind of 1. method using staphylococcus aureus biosynthesis nanogold, it is characterised in that：Comprise the following steps：

1) staphylococcus aureus liquid living and gold chloride are mixed, mixed liquor is reacted 4~6h；

2) upper step reaction solution is centrifuged, collects supernatant and continue 0.5~48h of reaction；

3) upper step reaction solution is centrifuged, collection, which precipitates, produces nanogold.
2. according to the method for claim 1, it is characterised in that：Staphylococcus aureus is dense in the step 1) mixed liquor Spend for 1 × 10⁸~1 × 10⁹CFU/mL。
3. according to the method for claim 1, it is characterised in that：In the step 1) mixed liquor concentration of gold chloride be 1 × 10^-2~1 × 10²mmol/L。
4. according to the method for claim 1, it is characterised in that：The temperature of step 1) the mixed liquor reaction is 15~37 ℃。
5. according to the method for claim 1, it is characterised in that：The rotating speed of the step 2) centrifugation is 3000~6000rpm.
6. according to the method for claim 1, it is characterised in that：The temperature of the step 2) reaction is 20~80 DEG C.
7. according to the method for claim 1, it is characterised in that：The rotating speed of the step 3) centrifugation be 10000~ 15000rpm。
8. according to the method for claim 1, it is characterised in that：The time of the step 3) centrifugation is 20~60min.
9. according to the method for claim 1, it is characterised in that：Prepared nanogold has biocompatibility.