CN106880842A - A kind of preparation method and application of copper sulfide nano material - Google Patents
A kind of preparation method and application of copper sulfide nano material Download PDFInfo
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Abstract
The invention discloses a kind of copper sulfide nano material preparation method near infrared absorption performance and its to Escherichia coli and the near infrared light bactericidal property of staphylococcus aureus, belong to metal nano material to prepare and biomedical applications field, the synthesis of the copper sulfide nano material uses immersion method, reaction condition is gentle, method is simple, by to temperature, the regulation of pH and enzyme protein dosage, successfully prepare uniform particle diameter, good dispersion, there is the copper sulfide nano material of strong absorbent energy in 1000 nm or so, under 980 nm near infrared lights, near infrared light effectively can be changed into heat energy by the material, Escherichia coli and staphylococcus aureus can be killed in several minutes, the unique photo-thermal sterilizing ability of the material, cheap cost can provide an alternative Therapeutic mode for clinical treatment skin disease.
Description
Technical field
Prepared the present invention relates to metal nano material and biomedical applications field, and in particular to a kind of BSA-CuS nanometers
The preparation method and application of composite.
Background technology
Either medicine, food, public health or other etc. every field, bacterium infection has become whole world face
The challenge faced.According to incompletely statistics, global food industry causes nearly 600,000,000,000 dollars of loss due to germ contamination every year.Another
On the one hand, many patients die from postoperative bacterium infection.According to the World Health Organization（WHO）Report, China have 2,000,000 people every year
Die from bacterium infection.At present, antibiotic is still the key agents for being clinically used for treating bacterium infection.It is existing so far from finding
The history of nearly 100 years, being capable of effectively bacteria growing inhibiting and breeding.However, the substantial amounts of use of antibiotic, abuse with
And irregular processing mode, bacterial dissociation is result in, occur in that drug resistance.At the same time, antibiotic is detected in underground water
Presence, seriously threaten life and the health of people.Another significant drawbacks of antibiotic are the development times long, use week
Phase is short, and existing antibiotic only reaches the mankind and uses 10-20.Therefore, a kind of new material is researched and developed to replace antibiotic to kill bacterium
And do not produce drug-fast bacteria extremely urgent.
In recent years, nano material is received more and more attention due to its unique physicochemical property, and many nano materials are
It is confirmed in experiment and can be used to kill bacterium.Mainly include Nano silver grain, golden nanometer particle, various metal oxides and oxidation stone
Black alkene etc..Wherein, Nano silver grain is a kind of material being most widely used.Just contained using silver-colored bowl, silver cup since ancient times
Dress Food Preservation.However, the shortcoming of Nano silver grain is once being exposed in air or water, silver ion begins to slow release
To in environment, and the conversion between chemistry and biology is carried out, very big harm is caused to the ecosystem.To the poison of a large amount of organisms
Property experiment be also demonstrated that minimal amount of silver will cause very big injury to body.Simultaneously because silver colloidal solution is by quiet
Electric exclusion principle synthesizes, very unstable in body, causes its anti-microbial property and dispersiveness to obtain very big slackening.Separately
On the one hand, graphene oxide and gold nano-material are affected by very big concern.Although they also have very in antibiosis
Big advantage, but body limits them to the non-biodegradable and unknown toxicity and the expensive cost of gold of graphene oxide
In further applying for clinical and laboratory.
Copper sulfide nano material, it is with low cost because its synthetic method is simple, the advantages of good biocompatibility, turn into
The focus of nano materials research.Cu in nano copper sulfate particle2+ The transition of d-d energy levels, makes it have very strong near infrared light and inhales
Receipts ability, can well convert light energy into heat energy.The CuS nano-particles average grain diameter that we synthesize, can in 50 nm or so
To be easily attached on bacterium surface and further be entered into inside bacterium by endocytosis.Under near infrared light,
The heat of generation can destroy the cell membrane and organelle of bacterium, and the apoptosis of bacterium is directly resulted in from most root.With antibiotic
Treatment compare, we provide a kind of simple, effective near-infrared method for disinfection.
The content of the invention
It is an object of the invention to provide a kind of preparation method of BSA-CuS nano composite materials, it is applied to near-infrared and kills
Bacterium, the doses of material that the method for disinfection is used is low, with low cost, and bactericidal effect substantially, does not almost have to ecological and human body environment
Influence.Can be used for the treatment to staphylococcus aureus and ETEC.
In order to reach above-mentioned purpose, solution of the invention is：
The preparation method preparation process of BSA-CuS nano composite materials is comprised the following steps：
(1) by Cu (NO that 5 mL concentration are 10 mM3)2Solution is added to 5 mL concentration for 5 mg/mL bovine serum albumin(BSA)s BSA
In solution, 30 min are stirred at room temperature, obtain BSA and Cu2+The mixed liquor of conjugate, solution is light blue color；
（2）By step（1）The pH of the mixed liquor of gained uses concentration that 3.0 are adjusted to for 95% salpeter solution, adds 0.5ml concentration
In for the thioacetyl amine aqueous solution of 0.2M, rear solution is shaken up in faint yellow, above-mentioned solution is placed in 90 DEG C of thermostat water baths and is incubated
1 h is educated, BSA-CuS nano composite materials are obtained.
The BSA-CuS nano materials that the preparation method of BSA-CuS nano composite materials is prepared are in light thermal sterilization
Using.
Application of the BSA-CuS nano materials in light thermal sterilization, comprises the following steps：
First, fresh staphylococcus aureus solution and ETEC solution are prepared, and adjusts bacterial density and be about 106
CFU/ml；
2nd, 24 10ml blake bottles are divided into tetra- groups of abcd, respectively numbering a1-a6、b1-b6、c1-c6、d1-d6, two groups of a, b each
2 ml step one gained staphylococcus aureus solution are separately added into bottle, 2 ml are separately added into two groups of each bottles of c, d
Step one gained ETEC solution, then, a1In add PBS and compareed, in a2-a6It is middle to add 2 ml respectively
Concentration is respectively the BSA-CuS solution of 10 ppm, 20 ppm, 30 ppm, 40 ppm, 50 ppm, b1-b6、c1-c6、d1-d6Respectively
Add and a1-a6The same amount of same substance of correspondence, sets a groups and c groups not irradiate group, and b groups and d groups are irradiation group；
3rd, to tetra- groups of abcd in step 2 after 37 DEG C of min of Constant temperature hatch 15, irradiation group b, d is swashed with 1.59 W near-infrareds
The min of light irradiation 20, then supplements after the inoculum of 2 ml in 37 DEG C of biochemical cultures in each blake bottle of tetra- groups of abcd
Continue to hatch 12 h in case；
4th, repeat step one is to step 33 times.
The present invention provides a kind of preparation method and applications of BSA-CuS nano composite materials and has the advantages that：
The sterilization dose for using is low, with low cost, and substantially, the pressure caused to environment is small, can be used for golden yellow for bactericidal effect
Staphylococcus and the treatment of ETEC.
Brief description of the drawings
Fig. 1 is the TEM figures of the BSA-CuS nano composite materials that the present invention is prepared；
Fig. 2 is the BSA-CuS nano composite materials for preparing ultraviolet spectrogram after near-infrared pre-irradiation is carried out of the invention；
Fig. 3 is photothermal deformation broken line graph of the BSA-CuS nano composite materials for preparing of the invention under various concentrations；
Fig. 4 is that the BSA-CuS nano composite materials that the present invention is prepared exist to staphylococcus aureus and ETEC
Agar plate bactericidal effect figure under the conditions of different disposal；
Fig. 5 is that the BSA-CuS nano composite materials that the present invention is prepared exist to staphylococcus aureus and ETEC
Solution turbidity comparison diagram under the conditions of different disposal；
Fig. 6 is that the BSA-CuS nano composite materials that the present invention is prepared exist to staphylococcus aureus and ETEC
Solution O D value curve maps under the conditions of different disposal；
Fig. 7 be the BSA-CuS nano composite materials that prepare of the present invention to staphylococcus aureus under the conditions of different disposal
Fluidic cell figure；
Fig. 8 is that the BSA-CuS nano composite materials that the present invention is prepared are thin under the conditions of different disposal to ETEC
Bacterium SEM schemes；
Fig. 9 be the BSA-CuS nano composite materials that prepare of the present invention to staphylococcus aureus under the conditions of different disposal
Bacterial fluorescence microscope figure, green represents viable bacteria, and red represents dead bacterium.
Specific embodiment
In order to technical scheme is explained further, the present invention is explained in detail below by specific embodiment
State.
Embodiment one
A kind of preparation method of BSA-CuS nano composite materials, comprises the following steps：
(1) by Cu (NO that 5 ml concentration are 10 mM3)2Solution adds to 5 mL concentration for 5 mg/mL bovine serum albumin(BSA)s
In BSA solution, 30 min being stirred at room temperature, obtaining generating the BSA-Cu suspensions of BSA-Cu conjugates, solution colour is in shallow
It is blue.
（2）By step（1）The pH of the BSA-Cu suspensions of gained uses concentration that 3.0 are adjusted to for 95% salpeter solution, adds
0.5ml concentration is the thioacetyl amine aqueous solution of 0.5 M, shakes up rear solution in faint yellow.Above-mentioned solution is placed in 90 DEG C of waters bath with thermostatic control
1 h is incubated naturally in pot, and BSA-CuS nano composite materials are obtained.
Embodiment two
The BSA-CuS nano composite materials of the gained of embodiment one are carried out into TEM signs, as a result as shown in figure 1, CuS nano-particles
Average grain diameter in 50 nm or so.
Embodiment three
The BSA-CuS nano composite materials of the gained of embodiment one are carried out into ultraviolet spectra inspection respectively before and after near infrared light
Survey, shown in result figure 2, obtained BSA-CuS nano composite materials show the present invention near infrared region all existing characteristics absworption peaks
The BSA-CuS nano composite materials of synthesis have the photothermal conversion efficiency of stabilization.
Example IV
The BSA-CuS nano composite materials of the various concentrations of the gained of embodiment one are carried out into photothermal deformation experiment, as a result such as Fig. 3 institutes
Show, as a result show, BSA-CuS nano composite materials have warming phenomenon, and the concentration of BSA-CuS nano composite materials is bigger,
Temperature rise effect is better.
Embodiment five
Fresh staphylococcus aureus solution and ETEC solution are prepared, regulation bacterial density is about 106 CFU/ml。
The above-mentioned staphylococcus aureus solution of 50 μ l is added in four 96 orifice plates each instrument connections, different control groups are set：A adds 50
μ l, the BSA-CuS near-infrareds of 50 ppm irradiate 5 min, and b adds 50 μ l, the BSA-CuS of 50 ppm not to irradiate, and c adds 50 μ l's
PBS near-infrareds irradiate 5 min, and d adds the PBS of 50 μ l not irradiate；Add 50 μ l above-mentioned in four 96 orifice plates each instrument connections
Escherichia coli solution, sets different control groups：E adds 50 μ l, the BSA-CuS near-infrareds of 50 ppm irradiate 5 min, and f adds 50 μ
L, the BSA-CuS of 50 ppm do not irradiate, and g adds the PBS near-infrareds of 50 μ l to irradiate 5 min, and h adds the PBS of 50 μ l not irradiate so
Afterwards by above-mentioned treated solution uniform application to agar plate, then 16h is cultivated in 37 DEG C of biochemical cultivation cases.Such as Fig. 4 institutes
Show, for staphylococcus aureus, a agar plates only occur in that several bacterium colonies of very little, are then overgrowed with b, c, d agar plate
Staphylococcus aureus.For Escherichia coli, do not occur colony growth situation on e agar plates, covered with f, g, h agar plate
Escherichia coli, illustrate that BSA-CuS nano composite materials of the present invention have apparent bactericidal effect under near-infrared irradiation.
Embodiment six：
Take 24 10ml blake bottles and be divided into tetra- groups of abcd, respectively numbering a1-a6、b1-b6、c1-c6、d1-d6, two groups of each bottles of a, b
The gained staphylococcus aureus solution of 2 ml embodiments five is separately added into son, 2 ml are separately added into two groups of each bottles of c, d
The gained ETEC solution of embodiment five, then, a1In add PBS and compareed, in a2-a6It is middle to add 2 respectively
Ml concentration is respectively the BSA-CuS solution of 10 ppm, 20 ppm, 30 ppm, 40 ppm, 50 ppm, b1-b6、c1-c6、d1-d6Point
Do not add and a1-a6The same amount of same substance of correspondence, sets a groups and c groups not irradiate group, and b groups and d groups are irradiation group；
Tetra- groups of abcd irradiates 20 min, then to irradiation group b, d after 37 DEG C of min of Constant temperature hatch 15 with 1.59 W near-infrared lasers
Continue to hatch 12 h in 37 DEG C of biochemical cultivation cases after supplementing the inoculum of 2 ml in each blake bottle of tetra- groups of abcd；
Repeat experiment three times.As shown in figure 5, in no near-infrared irradiation group, solution is all muddiness, and near-infrared irradiation group, it is golden yellow
Staphylococcus and ETEC start solution clarification occur respectively in 30 ppm, 20 ppm, illustrate that BSA-CuS of the present invention receives
Nano composite material does not have bactericidal effect under the irradiation of no near-infrared, and under near-infrared effect, concentration is 30 ppm
BSA-CuS nano composite materials just produce bactericidal effect to staphylococcus aureus, and concentration is BSA-CuS nanometers of 20 ppm multiple
Condensation material produces bactericidal effect to ETEC.
Embodiment seven
First 24 holes, are divided into tetra- groups of abcd, respectively numbering a in 96 orifice plates1-a6、b1-b6、c1-c6、d1-d6, two groups of a, b each
The gained staphylococcus aureus solution of 50 μ l embodiments five is separately added into hole, 50 μ l realities are separately added into two groups of each holes of c, d
Apply the gained ETEC solution of example five, then, a1In add PBS and compareed, in a2-a6It is middle to add 50 μ l respectively
Concentration is respectively the BSA-CuS solution of 10 ppm, 20 ppm, 30 ppm, 40 ppm, 50 ppm, b1-b6、c1-c6、d1-d6Respectively
Add and a1-a6The same amount of same substance of correspondence, sets a groups and c groups not irradiate group, and b groups and d groups are irradiation group.Every group
Test in triplicate, after hatching 15 min in 37 DEG C of constant temperature, irradiated with 1.59 W near-infrared lasers, per the min of hole 5, then often
Hole supplements the inoculum of 50 μ l, and 12 h are cultivated in 37 DEG C of biochemical cultivation cases, with each hole of ELIASA test at 450 nm
Absorbance (OD450 nm).As shown in fig. 6, scheming BSA-CuS nano composite materials that left concentration is 30 ppm just to golden yellow
Staphylococcus produces bactericidal effect, schemes the BSA-CuS nano composite materials that right concentration is 20 ppm and ETEC is produced
Bactericidal effect, illustrates that BSA-CuS nano composite materials of the present invention do not have bactericidal effect under the irradiation of no near-infrared, and near
Bactericidal effect is produced to staphylococcus aureus and ETEC under infrared effect.
Embodiment eight
The made staphylococcus aureus solution of 2 ml embodiments five is taken respectively, adds 2 ml inoculums, 37 DEG C of constant temperature trainings
12 h are supported, then different control groups are set：1. the BSA-CuS near-infrareds of 2 ml, 50 ppm are added to irradiate 40 min.2. 2 are added
Ml, the BSA-CuS of 50 ppm do not irradiate.3. the PBS near-infrareds of 2 ml are added to irradiate 40 min.4. add 2ml PBS not according to
Penetrate.Continue the h of Constant temperature hatch 12.Take 1 ml high speed centrifugations（10000 g）Disperseed with normal saline dilution after treatment removal nutrient solution
To 1 ml, it is centrifuged three times after 37 DEG C of hatching 1h, takes the 10 above-mentioned bacterium solutions of μ l and be added in fluidic cell pipe, is dispersed to physiological saline
1ml.The dead bacterium of work is dyeed using work dead Bacterial stain kit, flow cytometer is used after static 15 min of room temperature lucifuge
Detected.As seen from Figure 7, under near-infrared treatment with irradiation, the survival rate of bacterium is only for BSA-CuS nano composite materials of the present invention
Have 13.7%, well below the survival rate of other treatment conditions bacteriums, show that BSA-CuS nano composite materials are irradiated in near-infrared
Under the conditions of, there is good bactericidal effect to bacterium.
Embodiment nine
Take 1 ml and take the made ETEC solution of 2 ml embodiments five respectively, add 2 ml inoculums, 37 DEG C of perseverances
Temperature 12 h of culture, add the BSA-CuS near-infrareds of 2 ml, 50 ppm to irradiate 40 min, continue the h of Constant temperature hatch 12, are with concentration
35% glutaraldehyde carries out serial dehydration with the alcohol of various concentrations again after fixing 30min, uses transmission electron microscope special purpose copper
Net prepares sample and dries at room temperature, and bacterium pattern is observed under transmission electron microscope；As seen from Figure 8, large intestine angstrom
Uncommon Salmonella surface adhesion many nano particles, and the membrane structure of bacterium is destroyed, and situations such as being broken broken occurs, shows
ETEC is dead.
Embodiment ten
From embodiment eight live dead Bacterial stain kit the dead bacterium of work is dyeed after solution in take 10 μ l solution and be applied to
It is used for fluorescence microscope on slide, sequence number is 1. 2. 3. 4. 1. 2. 3. 4. corresponding with sequence number in example 8 respectively in Fig. 9, green
Viable bacteria is represented, red represents dead bacterium.Thus BSA-CuS nano composite materials of the present invention can be explained right under near-infrared treatment with irradiation
Bacterium has very strong bactericidal effect.
Therefore, the present invention provides a kind of near-infrared method for disinfection of BSA-CuS nano composite materials, with following notable effect
Really：
The sterilization dose for using is low, with low cost, and substantially, the pressure caused to environment is small, can be used for golden yellow for bactericidal effect
Staphylococcus and the treatment of ETEC.
Above-described embodiment and schema and non-limiting product form of the invention and style, any art it is common
Appropriate change or modification that technical staff is done to it, all should be regarded as not departing from patent category of the invention.
Claims (3)
- The preparation method of 1.BSA-CuS nano composite materials, it is characterised in that：Preparation process is comprised the following steps：(1) by Cu (NO that 5 mL concentration are 10 mM3)2Solution is added to 5 mL concentration for 5 mg/mL bovine serum albumin(BSA)s BSA In solution, 30 min are stirred at room temperature, obtain BSA and Cu2+The mixed liquor of conjugate, solution is light blue color；（2）By step（1）The pH of the mixed liquor of gained uses concentration that 3.0 are adjusted to for 95% salpeter solution, adds 0.5ml concentration In for the thioacetyl amine aqueous solution of 0.2M, rear solution is shaken up in faint yellow, above-mentioned solution is placed in 90 DEG C of thermostat water baths and is incubated 1 h is educated, BSA-CuS nano composite materials are obtained.
- 2. the BSA-CuS nanometers of material that the preparation method of BSA-CuS nano composite materials according to claim 1 is prepared Expect the application in light thermal sterilization.
- 3. application according to claim 2, it is characterised in that comprise the following steps：First, fresh staphylococcus aureus solution and ETEC solution are prepared, and adjusts bacterial density and be about 106 CFU/ ml；2nd, 24 10ml blake bottles are divided into tetra- groups of abcd, respectively numbering a1-a6、b1-b6、c1-c6、d1-d6, two groups of a, b each 2 ml step one gained staphylococcus aureus solution are separately added into bottle, 2 ml are separately added into two groups of each bottles of c, d Step one gained ETEC solution, then, a1In add PBS and compareed, in a2-a6It is middle to add 2 ml respectively Concentration is respectively the BSA-CuS solution of 10 ppm, 20 ppm, 30 ppm, 40 ppm, 50 ppm, b1-b6、c1-c6、d1-d6Respectively Add and a1-a6The same amount of same substance of correspondence, sets a groups and c groups not irradiate group, and b groups and d groups are irradiation group；3rd, to tetra- groups of abcd in step 2 after 37 DEG C of min of Constant temperature hatch 15, irradiation group b, d is swashed with 1.59 W near-infrareds The min of light irradiation 20, then supplements after the inoculum of 2 ml in 37 DEG C of biochemical cultures in each blake bottle of tetra- groups of abcd Continue to hatch 12 h in case；4th, repeat step one is to step 33 times.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108530470A (en) * | 2018-03-30 | 2018-09-14 | 上海师范大学 | A kind of copper complex of temperature triggered and its preparation method and application |
| CN109580607A (en) * | 2018-12-13 | 2019-04-05 | 泉州师范学院 | The preparation and its application of MXene-CuS nanocomposite |
| CN109809466A (en) * | 2019-03-27 | 2019-05-28 | 泉州师范学院 | A kind of preparation method and application of the copper sulfide nano material based on egg shell template |
| CN114617964A (en) * | 2022-03-17 | 2022-06-14 | 常州大学 | Enzyme-responsive photo-thermal nano material G @ CuS and preparation method thereof |
| CN114886918A (en) * | 2022-02-17 | 2022-08-12 | 上海交通大学医学院附属仁济医院 | Nano material capable of stimulating immune system to cooperate with antibiosis and preparation method thereof |
| CN116035033A (en) * | 2022-10-17 | 2023-05-02 | 中南大学 | Preparation method of synthetic CuS nanocomposite and sterilization application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108530470A (en) * | 2018-03-30 | 2018-09-14 | 上海师范大学 | A kind of copper complex of temperature triggered and its preparation method and application |
| CN109580607A (en) * | 2018-12-13 | 2019-04-05 | 泉州师范学院 | The preparation and its application of MXene-CuS nanocomposite |
| CN109580607B (en) * | 2018-12-13 | 2021-06-29 | 泉州师范学院 | Preparation and application of MXene-CuS nano composite material |
| CN109809466A (en) * | 2019-03-27 | 2019-05-28 | 泉州师范学院 | A kind of preparation method and application of the copper sulfide nano material based on egg shell template |
| CN109809466B (en) * | 2019-03-27 | 2021-06-29 | 泉州师范学院 | Preparation method and application of copper sulfide nano material based on eggshell template |
| CN114886918A (en) * | 2022-02-17 | 2022-08-12 | 上海交通大学医学院附属仁济医院 | Nano material capable of stimulating immune system to cooperate with antibiosis and preparation method thereof |
| CN114886918B (en) * | 2022-02-17 | 2023-12-08 | 上海交通大学医学院附属仁济医院 | Nanometer material capable of stimulating immune system to cooperatively resist bacteria and preparation method thereof |
| CN114617964A (en) * | 2022-03-17 | 2022-06-14 | 常州大学 | Enzyme-responsive photo-thermal nano material G @ CuS and preparation method thereof |
| CN116035033A (en) * | 2022-10-17 | 2023-05-02 | 中南大学 | Preparation method of synthetic CuS nanocomposite and sterilization application thereof |
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