CN108333164A - A kind of Nano diamond biology Raman microprobe and its preparation and application - Google Patents
A kind of Nano diamond biology Raman microprobe and its preparation and application Download PDFInfo
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- CN108333164A CN108333164A CN201710041570.9A CN201710041570A CN108333164A CN 108333164 A CN108333164 A CN 108333164A CN 201710041570 A CN201710041570 A CN 201710041570A CN 108333164 A CN108333164 A CN 108333164A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/65—Raman scattering
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/65—Raman scattering
- G01N2021/653—Coherent methods [CARS]
- G01N2021/656—Raman microprobe
Abstract
The invention discloses a kind of Nano diamond biology Raman microprobe and its preparations and application, belong to antimicrobial technology field.A kind of Nano diamond biology Raman microprobe, the Nano diamond biology Raman microprobe include Nano diamond and non-antibiotic class antibacterial material；For the Nano diamond biology Raman microprobe using Nano diamond as carrier, the Nano diamond and non-antibiotic class antibacterial material of carboxylated are combined by way of Electrostatic Absorption forms Nano diamond biology Raman microprobe.The Nano diamond biology Raman microprobe of the present invention is formed by the positive potential of non-antibiotic class antibacterial material and the negative electricity interdigit on Nano diamond surface by the combination of Electrostatic Absorption mode.The Nano diamond biology Raman microprobe can lossless observation non-antibiotic class antibacterial material and bacterial interactions process, react variety classes non-antibiotic antibacterial material system comprehensively influences caused by variety classes bacterium.
Description
Technical field
The present invention relates to antimicrobial technology field, more particularly, to a kind of Nano diamond biology Raman microprobe and its prepare and
Using.
Background technology
Since the first antibiotic is found, the mankind endure the puzzlement of its side effect to the fullest extent while largely utilizing antibiotic.
Bacterial resistance sex chromosome mosaicism grows in intensity, the global development strategy of World Health Organization tailor containment bacterial drug resistance, with
Postpone the propagation that bacterial drug resistance arrived and controlled on a large scale drug-resistant bacteria.But, it was reported that the drug resistance of bacterium is in always
Existing growth trend, and multidrug resistant and crossing drug resistant is presented in some bacteriums, and the drug resistance of bacterium is to treating clinical infection
Serious threat is constituted, some hospitals even the superbacteria that antibiotic can not be killed occur.Due to microorganism itself
Propagated, the bacterial drug resistance not instead of problem in some country or some area evolves into whole world facing most
One of urgent public health problem.We must find novel antibacterial substance to replace antibiotic to cope with the prestige of microorganism
The side of body.Also all in strongly searching and research and development novel antibacterial substance, these substances, lysozyme and antibacterial peptide are the past for international scientific man
Decades find the natural antimicrobial substance for having remarkable result, and it was found that quantity be also increasing year by year.
The antibacterial ability of lysozyme has just been widely studied since self-discovery, and abundant experimental results show that lysozyme has
As the potentiality of Substitutes For Antibiotic.Currently, lysozyme is applied to food preservation industry, disease research and clinical treatment neck
Domain.Carlos A.Rubi demonstrate lysozyme enterogastric diseases good therapeutic effect [Carlos A.Rubio.The
natural antimicrobial enzyme lysozyme is Up-regulated in Gastrointestinal
Infammatory Conditions.2014,3:73-92].In fact, before penicillin discovery, it is found in molten in nasal mucus
Bacterium enzyme, good anti-microbial property have been verified, and are found to study there are many lysozyme successively later.The antibacterial machine of lysozyme
Reason is than more visible, as a kind of hydrolase, the mainly acetylmuramic acid and Acetylglucos in hydrolytic bacteria whole cell peptidoglycan
Amine enzyme, by the formation of the Hydrolysis cell wall to peptide glycan so as to cause bacterial death.
In addition, antibacterial peptide is as another major class natural materials for being expected to substitute antibiotics, it is same attract extensive concern and
Research.The general length of antibacterial peptide be less than 100 amino acid residues, have it is amphipathic, most of antibacterial peptides contain more base
Amino acid and hydrophobic amino acid.Since its molecular weight is small, antibacterial peptide has low immunogenicity and higher thermal stability, and
Research shows that some antibacterial peptides keep good antibacterial activity in relatively low and higher pH.Further research verification, Duo Zhongkang
Bacterium peptide has the good performance for killing bacterium, and Zheng et al. verification R-Thanatin antibacterial polypeptides can inhibit the thin of bacterium
After birth is formed, to show good anti-microbial property.But due to focusing on novel antibacterial to the research emphasis of antibacterial peptide before
In the discovery of substance and the qualitative of its antibacterial effect, variety classes antibacterial peptide not deep enough to the research of antibacterial peptide Antibacterial Mechanism
It is less to the mechanism of action variance analysis of different bacterium, and then the actual application value of antibacterial peptide is influenced, limit facing for antibacterial peptide
Bed application potential.Therefore, it using a variety of observation and analysis means, studies and interacted between variety classes antibacterial peptide and different bacterium
Journey, it is significant for supplementing the research of antibacterial peptide Antibacterial Mechanism.
In numerous research approaches, by visible means, the interaction process between antibacterial material object bacterium is monitored,
It is a kind of important mode.In general, everybody uses Imaging-PAM, antibacterial peptide is marked with fluorescent molecular, for observing antibacterial
Peptide and bacterial interactions realize the visualization to entire sterilization process.But there is much urgently to be resolved hurrily ask in fluorescent technique
Topic, if stability is poor, photobleaching and the interference of archebiosis fluorescence etc. significantly limit the application of Imaging-PAM.And it draws
Graceful imaging technique, because having Noninvasive, the advantages such as spontaneous and high-resolution, before the wide application during organism is imaged
Scape is constantly excavated and is developed.
Therefore, the present invention provides a kind of Nano diamond biology Raman microprobe, using Raman image technology, realizes to non-anti-
The visualization of raw element class antibacterial material and bacterial interactions process, it is for non-antibiotic class antibacterial Quality Research and entirely anti-
Bacterium field has great importance.
Invention content
The first purpose of the invention is to provide a kind of Nano diamond biology Raman microprobes；The probe may be implemented to non-
The positioning of antibiotics antibacterial material.
Second object of the present invention is to provide a kind of preparation method of Nano diamond biology Raman microprobe；This method letter
It is single, it is easily operated.
Third object of the present invention is to provide a kind of applications of Nano diamond biology Raman microprobe；The application is with Raman
Based on imaging technique, the observation to non-antibiotic class antibacterial material and bacterial interactions process is realized.
In order to achieve the above objectives, the present invention uses following technical scheme：
A kind of Nano diamond biology Raman microprobe, the Nano diamond biology Raman microprobe include Nano diamond and non-
Antibiotics antibacterial material；
The Nano diamond biology Raman microprobe is using Nano diamond as carrier, the Nano diamond of carboxylated and non-anti-
Raw element class antibacterial material is combined by way of Electrostatic Absorption forms Nano diamond biology Raman microprobe.
Further, grain size >=50nm of the Nano diamond；The non-antibiotic class antibacterial material is thanatin, bacteriolyze
Enzyme or cecropin.
A kind of preparation method of Nano diamond biology Raman microprobe, includes the following steps：
1) Nano diamond carboxylated
With mixed acid carboxylation reaction occurs for Nano diamond, obtains the Nano diamond of carboxylated；
2) Electrostatic Absorption combination non-antibiotic class antibacterial material
Under 30-40 DEG C of water bath condition, the Nano diamond of carboxylated passes through Electrostatic Absorption non-antibiotic class antibacterial
Matter, in conjunction with formation Nano diamond biology Raman microprobe.
Further, step 1) is realized especially by following steps：
1) Nano diamond is mixed with mixed acid, then stirs 12-24h at 60-85 DEG C, obtain the first mixture；It will
Extra mixed acid is sucked out after being cooled to room temperature in first mixture, and sodium hydroxide then is added into first mixture,
50-120min is stirred at 60-85 DEG C, obtains the second mixture；The pH value for measuring second mixture, then uses deionized water
It is cleaned multiple times, its pH value is made to be in neutrality, obtain the Nano diamond of carboxylated；
Further, step 2) is realized especially by following steps：The Nano diamond of the carboxylated is distributed in water
Ultrasonic 5-20min obtains the Nano diamond aqueous solution of carboxylated；By the Nano diamond aqueous solution and non-antibiotic class of carboxylated
Antibacterial material aqueous solution is mixed, and 1-2h is stirred under 30-40 DEG C of water bath condition, obtains third mixture；The third is mixed
It closes object and carries out centrifugal treating, obtain Nano diamond biology Raman microprobe.
Further, the mixed acid is the mixture of sulfuric acid and nitric acid；The volume ratio of sulfuric acid and nitric acid is 2- in mixed acid
4:1.Nano diamond is carried out carboxylated by the mixed acid, to make its uniform surface.
Further, the Nano diamond is the diamond dust of high temperature and pressure synthesis.
Further, a concentration of 1-5mg/mL of the Nano diamond aqueous solution of the carboxylated (is, for example,：1,2,3,4 or
5mg/mL etc.)；A concentration of 200-600 μm of ol/L of the non-antibiotic class antibacterial material aqueous solution is (for example,：1,2,3,4 or
5mg/mL etc.).
Further, the volume ratio of the Nano diamond aqueous solution of the carboxylated and non-antibiotic class antibacterial material aqueous solution
It is 1：1-3；Preferably, the volume of the Nano diamond aqueous solution of the carboxylated and non-antibiotic class antibacterial material aqueous solution
Than being 1：1.
A kind of application of Nano diamond biology Raman microprobe, includes the following steps：
1) bacteria samples are prepared:By prepared Nano diamond biology Raman microprobe solution and bacterial action 0.5-
10min, the glutaraldehyde for being 2%-5% with mass percent are handled overnight, then use mass percent be 20%, 40%, 60%,
80% and 100% ethanol solution serial dehydration, obtains bacteria samples；In ethanol by institute's bacterium storage；
2) Raman image:The bacteria samples are dropped in and are carried on thin slice, microscopical 100 times of confocal is subsequently placed in
On hydroscope, with 532nm excitation wavelength light sources, Raman scanning, observation are carried out using line scan mode.
Further, the bacterium is Escherichia coli, bacillus subtilis or staphylococcus aureus etc..
It is further noted that if not otherwise specified, any range recorded in the present invention includes end value and end value
Between any numerical value and the arbitrary subrange that is constituted with any number between end value or end value.
Beneficial effects of the present invention are as follows：
1, Nano diamond biology Raman microprobe of the invention, by the positive potential and nanometer of non-antibiotic class antibacterial material
The negative electricity interdigit of diamond surface is formed by the combination of Electrostatic Absorption mode.The Nano diamond biology Raman microprobe is bacterium
The visualization of vital movement provides new method in system.
2, the preparation method of Nano diamond biology Raman microprobe of the invention is simple and convenient, easily operated.
3, the Raman image technology that the present invention uses, can effectively utilize Nano diamond biology Raman microprobe, lossless sight
Non-antibiotic class antibacterial material and bacterial interactions process are surveyed, reacts variety classes non-antibiotic antibacterial material system pair comprehensively
Influence caused by variety classes bacterium.
Description of the drawings
Specific embodiments of the present invention will be described in further detail below in conjunction with the accompanying drawings.
Fig. 1：Show the preparation flow figure of Nano diamond-thanatin Raman microprobe.
Fig. 2：Show the infrared spectrogram that thanatin is successfully adsorbed on Nano diamond.
Fig. 3：Show the grain size distribution of Nano diamond-thanatin Raman microprobe.
Fig. 4：Show the Zeta potential figure of Nano diamond-thanatin Raman microprobe.
Fig. 5：Show Nano diamond-thanatin Raman microprobe using Raman image visualization thanatin and withered grass gemma
The schematic diagram of bacillus interaction process.
Fig. 6：Under conditions of scanning electron microscope, Nano diamond, Nano diamond-thanatin Raman microprobe respectively with withered grass
The schematic diagram of bacillus interaction process.
Fig. 7：Show Nano diamond-lysozyme Raman microprobe using Raman image visualization lysozyme and withered grass gemma
The schematic diagram of bacillus interaction process.
Fig. 8：Under conditions of scanning electron microscope, Nano diamond, Nano diamond-lysozyme Raman microprobe respectively with large intestine
The schematic diagram of bacillus interaction process.
Fig. 9：(a) antibacterial effect of the Nano diamond-thanatin Raman microprobe to Escherichia coli is shown；(b) it shows
Antibacterial effect of the Nano diamond-thanatin Raman microprobe to bacillus subtilis.
Figure 10：(a) antibacterial effect of the Nano diamond-lysozyme Raman microprobe to Escherichia coli is shown；(b) it shows
Antibacterial effect of the Nano diamond-lysozyme Raman microprobe to bacillus subtilis.
Specific implementation mode
In order to illustrate more clearly of the present invention, with reference to preferred embodiment, the present invention is described further.Ability
Field technique personnel should be appreciated that following specifically described content is illustrative and be not restrictive, this should not be limited with this
The protection domain of invention.
Embodiment 1
1) Nano diamond carboxylated
Be by 0.5g grain sizes >=(mixed acid is that volume ratio is 3 with mixed acid for the Nano diamond of 50nm:1 sulfuric acid and nitre
Acid) mixing, it is then stirred at 70 DEG C for 24 hours, obtains the first mixture；It is extra to be sucked out after first mixture is cooled to room temperature
Then the sodium hydrate aqueous solution of a concentration of 0.1mol/L of 10mL is added, at 70 DEG C in mixed acid into first mixture
60min is stirred, the second mixture is obtained；The pH value for measuring second mixture, is then cleaned multiple times with deionized water, is made
Its pH value is in neutrality, and obtains the Nano diamond of carboxylated；
2) Electrostatic Absorption combination thanatin
The Nano diamond of the carboxylated is distributed to ultrasound 10min in water, obtains the carboxylated of a concentration of 10mg/mL
Nano diamond aqueous solution；The Nano diamond aqueous solution of the carboxylated of a concentration of 10mg/mL of 100uL and 100 μ L is a concentration of
200 μm of ol/L thanatin aqueous solutions are mixed, and are stirred 1h under 37 DEG C of water bath conditions, are obtained third mixture；By the third
Mixture carries out centrifugal treating 3min under conditions of 1200rpm, obtains Nano diamond-thanatin Raman microprobe.
Characterization result：
With Fourier transformation infrared spectrometer (Excalibur 3100, Varian, America), verifies thanatin and receive
Simple suction-operated between rice diamond；The results are shown in Figure 2, and a, b and c curve indicate thanatin, Nano diamond respectively in Fig. 2
With the absorption curve of Nano diamond-thanatin Raman microprobe；It obviously observes and derives from Nano diamond-thanatin (c)
Thanatin polypeptide is located at 1510-1580cm-1The peaks amide II and 1600-1700cm at place-1The amide I peaks at place.In comparison with death
The infrared spectrum of element (a) and Nano diamond (b), successful load of the thanatin on Nano diamond, and by c curves
Amide I peaks and the peaks amide II are verified, and the combination is non-covalent.
In addition, being detected using Zetasizer instrument (3000HS, Malvern Instruments, Malvern, England)
The particle diameter distribution (Fig. 3) and surface zeta potential current potential (Fig. 4) of the Raman microprobe of Nano diamond-thanatin；Fig. 3 experimental results are aobvious
Show, after modifying thanatin, the grain size of Nano diamond increases to 271.6nm by 102.2nm, this shows thanatin in nanometer Buddha's warrior attendant
The successful load on stone surface；Fig. 4 potentiometric analysis shows that the current potential on Nano diamond surface is by -15.27mV in conjunction with after thanatin
It is changed into 6.12mV, is caused by the positive potential of the thanatin of area load.This result is corresponding with above-mentioned infrared results,
Show to be combined by electrostatic adsorption between Nano diamond and thanatin.
3) Raman is visually realized
Prepared Nano diamond-thanatin Raman microprobe solution and bacillus subtilis are acted on into 0.5-10min, used
The glutaraldehyde that mass percent is 2% is handled overnight, and it is 20%, 40%, 60%, 80% and 100% then to use mass percent
Ethanol solution serial dehydration, obtain bacillus subtilis sample；By the bacillus subtilis sample drop on carrying thin slice, so
It is placed on the microscopical 100 times of hydroscopes of confocal, with 532nm excitation wavelength light sources, is drawn using line scan mode
Graceful scanning, observation.
Fig. 5：(a) and (c) be respectively bacillus subtilis Raman image figure and Raman spectrogram；(b) it is nanometer Buddha's warrior attendant
The Raman image figure of stone-thanatin Raman microprobe and bacillus subtilis interaction；(d) it is that Nano diamond-thanatin is drawn
The Raman spectrogram of graceful probe.
The Raman image figure for comparing Fig. 5 (a) and Fig. 5 (b) can be seen that action time by 3min, and Fig. 5's (b) is withered
There are a large amount of Nano diamond-thanatin Raman microprobe signals in careless Bacillus surface, shows bacillus subtilis surface aggregation
A large amount of diamond-thanatin Raman microprobes.Compare Fig. 5 (c) and Fig. 5 (d) Raman spectrograms, it can be seen that the spectral peak of Fig. 5 (d) goes out
Now apparent diamond nano signal, and Fig. 5 (c) is without clear signal.It these results suggest that, which visits
Needle can be efficiently used for the interaction process between observation thanatin and bacillus subtilis.
4) scanning electron microscope visually supplements Raman
In order to further verify and visualize the interaction process between non-antibiotic class antibacterial material and bacterium, use
The means of scanning electron microscope are observed, to supplement Raman image.Bacillus subtilis sample drop is added on silicon chip and is dried in the air
Dry, metal spraying is placed under scanning electron microscope, is observed.
Fig. 6：(a) and (b) is respectively Nano diamond and the scanning electron microscope (SEM) photograph of bacillus subtilis；(c) and (d) is respectively
The scanning electron microscope (SEM) photograph of Nano diamond and bacillus subtilis effect 1min and 3min；(e) and (f) is respectively Nano diamond-
The scanning electron microscope (SEM) photograph of thanatin Raman microprobe and bacillus subtilis effect 1min and 3min；It can be seen that from (c) and (d)
Nano diamond is very poor to bacillus subtilis adhesion, and through acting on after a period of time, Nano diamond is still only individual to be inhaled
It is attached on bacillus subtilis；As can be seen that Nano diamond-thanatin Raman microprobe is for withered grass bud from figure (e) and (f)
The adhesive force of spore bacillus is very strong, acts on 1min, just a large amount of aggregation Nano diamond-thanatin Ramans are visited on bacillus subtilis surface
Needle；3min is acted on, dead, thalli morphology change, intracellular organic matter outflow, the shape of only remaining epicyte occurs in bacillus subtilis
State.This shows that diamond-thanatin Raman microprobe to the anti-microbial property of bacillus subtilis is then destroyed by adsorption
The mode of bacterium internal charge balance plays a role, rather than the mode for destroying cell wall structure is realized.It these results suggest that, it should
Nano diamond biology Raman microprobe can be efficiently used for the interaction process between observation thanatin and bacterium.
Embodiment 2
1) Nano diamond carboxylated
Be by 0.5g grain sizes >=(mixed acid is that volume ratio is 3 with mixed acid for the Nano diamond of 50nm:1 sulfuric acid and nitre
Acid) mixing, it is then stirred at 70 DEG C for 24 hours, obtains the first mixture；It is extra to be sucked out after first mixture is cooled to room temperature
Then the sodium hydrate aqueous solution of a concentration of 0.1mol/L of 10mL is added, at 70 DEG C in mixed acid into first mixture
60min is stirred, the second mixture is obtained；The pH value for measuring second mixture, is then cleaned multiple times with deionized water, is made
Its pH value is in neutrality, and obtains the Nano diamond of carboxylated；
2) Electrostatic Absorption binding lysozyme
The Nano diamond of the carboxylated is distributed to ultrasound 10min in water, obtains the carboxylated of a concentration of 10mg/mL
Nano diamond aqueous solution；The Nano diamond aqueous solution of the carboxylated of a concentration of 10mg/mL of 100uL and 100 μ L is a concentration of
600 μm of ol/L Lysozyme in Aqueous Solution are mixed, and 1h is stirred under 37 DEG C of water bath conditions, obtain third mixture；By the third
Mixture carries out centrifugal treating 3min under conditions of 1200rpm, obtains Nano diamond-lysozyme Raman microprobe.
3) Raman is visually realized
The solution for preparing above-mentioned Nano diamond-lysozyme Raman microprobe first acts on 0.5- with different bacterium
10min, with mass percent be 2% glutaraldehyde fix overnight processing, after respectively use mass percent be 20%, 40%, 60%,
80% and 100% ethanol solution serial dehydration, obtains bacteria samples, is stored in 100% ethyl alcohol.Then bacterium is added dropwise
It carries on thin slice, is placed on the microscopical 100 times of hydroscopes of confocal, with 532nm excitation wavelength light sources, using line scan mode
Carry out Raman scanning, observation.
Fig. 7：(a) it is the Raman image figure of Escherichia coli；(b) it is Nano diamond-lysozyme Raman microprobe and large intestine bar
The Raman image figure of bacterium interaction；(c) A is the Raman spectrogram of Escherichia coli in, and B is Nano diamond-lysozyme Raman
The Raman spectrogram of probe.
The Raman image figure for comparing Fig. 7 (a) and 7 (b) can be seen that action time by 3min, the large intestine bar of 7 (b)
There are a large amount of Nano diamond-lysozyme Raman microprobe signals in bacterium surface, shows that surface of E. coli assembles a large amount of nanometer Buddha's warrior attendants
Stone-lysozyme Raman microprobe.Compare A curves and B curves in Fig. 7 (c) and can be seen that apparent Buddha's warrior attendant occurs in the spectral peak of B curves
Stone nanowire signal, and A curves are without clear signal.It these results suggest that, which can be effectively
For observing the interaction process between lysozyme and Escherichia coli.
4) scanning electron microscope visually supplements Raman
In order to further verify and visualize the interaction process between non-antibiotic class antibacterial material and bacterium, use
The means of scanning electron microscope are observed, to supplement Raman image.E. coli SampLes are added dropwise and are dried on silicon chip, are sprayed
Gold is placed under scanning electron microscope, is observed.
Fig. 8：(a) it is the scanning electron microscope (SEM) photograph of Escherichia coli；(b) it is sweeping after Escherichia coli and Nano diamond effect 3min
Retouch electron microscope；(c) it is the scanning electron microscope (SEM) photograph of Escherichia coli and Nano diamond-lysozyme Raman microprobe；(d) it Escherichia coli and receives
Scanning electron microscope (SEM) photograph after rice diamond-lysozyme Raman microprobe effect 3min.
From Fig. 8 (b) as can be seen that through acting on after a period of time, Nano diamond is still only adsorbed on individually large intestine bar
On bacterium；And Nano diamond-lysozyme Raman microprobe is very strong for the adhesive force of Escherichia coli, effect a period of time, large intestine bar
Nano diamond-lysozyme is largely assembled on bacterium surface, and death occur in Escherichia coli, and phage structure is destroyed, and cell wall goes out
Now rupture dissolved phenomenon.This show Nano diamond-lysozyme to the anti-microbial property of Escherichia coli be by adsorption,
Then cell wall hydrolysis destroys cell wall structure, this is different from the mode of action of above-mentioned thanatin.It these results suggest that, this is received
Rice diamond biology Raman microprobe can be efficiently used for the interaction process between observation lysozyme and Escherichia coli, and
It can be used for studying different types of non-antibiotic class antibacterial material antibacterial mechanisms.
Embodiment 3
1) Nano diamond carboxylated
Be by 0.5g grain sizes >=(mixed acid is that volume ratio is 3 with mixed acid for the Nano diamond of 50nm:1 sulfuric acid and nitre
Acid) mixing, it is then stirred at 70 DEG C for 24 hours, obtains the first mixture；It is extra to be sucked out after first mixture is cooled to room temperature
Then the sodium hydrate aqueous solution of a concentration of 0.1mol/L of 10mL is added, at 70 DEG C in mixed acid into first mixture
60min is stirred, the second mixture is obtained；The pH value for measuring second mixture, is then cleaned multiple times with deionized water, is made
Its pH value is in neutrality, and obtains the Nano diamond of carboxylated；
2) Electrostatic Absorption combination cecropin
The Nano diamond of the carboxylated is distributed to ultrasound 10min in water, obtains the carboxylated of a concentration of 10mg/mL
Nano diamond aqueous solution；The Nano diamond aqueous solution of the carboxylated of a concentration of 10mg/mL of 100uL and 100 μ L is a concentration of
400 μm of ol/L cecropin aqueous solutions are mixed, and are stirred 1h under 37 DEG C of water bath conditions, are obtained third mixture；By the third
Mixture carries out centrifugal treating 3min under conditions of 1200rpm, obtains Nano diamond-cecropin Raman microprobe.
Experimental example 4
Nano diamond-thanatin Raman microprobe detects the antibacterial effect of Escherichia coli
Experimental group：Escherichia coli are selected, in the culture medium of peptone, beef extract and sodium chloride composition, in 37 DEG C of cultures
Then 18h uses phosphate buffer (PBS solution) by cultured Escherichia coli, rinses and centrifuge three times.It will be thin after flushing
Bacterium is divided into three groups, then the PBS solution (a concentration of 1mg/mL), embodiment 1 of Nano diamond in embodiment 1 is used to prepare respectively
The PBS solution (a concentration of 1mg/mL) of Nano diamond-thanatin Raman microprobe and Nano diamond-thanatin Raman microprobe
The consistent PBS solution (a concentration of 40 μm of ol/L) containing thanatin of middle death cellulose content cultivates 1h.By above-mentioned culture solution after culture
Dilution 104Times, it is sufficiently stirred, the culture solution after 20 μ L dilutions is then taken to be added on the agar of culture dish.By culture dish 37
12-16h is cultivated at DEG C, uses colony counting method to evaluate anti-microbial property later.
Control group：Escherichia coli are selected, in the culture medium of peptone, beef extract and sodium chloride composition, in 37 DEG C of cultures
Then cultured Escherichia coli are rinsed and are centrifuged three times with PBS solution by 18h.Escherichia coli after flushing are directly added into
1h is cultivated in PBS solution, and above-mentioned culture solution is diluted 10 later4Times, it is sufficiently stirred, the culture solution after 20 μ L dilutions is then taken to add
Enter onto the agar of culture dish.Culture dish is cultivated into 12-16h at 37 DEG C, uses colony counting method to evaluate anti-microbial property later.
Experimental group and control group result are compared in conjunction with Fig. 9 a.Nano diamond, Escherichia coli survival rate are added in PBS
It is 99.02 ± 0.28%, shows that Nano diamond itself does not have lethal effect to bacterium at this concentration, verify Nano diamond
It is suitable as bioprobe；Nano diamond-thanatin Raman microprobe is added in PBS, the survival rate of Escherichia coli is 45.81
± 2.07%, show that Nano diamond-thanatin Raman microprobe can effectively kill Escherichia coli under the concentration；And
The thanatin consistent with dead cellulose content in Nano diamond-thanatin Raman microprobe, the survival rate of Escherichia coli are added in PBS
It is 39.93 ± 4.24%.This shows that the thanatin being carried on Nano diamond shows good anti-microbial property, and same in solution
The thanatin effect of concentration is close.The above experiment show, using Nano diamond as when bioprobe, in nanometer Buddha's warrior attendant
Thanatin is loaded on stone will not change the anti-microbial property of thanatin itself, for answering for the Nano diamond-thanatin Raman microprobe
With laying the foundation.
Embodiment 5
Nano diamond-thanatin Raman microprobe detects the antibacterial effect of bacillus subtilis
Experimental group：Bacillus subtilis is selected, in the culture medium of peptone, beef extract and sodium chloride composition, in 37 DEG C
Culture for 24 hours, then uses phosphate buffer (PBS solution) by cultured bacillus subtilis, rinses and centrifuge three times.It will punching
Bacterium after washing is divided into three groups, then uses the PBS solution (a concentration of 1mg/mL) of Nano diamond in embodiment 1 respectively, implements
The PBS solution (a concentration of 1mg/mL) of Nano diamond-thanatin Raman microprobe prepared by example 1 and Nano diamond-thanatin
The consistent PBS solution (a concentration of 40 μm of ol/L) containing thanatin of dead cellulose content cultivates 1h in Raman microprobe.It will will train later
Nutrient solution dilution 104Times, it is sufficiently stirred, 20 μ L is then taken to be added on the agar of culture dish.Culture dish is cultivated into 12- at 37 DEG C
16h uses colony counting method to evaluate anti-microbial property later.
Control group：Escherichia coli are selected, in the culture medium of peptone, beef extract and sodium chloride composition, in 37 DEG C of cultures
Then cultured bacillus subtilis is rinsed and is centrifuged three times with PBS solution by 18h.Bacterium after flushing is directly added into
1h is cultivated in PBS solution, culture solution will be diluted 10 later4Times, it is sufficiently stirred, 20 μ L is then taken to be added to the agar of culture dish
On.Culture dish is cultivated into 12-16h at 37 DEG C, uses colony counting method to evaluate anti-microbial property later.
Experimental group and control group result are compared in conjunction with Fig. 9 b.Nano diamond is added in PBS, bacillus subtilis is deposited
Motility rate is 100.02 ± 3.67%, shows that Nano diamond itself does not have lethal effect to bacillus subtilis at this concentration,
Verification Nano diamond is suitable as bioprobe；Nano diamond-thanatin Raman microprobe, withered grass gemma are added in PBS
The survival rate of bacillus is 65.58 ± 2.42%, shows that Nano diamond-thanatin Raman microprobe can be effectively under the concentration
Kill bacillus subtilis；And it is added in PBS and consistent dead of death cellulose content in Nano diamond-thanatin Raman microprobe
Element is died, the survival rate of bacillus subtilis is 60.58 ± 2.42%.This shows that the thanatin being carried on Nano diamond shows
Good anti-microbial property is approached with solution with the thanatin effect of concentration.The above experiment show, by Nano diamond
When as bioprobe, load thanatin will not change antibacterial of the thanatin to bacillus subtilis itself on Nano diamond
Performance lays the foundation for the application of the probe.
Experimental example 6
Nano diamond-lysozyme Raman microprobe detects the antibacterial effect of Escherichia coli
Experimental group：Escherichia coli are selected, in the culture medium of peptone, beef extract and sodium chloride composition, in 37 DEG C of cultures
Then 18h uses phosphate buffer (PBS solution) by cultured Escherichia coli, rinses and centrifuge three times.It will be thin after flushing
Bacterium is divided into three groups, then the PBS solution (a concentration of 1mg/mL), embodiment 1 of Nano diamond in embodiment 1 is used to prepare respectively
The PBS solution (a concentration of 1mg/mL) of Nano diamond-lysozyme Raman microprobe and Nano diamond-lysozyme Raman microprobe
The PBS solution (a concentration of 80 μm of ol/L) of the consistent lysozyme of middle death cellulose content cultivates 1h.Culture solution is diluted 10 later4
Times, it is sufficiently stirred, 20 μ L is then taken to be added on the agar of culture dish.Culture dish is cultivated into 12-16h at 37 DEG C, is adopted later
Anti-microbial property is evaluated with colony counting method.
Control group：Escherichia coli are selected, in the culture medium of peptone, beef extract and sodium chloride composition, in 37 DEG C of cultures
Then cultured Escherichia coli are rinsed and are centrifuged three times with PBS solution by 18h.Bacterium after flushing is directly added into PBS
1h is cultivated in solution, and culture solution is diluted 10 later4Times, it is sufficiently stirred, 20 μ L is then taken to be added on the agar of culture dish.It will
Culture dish cultivates 12-16h at 37 DEG C, uses colony counting method to evaluate anti-microbial property later.
Experimental group and control group result are compared in conjunction with Figure 10 a.Nano diamond, Escherichia coli survival are added in PBS
Rate is 101.02 ± 2.88%, shows that Nano diamond itself does not have lethal effect to bacterium at this concentration, verifies nanogold
Hard rock is suitable as bioprobe；Nano diamond-lysozyme Raman microprobe is added in PBS, the survival rate of Escherichia coli is
59.79 ± 4.05%, show that Nano diamond-lysozyme Raman microprobe can effectively kill Escherichia coli under the concentration；
And the lysozyme consistent with lysozyme content in Nano diamond-lysozyme Raman microprobe is added in PBS, Escherichia coli are deposited
Motility rate is 64.64 ± 1.28%.This shows that the lysozyme being carried on Nano diamond shows good anti-microbial property, with solution
In with concentration bacteriolyze enzyme effect it is close.The above experiment show, using Nano diamond as when bioprobe, in nanometer
Lysozyme is loaded on diamond will not change the anti-microbial property of lysozyme itself, lay the foundation for the application of the probe.
Experimental example 7
Nano diamond-lysozyme Raman microprobe detects the antibacterial effect of bacillus subtilis
Experimental group：Bacillus subtilis is selected, in the culture medium of peptone, beef extract and sodium chloride composition, in 37 DEG C
Culture for 24 hours, then uses phosphate buffer (PBS solution) by cultured bacillus subtilis, rinses and centrifuge three times.It will punching
Bacterium after washing is divided into three groups, then uses the PBS solution (a concentration of 1mg/mL) of Nano diamond in embodiment 1 respectively, implements
The PBS solution (a concentration of 1mg/mL) of Nano diamond-lysozyme Raman microprobe prepared by example 1 and Nano diamond-lysozyme
The consistent PBS solution (a concentration of 80 μm of ol/L) containing thanatin of dead cellulose content cultivates 1h in Raman microprobe.It then will culture
Liquid dilution 104Times, it is sufficiently stirred, the culture solution after 20 μ L dilutions is then taken to be added on the agar of culture dish.Culture dish is existed
12-16h is cultivated at 37 DEG C, uses colony counting method to evaluate anti-microbial property later.
Control group：Escherichia coli are selected, in the culture medium of peptone, beef extract and sodium chloride composition, in 37 DEG C of cultures
Then cultured bacillus subtilis is rinsed and is centrifuged three times with PBS solution by 18h.Bacterium after flushing is directly added into
1h is cultivated in PBS solution, and culture solution is diluted 10 later4Times, it is sufficiently stirred, the culture solution after 20 μ L dilutions is then taken to be added to
On the agar of culture dish.Culture dish is cultivated into 12-16h at 37 DEG C, uses colony counting method to evaluate anti-microbial property later.
Experimental group and control group result are compared in conjunction with Figure 10 b.Nano diamond, bacillus subtilis are added in PBS
Survival rate is 96.96 ± 2.73%, shows that Nano diamond itself does not have lethal effect to bacillus subtilis at this concentration,
Verification Nano diamond is suitable as bioprobe；Nano diamond-lysozyme Raman microprobe, withered grass gemma are added in PBS
The survival rate of bacillus is 53.15 ± 2.58%, shows that Nano diamond-lysozyme Raman microprobe can be effectively under the concentration
Kill bacillus subtilis；And it is added in PBS consistent with lysozyme content in Nano diamond-lysozyme Raman microprobe molten
The survival rate of bacterium enzyme, bacillus subtilis is 54.46 ± 1.33%.This shows that the lysozyme being carried on Nano diamond shows
Good anti-microbial property is approached with solution with the bacteriolyze enzyme effect of concentration.The above experiment show, by Nano diamond
When as bioprobe, load lysozyme will not change antibacterial of the lysozyme to bacillus subtilis itself on Nano diamond
Performance lays the foundation for the application of the probe.
Obviously, the above embodiment of the present invention be only to clearly illustrate example of the present invention, and not be pair
The restriction of embodiments of the present invention may be used also on the basis of the above description for those of ordinary skill in the art
To make other variations or changes in different ways, all embodiments can not be exhaustive here, it is every to belong to this hair
Row of the obvious changes or variations that bright technical solution is extended out still in protection scope of the present invention.
Claims (10)
1. a kind of Nano diamond biology Raman microprobe, it is characterised in that：The Nano diamond biology Raman microprobe includes nanometer
Diamond and non-antibiotic class antibacterial material；
The Nano diamond biology Raman microprobe is using Nano diamond as carrier, the Nano diamond and non-antibiotic of carboxylated
Class antibacterial material is combined by way of Electrostatic Absorption and forms Nano diamond biology Raman microprobe.
2. Nano diamond biology Raman microprobe according to claim 1, which is characterized in that the grain of the Nano diamond
Diameter >=50nm；The non-antibiotic class antibacterial material is thanatin, lysozyme or cecropin.
3. a kind of preparation method of Nano diamond biology Raman microprobe as claimed in claim 1 or 2, which is characterized in that including
Following steps：
1) Nano diamond carboxylated
With mixed acid carboxylation reaction occurs for Nano diamond, obtains the Nano diamond of carboxylated；
2) Electrostatic Absorption combination non-antibiotic class antibacterial material
Under 30-40 DEG C of water bath condition, the Nano diamond of carboxylated passes through Electrostatic Absorption non-antibiotic class antibacterial material, knot
Conjunction forms Nano diamond biology Raman microprobe.
4. preparation method according to claim 3, which is characterized in that step 1) is realized especially by following steps：
1) Nano diamond is mixed with mixed acid, then stirs 12-24h at 60-85 DEG C, obtain the first mixture；It will be described
Extra mixed acid is sucked out in first mixture after being cooled to room temperature, sodium hydroxide then is added into first mixture, in 60-
50-120min is stirred at 85 DEG C, obtains the second mixture；The pH value for measuring second mixture, is then carried out with deionized water
It is cleaned multiple times, its pH value is made to be in neutrality, obtain the Nano diamond of carboxylated.
5. preparation method according to claim 3, which is characterized in that step 2) is realized especially by following steps：By institute
The Nano diamond for stating carboxylated is distributed to ultrasound 5-20min in water, obtains the Nano diamond aqueous solution of carboxylated；By carboxylated
Nano diamond aqueous solution and non-antibiotic class antibacterial material aqueous solution mixed, stirred under 30-40 DEG C of water bath condition
1-2h obtains third mixture；The third mixture is subjected to centrifugal treating, obtains Nano diamond biology Raman microprobe.
6. preparation method according to claim 3, which is characterized in that the mixed acid is the mixture of sulfuric acid and nitric acid；
The volume ratio of sulfuric acid and nitric acid is 2-4 in mixed acid:1.
7. preparation method according to claim 5, which is characterized in that the Nano diamond aqueous solution of the carboxylated
A concentration of 1-5mg/mL；A concentration of 200-600 μm of ol/L of the non-antibiotic class antibacterial material aqueous solution.
8. preparation method according to claim 5, which is characterized in that the Nano diamond aqueous solution of the carboxylated and non-
The volume ratio of antibiotics antibacterial material aqueous solution is 1:1-3.
9. a kind of application of Nano diamond biology Raman microprobe as claimed in claim 1 or 2, which is characterized in that including as follows
Step：
1) bacteria samples are prepared:By prepared Nano diamond biology Raman microprobe solution and bacterial action 0.5-10min, use
The glutaraldehyde of 2%-5% is handled overnight, then with 20%, 40%, 60%, 80% and 100% ethanol solution serial dehydration, obtain
To bacteria samples；
2) Raman image:The bacteria samples are dropped in and are carried on thin slice, the microscopical 100 times of hydroscopes of confocal are subsequently placed in
On, with 532nm excitation wavelength light sources, Raman scanning, observation are carried out using line scan mode.
10. application according to claim 9, which is characterized in that the bacterium is bacillus subtilis, Escherichia coli or gold
Staphylococcus aureus.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111208066A (en) * | 2018-11-22 | 2020-05-29 | 清华大学 | Biological detection device and method |
| CN111328831A (en) * | 2020-03-06 | 2020-06-26 | 康特善利(上海)生物科技有限公司 | Antibacterial and antivirus material and application thereof |
| CN111362264A (en) * | 2020-03-19 | 2020-07-03 | 吉林大学 | Oxygen-containing nano-diamond for antibacterial treatment and preparation method thereof |
-
2017
- 2017-01-20 CN CN201710041570.9A patent/CN108333164A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111208066A (en) * | 2018-11-22 | 2020-05-29 | 清华大学 | Biological detection device and method |
| CN111208066B (en) * | 2018-11-22 | 2021-07-30 | 清华大学 | Biological detection device and method |
| CN111328831A (en) * | 2020-03-06 | 2020-06-26 | 康特善利(上海)生物科技有限公司 | Antibacterial and antivirus material and application thereof |
| CN111362264A (en) * | 2020-03-19 | 2020-07-03 | 吉林大学 | Oxygen-containing nano-diamond for antibacterial treatment and preparation method thereof |
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Application publication date: 20180727 |