CN109283333A - A method of chiral dimer is above converted based on golden shell-to Drug Resistance of E. coli quantitative analysis - Google Patents
A method of chiral dimer is above converted based on golden shell-to Drug Resistance of E. coli quantitative analysis Download PDFInfo
- Publication number
- CN109283333A CN109283333A CN201811105858.9A CN201811105858A CN109283333A CN 109283333 A CN109283333 A CN 109283333A CN 201811105858 A CN201811105858 A CN 201811105858A CN 109283333 A CN109283333 A CN 109283333A
- Authority
- CN
- China
- Prior art keywords
- polymyxin
- golden shell
- antibody
- drug
- golden
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
Abstract
A method of chiral dimer is above converted based on golden shell-to Drug Resistance of E. coli quantitative analysis, belongs to material chemistry technical field.The method comprises the steps of firstly, preparing golden shell nanoparticles, in the nanoparticle surface modified polymyxin B antibody of golden shell, in up-conversion nanoparticles surface modification polymyxin B, the up-conversion nanoparticles of polymyxin B modification are mixed from the Escherichia coli of different drug-resistant intensities respectively again, after reacting a period of time, the golden shell nanoparticle of polymyxin B antibody modification is added, by antigen-antibody reaction, forms golden shell and unbonded upper conversion forms dimeric structure.The measurement to Escherichia coli drug-resistant intensity may be implemented by fluorescence and circular dichroism.It is uniform that the present invention has prepared structure, the golden shell-of good biocompatibility above converts chiral dimer structure, providing can be by fluorescence and CD signal while the method for detecting the resistance to polymyxin B degree of Escherichia coli, establish the standard curve between the resistance to polymyxin B concentration of Escherichia coli and fluorescence signal and CD signal, good, the advantage that detection limit is low, the used time is short with high sensitivity, selectivity.
Description
Technical field
The present invention relates to a kind of above to convert chiral dimer based on golden shell-to the side of Drug Resistance of E. coli quantitative analysis
Method belongs to material chemistry technical field.
Background technique
To antibacterials it is improper using accelerate drug-resistant bacteria the whole world propagate, these bacteriums become it is sanitarian most
One of big threat.For a long time, people are researching and developing always the detection technique for bacterium in food, medical treatment and public environment.
Conventional method, such as minimal inhibitory concentration (MIC) and polymerase chain reaction (PCR), respectively according to its phenotype and genotype detection and
Drug-resistant bacteria is identified, although these methods are effective, laborious and time-consumings.With the quick hair of nano science and nanotechnology
Exhibition, nano material probe have been used for Rapid identification bacterial pathogens.By fluorescence, Surface enhanced Raman scattering and colorimetric nanometer
Sensor etc., previous studies concentrate on the specific detection and imaging of bacterium infection.However, the commonly used of antibacterials makes
These diagnostic tools are obtained to be restricted in terms of Clinical practicability.Recently the optical activities of nanoscale widely grind
Study carefully, be not focusing only in the preparation of chiral structure, and also focuses on plasma circular dichroism (CD) light in visible-range
The high s/n ratio of spectrum shows great potential to the detection of biomolecule.
Polymyxin B is a kind of wide spectrum cationic polypeptide antibiotic, is typically considered last one of bacterial-infection resisting
Defence line, especially gram-negative pathogens.However, nearest the study found that the drug resistant gene mcr-1 that plasmid carries can be very
It is easily shifted between different pathogen, so as to cause the development of antibody-resistant bacterium.Lipid A group in lipopolysaccharides (LPS)
It is the Key Metabolic factor in polymyxins resistance mechanism that N- ethyl hexanol is amine-modified, because encoding Factor reduces polymyxins
Affinity between bacterium surface.Strategy of the exploitation based on nano material is to detect polymyxins tolerant bacteria effectively to meet
Advanced clinical demand is still a challenge.Drug Resistance of E. coli is quantified so developing a kind of realize based on chiral assembly
It analyzes particularly important.
Summary of the invention
Chiral dimer is above converted based on golden shell-the object of the present invention is to provide one kind quantitatively to divide Drug Resistance of E. coli
The method of analysis.
Technical solution of the present invention prepares golden shell nanoparticle first, in the nanoparticle surface modified more Acarasiales of golden shell
The antibody of plain B, in up-conversion nanoparticles surface modification polymyxin B, then the up-conversion nanoparticles that polymyxin B is modified
It is mixed respectively from the Escherichia coli of different drug-resistant intensities, after reacting a period of time, the gold of polymyxin B antibody modification is added
Core/shell nanoparticles form golden shell and unbonded upper conversion form dimeric structure by antigen-antibody reaction.Pass through fluorescence
The measurement to Escherichia coli drug-resistant intensity may be implemented with circular dichroism.
A method of chiral dimer is above converted based on golden shell-to Drug Resistance of E. coli quantitative analysis, and steps are as follows:
(1) synthesis of gold nanoparticle: the gold nanoparticle for being 10 ± 2nm using tannic acid reduction gold chloride synthesis diameter；
(2) preparation of golden shell nanoparticle: step (1) is prepared into gained gold nanoparticle and is dissolved in polyvinylpyrrolidonesolution solution
In, silver nitrate and ascorbic acid solution are added, golden contracted payment nano particle is synthesized；Add chlorauric acid solution preparation golden shell nanometer
Particle；
(3) up-conversion nanoparticles surface modification polymyxin B: being coupled cysteine on up-conversion nanoparticles surface first,
It is reacted by amino-carboxyl, up-conversion nanoparticles are connected with polymyxin B；
Up-conversion nanoparticles: it is bought by Beijing Oneder Hightech Co., Ltd.；
(4) the nanoparticle surface modified polymyxin B antibody of golden shell: by Au-S key, make golden shell nanoparticle obtained by step (2)
It is connected with polymyxin B antibody；
(5) golden shell-above converts the preparation of chiral dimer: by antigen-antibody reaction, preparing golden shell-and above converts chiral dimerization
Body；
(6) bacterial origin and condition of culture: taking drug-resistant intensity is respectively five plants of resistance to polymyxin Bs of 4,8,16,20,24mg/mL
Coli strain；
The coli strain of resistance to polymyxin B (E15017, drug-resistant intensity 24mg/mL), polymyxin B sensitive E. coli bacterium
Strain (MG1655, drug-resistant intensity 4mg/mL) is provided by Zhejiang University.The coli strain of resistance to polymyxin B (EYAC08-25-1,
Drug-resistant intensity 20mg/mL and GZP11-11, drug-resistant intensity 16mg/mL) it is provided by China Agricultural University.The large intestine of resistance to polymyxin B
Bacillus strain (JN-PBR-1, drug-resistant intensity 8mg/mL) is obtained from Southern Yangtze University.By the single bacterium colony inoculation on solid agar plate
Into 10mL LB culture medium, and the overnight incubation at 37 DEG C of constant oscillation 200rpm.Obtain the bacterium of exponential phase of growth, OD600
For 0.7(5 × 107CFU/mL).Then, bacterium is diluted to final concentration of 1 × 10 by PBS buffer solution (pH 7.4) 3
CFU/mL is used for following experiment.
(7) golden shell-above converts detection of the chiral dimer to Drug Resistance of E. coli: the difference of step (6) culture is resistance to
The up-conversion nanoparticles for the polymyxin B modification that the Escherichia coli of medicine degree and step (3) obtain are incubated for 8 h jointly.Due to
Drug-resistant intensity is different, and bacterial membrane is different from polymyxin B affinity, can be examined by the variation of bacterium film surface up-conversion fluorescence
Survey drug-resistant intensity；At this point, adding the golden shell of the polymyxin B antibody modification in step (4), pass through antigen-antibody reaction, gold
Shell forms dimer in conjunction with unbonded up-conversion nanoparticles, generates CD signal, passes through bacterium film surface up-conversion fluorescence
Variation can detecte drug-resistant intensity.
(8) golden shell-above converts the characterization that chiral dimer detects Drug Resistance of E. coli, establishes standard curve: will walk
Suddenly the bacterium in (7) carries out fluorescence signal characterization, and establishes standard curve.Meanwhile it being centrifuged removal bacterium, supernatant is subjected to CD letter
Number characterization, establish standard curve.
Specific step is as follows:
(1) golden shell-above converts the preparation of chiral dimer
A, the synthesis of gold nanoparticle: the gold nanoparticle for being 10 ± 2 nm using tannic acid reduction gold chloride synthesis diameter, it will
The aqueous solution of chloraurate of the tannin aqueous acid of 0.1 mL 1% and 80 mL 0.0125% are heated to 60 DEG C, are then being vigorously stirred
It is lower that tannic acid solution is added rapidly in chlorauric acid solution.2 h are reacted, constant to color, solution is cooled to room temperature, and then will
Solution storage is spare at 4 DEG C.
B, the preparation of golden shell nanoparticle: firstly, the gold nanoparticle of above-mentioned preparation is centrifuged 10 at 13,000 rpm
Min, precipitation concentration 5 are resuspended in phosphate buffer (10 mM, pH 7.4) again, take 1 mL that 1 μ L, 10 mM mPEG-SH is added
(MW=5000) AuNP-PEG is obtained, then it is sufficiently mixed with 0.5 mL, 1% polyvinylpyrrolidonesolution solution, is then distinguished
0.1 M ascorbic acid solution of 40 μ L, 20 mM silver nitrate and 20 μ L is added, at room temperature uniformly mixing, after reacting 10min,
5 min are centrifuged under 6000 rpm, precipitating is resuspended in 1 mL, 1% polyvinylpyrrolidonesolution solution, obtains golden contracted payment nanoparticle
Son.Finally, 200 μ L, 10 mM chlorauric acid solution is added in the above solution, it is stirred to react 10min at room temperature, obtains golden shell and receives
Rice grain.
C, up-conversion nanoparticles surface modification polymyxin B:
The preparation of up-conversion nanoparticles UCNP: being bought by Beijing Oneder Hightech Co., Ltd., is 10 mM with concentration,
The Tris buffer that pH is 7.4 uses after diluting 100 times；
Water-soluble UCNP Tris buffer (10mM, pH7.4) is diluted to 10 nM.Then cysteine Cys is added, makes
The molar concentration rate of Cys:UCNP is that 100:1 is functionalized UCNP, super with the super filter tube of 30kDa molecular weight after reacting at room temperature 4 h
Filter is to remove the Cys not being coupled.4mg polymyxin B is dissolved in 1mL deionized water, and addition 4 μ L, 2.5% glutaraldehyde (>
98%) 30 min are activated, finally, the Cys-UCNP solution of 20 nM is being reacted at room temperature 6 h with the polymyxin B activated.It is logical
Ultrafiltration (30kDa molecular weight) is crossed to purify final product and be stored in spare at 4 DEG C.
D, the nanoparticle surface modified polymyxin B antibody of golden shell: by 10 times of golden shell nanoparticle concentration of preparation, then
Polymyxin B antibody is added, with polymyxin B antibody: the molar concentration rate of golden shell is that 100:1 is modified.Mixture is existed
6h is reacted at room temperature.By the golden shell of modification in 6000 rpm centrifugation 5min to remove any excessive polymyxin B antibody, then
It is resuspended in water.
E, golden shell-above converts the preparation of chiral dimer: the UCNP and polymyxin B that the polymyxin B of purifying is modified
The golden shell nano particle of antibody modification is mixed 2h with the molar concentration rate of 1:1 in Tris buffer (pH 7.4), then will
Mixture is centrifuged 10 min at 4000 rpm and is resuspended in spare in PBS.
(2) golden shell-above converts detection of the chiral dimer to Drug Resistance of E. coli
A, bacterial origin and condition of culture: the coli strain of resistance to polymyxin B (E15017, drug-resistant intensity 24mg/mL), it is mostly viscous
Rhzomorph B sensitive E. coli bacterial strain (MG1655, drug-resistant intensity 4mg/mL) is provided by Zhejiang University.The large intestine of resistance to polymyxin B
Bacillus strain (EYAC08-25-1, drug-resistant intensity 20mg/mL and GZP11-11, drug-resistant intensity 16mg/mL) is by China Agricultural University
It provides.The coli strain of resistance to polymyxin B (JN-PBR-1, drug-resistant intensity 8mg/mL) is obtained from Southern Yangtze University.By solid agar
Single bacterium colony on plate is inoculated into 10mL LB culture medium, and the overnight incubation at 37 DEG C of constant oscillation 200rpm.Referred to
The bacterium in number growth period, OD600For 0.7(5 × 10 7CFU/mL).Then, by PBS buffer solution (pH 7.4) that bacterium is dilute
Release final concentration of 1 × 103CFU/ mL is used for following experiment.
B, golden shell-above converts detection of the chiral dimer to Drug Resistance of E. coli: the difference of step (2, a) culture is resistance to
The up-conversion nanoparticles for the polymyxin B modification that the Escherichia coli of medicine degree and step (1, c) obtain are incubated for 8 h jointly.By
In drug-resistant intensity difference, bacterial membrane is different from polymyxin B affinity, can be with by the variation of bacterium film surface up-conversion fluorescence
Detect drug-resistant intensity；At this point, the golden shell of the polymyxin B antibody modification in step (1, d) is added, it is anti-by Ag-Ab
It answers, golden shell forms dimer in conjunction with unbonded up-conversion nanoparticles, generates CD signal.
(3) golden shell-above converts the characterization that chiral dimer detects Drug Resistance of E. coli: chiral dimer was formed
Cheng Jinhang Electronic Speculum characterization；Bacterial drug resistance is detected and carries out fluorescence signal and CD characterization, and establishes standard curve.
Biological material specimens explanation: the coli strain of resistance to polymyxin B (E15017, drug-resistant intensity 24mg/mL), it is mostly viscous
Rhzomorph B sensitive E. coli bacterial strain (MG1655, drug-resistant intensity 4mg/mL) is provided by Zhejiang University.The large intestine of resistance to polymyxin B
Bacillus strain (EYAC08-25-1, drug-resistant intensity 20mg/mL and GZP11-11, drug-resistant intensity 16mg/mL) is by China Agricultural University
It provides.The coli strain of resistance to polymyxin B (JN-PBR-1, drug-resistant intensity 8mg/mL) is obtained from Southern Yangtze University.
Beneficial effects of the present invention: the present invention has prepared that structure is uniform, and above conversion is chiral for the golden shell-of good biocompatibility
Dimeric structure, providing can be built by fluorescence and CD signal while the method for detecting the resistance to polymyxin B degree of Escherichia coli
The standard curve between the resistance to polymyxin B concentration of Escherichia coli and fluorescence signal and CD signal has been found, there is high sensitivity, selection
Property it is good, detection limit is low, advantage that the used time is short, has extraordinary actual application prospect.
Detailed description of the invention
Fig. 1 is the transmission electron microscope picture that golden shell-of the invention above converts chiral dimer.A, up-conversion nanoparticles；B, gold
Core-shell nanoparticles；C, golden shell-above converts chiral dimer.
Fig. 2 is the fluorescence spectra and standard curve that golden shell-of the invention above converts chiral dimer detection drug-fast bacteria.a,
The fluorescence spectra of dimer；B, fluorescence standard curve.
Fig. 3 is the CD spectrogram and standard curve that golden shell-of the invention above converts chiral dimer detection drug-fast bacteria.A, two
The CD spectrogram of aggressiveness；B, CD standard curve.
Specific embodiment
1 golden shell of embodiment-above converts the preparation of chiral dimer
All glass apparatus are all impregnated with chloroazotic acid, and are cleaned with distilled water, are dried spare.Water used in experiment is 18.2
The Milli-Q ultrapure water of M Ω.
(1) synthesis of gold nanoparticle: the Jenner's grain of rice for being 10 ± 2 nm using tannic acid reduction gold chloride synthesis diameter
The aqueous solution of chloraurate of the tannin aqueous acid of 0.1 mL 1% and 80 mL 0.0125% is heated to 60 DEG C, then in play by son
Tannic acid solution is added rapidly in chlorauric acid solution under strong stirring.2 h are reacted, constant to color, solution is cooled to room temperature,
Then solution storage is spare at 4 DEG C.
(2) preparation of golden shell nanoparticle: firstly, the gold nanoparticle of above-mentioned preparation is centrifuged 10 at 13,000 rpm
Min, precipitation concentration 5 are resuspended in phosphate buffer (10 mM, pH 7.4) again, take 1 mL that 1 μ L, 10 mM mPEG-SH is added
(Mw=5000) obtain AuNP-PEG, then it is sufficiently mixed with 0.5 mL, 1% polyvinylpyrrolidonesolution solution, then distinguish
0.1 M ascorbic acid solution of 40 μ L, 20 mM silver nitrate and 20 μ L is added, at room temperature uniformly mixing, after reacting 10min,
5 min are centrifuged under 6000 rpm, precipitating is resuspended in 1 mL, 1% polyvinylpyrrolidonesolution solution, obtains golden contracted payment nanoparticle
Son.Finally, 200 μ L, 10 mM chlorauric acid solution is added in the above solution, it is stirred to react 10min at room temperature, obtains golden shell and receives
Rice grain.As shown in Figure 1 b, the golden shell nanoparticle structure of preparation is uniform, favorable dispersibility.
(3) preparation of up-conversion nanoparticles: being bought by Beijing Oneder Hightech Co., Ltd., is 10 with concentration
The Tris buffer that mM, p H are 7.4 uses after diluting 100 times.Electron microscope is as shown in 1a.
(4) up-conversion nanoparticles surface modification polymyxin B: by water-soluble UCNP with Tris buffer (10mM,
PH7.4 10 nM) are diluted to.Then Cys is added, so that the molar concentration rate 100:1 of Cys:UCNP is functionalized UCNP, room temperature
After reacting 4 h, with the super filter tube ultrafiltration of 30kDa molecular weight to remove the Cys not being coupled.4mg polymyxin B is dissolved in 1mL
In deionized water, and 4 μ L, 2.5% glutaraldehyde (> 98%) 30 min activation is added, finally, by the Cys-UCNP solution of 20 nM
6 h are being reacted at room temperature with the polymyxin B activated.Final product is purified and is stored in by ultrafiltration (30 kDa molecular weight)
It is spare at 4 DEG C.
(5) the nanoparticle surface modified polymyxin B antibody of golden shell: by 10 times of golden shell nanoparticle concentration of preparation, so
Polymyxin B antibody is added afterwards, with polymyxin B antibody: the molar concentration rate of golden shell is that 100:1 is modified.By mixture
6h is reacted at room temperature.By the golden shell of modification in 6000 rpm centrifugation 5min to remove any excessive polymyxin B antibody, so
After be resuspended in water.
(6) golden shell-above converts the preparation of chiral dimer: the UCNP and polymyxin B that the polymyxin B of purifying is modified
The golden shell nano particle of antibody modification is mixed 2h with the molar concentration rate of 1:1 in Tris buffer (pH 7.4), then will
Mixture is centrifuged 10 min at 4000 rpm and is resuspended in spare in PBS.As illustrated in figure 1 c, chiral dimer yield it is high,
Structural integrity, favorable dispersibility.
2 golden shell of embodiment-above converts detection of the chiral dimer to Drug Resistance of E. coli
(1) bacterial origin and condition of culture: the coli strain of resistance to polymyxin B (E15017, drug-resistant intensity 24mg/mL) is more
Colistin B sensitive E. coli bacterial strain (MG1655, drug-resistant intensity 4mg/mL) is provided by Zhejiang University.Resistance to polymyxin B is big
Enterobacteria bacterial strain (EYAC08-25-1, drug-resistant intensity 20mg/mL and GZP11-11, drug-resistant intensity 16mg/mL) is big by Chinese agriculture
It learns and provides.The coli strain of resistance to polymyxin B (JN-PBR-1, drug-resistant intensity 8mg/mL) is obtained from Southern Yangtze University.By solid fine jade
Single bacterium colony on rouge plate is inoculated into 10mL LB culture medium, and the overnight incubation at 37 DEG C of constant oscillation 200rpm.It obtains
The bacterium of exponential phase of growth, OD600For 0.7(5 × 107CFU/mL).Then, by PBS buffer solution (pH 7.4) by bacterium
It is diluted to final concentration of 1 × 103CFU/ mL is used for following experiment.
(2) golden shell-above converts detection of the chiral dimer to Drug Resistance of E. coli: the difference of step (1) culture is resistance to
The up-conversion nanoparticles for the polymyxin B modification that the Escherichia coli of medicine degree and 1 step of embodiment (4) obtain are incubated for 8 jointly
h.Due to drug-resistant intensity difference, bacterial membrane is different from polymyxin B affinity, passes through the variation of bacterium film surface up-conversion fluorescence
It can detecte drug-resistant intensity, as shown in Figure 2 a, with increasing for Escherichia coli drug-resistant intensity, the knot of bacterial membrane and polymyxins
Resultant force weakens, and fluorescence signal is caused constantly to reduce；At this point, adding the polymyxin B antibody modification in 1 step of embodiment (5)
Golden shell dimer is formed in conjunction with unbonded up-conversion nanoparticles by antigen-antibody reaction, generate CD signal.Such as
Shown in Fig. 3 a, due to increasing for Escherichia coli drug-resistant intensity, the binding force of bacterial membrane and polymyxins weakens, and causes increasingly
Mostly free up-conversion nanoparticles and golden shell are assembled into dimer, and then CD signal enhancing.
3 golden shell of embodiment-above converts the characterization that chiral dimer detects Drug Resistance of E. coli: by chiral dimer shape
Electronic Speculum characterization is carried out at process；Bacterial drug resistance is detected and carries out fluorescence signal and CD characterization, and establishes standard song respectively
Line.As shown by figures 2 b and 3b, the drug-resistant intensity of Escherichia coli is strong with the ratio, bacterial population and fluorescence of bacterial population and CD signal respectively
Good linear relationship is presented in the ratio of degree.Illustrate that the method can be used for the detection of Escherichia coli drug-resistant intensity.
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
Claims (2)
1. a kind of chiral dimer of above being converted based on golden shell-is to the method for Drug Resistance of E. coli quantitative analysis, it is characterized in that step
It is rapid as follows:
(1) synthesis of gold nanoparticle: the gold nanoparticle for being 8-12nm using tannic acid reduction gold chloride synthesis diameter；
(2) preparation of golden shell nanoparticle: step (1) is prepared into gained gold nanoparticle and is dissolved in polyvinylpyrrolidonesolution solution
In, silver nitrate and ascorbic acid solution are added, golden contracted payment nano particle is synthesized, then adds chlorauric acid solution preparation golden shell
Nanoparticle；
(3) up-conversion nanoparticles surface modification polymyxin B: being coupled cysteine on up-conversion nanoparticles surface first,
It is reacted by amino-carboxyl, up-conversion nanoparticles are connected with polymyxin B；
(4) the nanoparticle surface modified polymyxin B antibody of golden shell: by Au-S key, make golden shell nanoparticle obtained by step (2)
It is connected with polymyxin B antibody；
(5) golden shell-above converts the preparation of chiral dimer: by antigen-antibody reaction, preparing golden shell-and above converts chiral dimerization
Body；
(6) bacterial origin and condition of culture: taking drug-resistant intensity is respectively five plants of resistance to polymyxin Bs of 4,8,16,20,24mg/mL
Coli strain；
Single bacterium colony is inoculated into 10mL LB culture medium, and the overnight incubation at 37 DEG C of constant oscillation 200rpm；Obtain index
Growth period 5 × 107The bacterium of CFU/mL, OD600It is 0.7, then, is diluted bacterium by the PBS buffer solution of pH 7.4
To final concentration of 1 × 10 3CFU/mL is used for following experiment；
(7) golden shell-above converts detection of the chiral dimer to Drug Resistance of E. coli: the different drug resistance journeys that step (6) are cultivated
The up-conversion nanoparticles for the polymyxin B modification that the Escherichia coli of degree and step (3) obtain are incubated for 8 h jointly；Step is added
(4) golden shell of the polymyxin B antibody modification in, by antigen-antibody reaction, golden shell and unbonded up-conversion nanoparticles
In conjunction with dimer is formed, CD signal is generated；Drug-resistant intensity is detected by the variation of bacterium film surface up-conversion fluorescence；
(8) golden shell-above converts the characterization that chiral dimer detects Drug Resistance of E. coli, establishes standard curve: by step (7)
In bacterium carry out fluorescence signal characterization, and establish standard curve；Meanwhile it being centrifuged removal bacterium, supernatant is subjected to CD signal table
Sign, establishes standard curve.
2. chiral dimer is above converted based on golden shell-according to claim 1 to the side of Drug Resistance of E. coli quantitative analysis
Method, it is characterised in that specific step is as follows:
(1) synthesis of gold nanoparticle: the gold nanoparticle for being 8-12nm using tannic acid reduction gold chloride synthesis diameter, it will
The tannin aqueous acid of 0.1mL 1% and the aqueous solution of chloraurate of 80mL 0.0125% are heated to 60 DEG C, then with vigorous stirring
Tannic acid solution is added rapidly in chlorauric acid solution, reacts 2h, constant to color, solution is cooled to room temperature, then will be molten
Liquid be stored in 4 DEG C it is spare；
(2) preparation of golden shell nanoparticle: firstly, gold nanoparticle prepared by step (1) is centrifuged 10 at 13000 rpm
Min, precipitation concentration 5 are resuspended in again in the phosphate buffer of pH 7.4,10mM, take 1mL that 1 μ L, 10 mM mPEG-SH is added,
MW=5000, obtain AuNP-PEG；It is sufficiently mixed with 0.5 mL, 1% polyvinylpyrrolidonesolution solution again, is then distinguished
0.1 M ascorbic acid solution of 40 μ L, 20 mM silver nitrate and 20 μ L is added, at room temperature uniformly mixing, after reacting 10min,
5 min are centrifuged under 6000 rpm, precipitating is resuspended in 1 mL, 1% polyvinylpyrrolidonesolution solution, obtains golden contracted payment nanoparticle
Son；Finally, 200 μ L, 10 mM chlorauric acid solution is added in the above solution, it is stirred to react 10min at room temperature, obtains golden shell and receives
Rice grain；
(3) up-conversion nanoparticles surface modification polymyxin B:
The preparation of up-conversion nanoparticles: taking up-conversion nanoparticles UCNP, is 10 mM with concentration, and the Tris that pH is 7.4 is buffered
Liquid uses after diluting 100 times；
Up-conversion nanoparticles modify polymyxin B: the Tris buffer of water-soluble UCNP pH7.4,10mM are diluted to
10 nM；Then cysteine Cys is added, makes the molar concentration rate 100:1 of Cys:UCNP, after reacting at room temperature 4 h, uses 30kDa
The super filter tube ultrafiltration of molecular weight is to remove the Cys not being coupled；4mg polymyxin B is dissolved in 1mL deionized water, and is added 4
2.5% glutaraldehyde of μ L carries out 30 min activation, finally, the Cys-UCNP solution of 20 nM is existed with the polymyxin B activated
6 h are reacted at room temperature, purified final product by the ultrafiltration of 30kDa molecular weight and are stored in is spare at 4 DEG C；
(4) the nanoparticle surface modified polymyxin B antibody of golden shell: by 10 times of golden shell nanoparticle concentration of step (2) preparation,
Then polymyxin B antibody is added, with polymyxin B antibody: golden shell nano particle molar concentration rate is that 100:1 is modified；
Mixture is reacted into 6h at room temperature；By the golden shell after modification in 6000 rpm centrifugation 5min to remove any excessive more Acarasiales
Plain B antibody, is then resuspended in water；
(5) golden shell-above converts the preparation of chiral dimer: the UCNP and polymyxin B antibody that the polymyxin B of purifying is modified
The golden shell nano particle of modification is mixed 2h with the molar concentration rate of 1:1 in pH 7.4, Tris buffer, then by mixture
It is centrifuged 10 min at 4000 rpm and is resuspended in spare in PBS；
(6) bacterial origin and condition of culture: taking drug-resistant intensity is respectively five plants of resistance to polymyxin Bs of 4,8,16,20,24mg/mL
Coli strain；
Single bacterium colony on solid agar plate is inoculated into 10mL LB culture medium, and at 37 DEG C of constant oscillation 200rpm
Overnight incubation；
Obtain exponential phase of growth 5 × 107The bacterium of CFU/mL, OD600It is 0.7, then, is buffered by the PBS of pH 7.4
Bacterium is diluted to final concentration of 1 × 10 by liquid 3CFU/ mL is used for following experiment；
(7) golden shell-above converts detection of the chiral dimer to Drug Resistance of E. coli: the difference for taking 1 mL step (6) to cultivate is resistance to
The up-conversion nanoparticles for the polymyxin B modification that the Escherichia coli of medicine degree and 100 μ L steps (3) obtain are incubated for 8 h jointly,
The golden shell of the polymyxin B antibody modification in 100 μ L steps (4) is added, after being mixed 2h, is centrifuged 5 at 2000 rpm
Min, precipitating are resuspended in 100 μ L PBS buffer solution and measure up-conversion fluorescence, due to drug-resistant intensity difference, bacterial membrane and polymyxins
B affinity is different, detects drug-resistant intensity by the variation of bacterium film surface up-conversion fluorescence；
Meanwhile supernatant being taken to measure CD signal, by antigen-antibody reaction, golden shell is in conjunction with unbonded up-conversion nanoparticles
Dimer is formed, CD signal is generated；
(8) golden shell-above converts the characterization that chiral dimer detects Drug Resistance of E. coli, establishes standard curve: by step (7)
In bacterium carry out fluorescence signal characterization, and establish standard curve；Meanwhile it being centrifuged removal bacterium, supernatant is subjected to CD signal table
Sign, establishes standard curve.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811105858.9A CN109283333B (en) | 2018-09-21 | 2018-09-21 | Method for quantitatively analyzing drug resistance of escherichia coli based on gold shell-up-conversion chiral dimer |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811105858.9A CN109283333B (en) | 2018-09-21 | 2018-09-21 | Method for quantitatively analyzing drug resistance of escherichia coli based on gold shell-up-conversion chiral dimer |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109283333A true CN109283333A (en) | 2019-01-29 |
| CN109283333B CN109283333B (en) | 2021-11-12 |
Family
ID=65181870
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811105858.9A Active CN109283333B (en) | 2018-09-21 | 2018-09-21 | Method for quantitatively analyzing drug resistance of escherichia coli based on gold shell-up-conversion chiral dimer |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109283333B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109856099A (en) * | 2019-03-15 | 2019-06-07 | 浙江工业大学 | A method of based on alcoholic strength in upconverting fluorescent material detection wine |
| CN109900668A (en) * | 2019-03-15 | 2019-06-18 | 浙江工业大学 | A method of based on the test strip containing upconversion fluorescence nano material detection wine in alcoholic strength |
Citations (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013052484A1 (en) * | 2011-10-03 | 2013-04-11 | Immunomedics, Inc. | Dye conjugated peptides for fluorescent imaging |
| CN103157811A (en) * | 2013-03-13 | 2013-06-19 | 江南大学 | Preparing method of gold-silver core-shell structure - gold dimer chirality assembly body |
| CN104342155A (en) * | 2014-10-13 | 2015-02-11 | 江南大学 | Preparation method of pyramid assembly structure simultaneously having fluorescent, magnetic and chiral signals |
| CN105372213A (en) * | 2015-09-29 | 2016-03-02 | 江南大学 | Method for detecting ochratoxin A (OTA) based on luminescence resonance energy transfer between up-conversion luminescence nano-material and gold nano-rods |
| CN105842464A (en) * | 2016-06-12 | 2016-08-10 | 吉林大学 | Device for jointly quantitatively detecting uNGAL (urinary Neutrophil Gelatinase Associated Lipocalin) and uCr (urinary Creatinine) based on up-converting phosphor technology and preparation method of device |
| CN106086173A (en) * | 2016-06-14 | 2016-11-09 | 西安交通大学 | A kind of quick bacteria detection method based on up-conversion fluorescence Resonance energy transfer |
| CN106290898A (en) * | 2016-07-29 | 2017-01-04 | 江南大学 | A kind of gold up-conversion nanoparticles trimer preparation method and application thereof |
| CN106290873A (en) * | 2016-07-28 | 2017-01-04 | 江南大学 | A kind of based on preparation and the application with transformed space tetrahedral structure on the gold of Raman and fluorescent dual signal |
| CN106281303A (en) * | 2016-08-10 | 2017-01-04 | 江南大学 | A kind of preparation method of the chirality self assembly superstructure of propeller-like gold nanorods up-conversion nanoparticles |
| CN106323882A (en) * | 2016-09-20 | 2017-01-11 | 江南大学 | Method based on gold-graphene nano-assembly for chiral signal ultrasensitive detection of Mucin-1 |
| CN106498047A (en) * | 2016-10-21 | 2017-03-15 | 江南大学 | Method based on tetrahedral dual signal in situ detection intracellular microRNA of golden up-conversion nanoparticles |
| US20180133318A1 (en) * | 2015-03-03 | 2018-05-17 | Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College | Enhanced plasmonic nanoparticles for cancer therapy and diagnostics |
| CN108333342A (en) * | 2018-05-14 | 2018-07-27 | 广东药科大学 | A kind of quickly detection remaining method of Tetracyclines in Milk |
-
2018
- 2018-09-21 CN CN201811105858.9A patent/CN109283333B/en active Active
Patent Citations (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013052484A1 (en) * | 2011-10-03 | 2013-04-11 | Immunomedics, Inc. | Dye conjugated peptides for fluorescent imaging |
| CN103157811A (en) * | 2013-03-13 | 2013-06-19 | 江南大学 | Preparing method of gold-silver core-shell structure - gold dimer chirality assembly body |
| CN104342155A (en) * | 2014-10-13 | 2015-02-11 | 江南大学 | Preparation method of pyramid assembly structure simultaneously having fluorescent, magnetic and chiral signals |
| US20180133318A1 (en) * | 2015-03-03 | 2018-05-17 | Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College | Enhanced plasmonic nanoparticles for cancer therapy and diagnostics |
| CN105372213A (en) * | 2015-09-29 | 2016-03-02 | 江南大学 | Method for detecting ochratoxin A (OTA) based on luminescence resonance energy transfer between up-conversion luminescence nano-material and gold nano-rods |
| CN105842464A (en) * | 2016-06-12 | 2016-08-10 | 吉林大学 | Device for jointly quantitatively detecting uNGAL (urinary Neutrophil Gelatinase Associated Lipocalin) and uCr (urinary Creatinine) based on up-converting phosphor technology and preparation method of device |
| CN106086173A (en) * | 2016-06-14 | 2016-11-09 | 西安交通大学 | A kind of quick bacteria detection method based on up-conversion fluorescence Resonance energy transfer |
| CN106290873A (en) * | 2016-07-28 | 2017-01-04 | 江南大学 | A kind of based on preparation and the application with transformed space tetrahedral structure on the gold of Raman and fluorescent dual signal |
| CN106290898A (en) * | 2016-07-29 | 2017-01-04 | 江南大学 | A kind of gold up-conversion nanoparticles trimer preparation method and application thereof |
| CN106281303A (en) * | 2016-08-10 | 2017-01-04 | 江南大学 | A kind of preparation method of the chirality self assembly superstructure of propeller-like gold nanorods up-conversion nanoparticles |
| CN106323882A (en) * | 2016-09-20 | 2017-01-11 | 江南大学 | Method based on gold-graphene nano-assembly for chiral signal ultrasensitive detection of Mucin-1 |
| CN106498047A (en) * | 2016-10-21 | 2017-03-15 | 江南大学 | Method based on tetrahedral dual signal in situ detection intracellular microRNA of golden up-conversion nanoparticles |
| CN108333342A (en) * | 2018-05-14 | 2018-07-27 | 广东药科大学 | A kind of quickly detection remaining method of Tetracyclines in Milk |
Non-Patent Citations (3)
| Title |
|---|
| XU S. 等: ""Upconversion nanophosphores for bioimaging"", 《TRENDS IN ANALYTICAL CHEMISTRY》 * |
| 张慧中 等: ""稀土上转换材料活体干细胞示踪技术的研究进展"", 《组织工程与重建外科杂志》 * |
| 杨雯雯 等: ""碘介导金包银核壳纳米粒子的合成及SERS应用"", 《武汉工程大学学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109856099A (en) * | 2019-03-15 | 2019-06-07 | 浙江工业大学 | A method of based on alcoholic strength in upconverting fluorescent material detection wine |
| CN109900668A (en) * | 2019-03-15 | 2019-06-18 | 浙江工业大学 | A method of based on the test strip containing upconversion fluorescence nano material detection wine in alcoholic strength |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109283333B (en) | 2021-11-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Chandra et al. | One step synthesis of functionalized carbon dots for the ultrasensitive detection of Escherichia coli and iron (III) | |
| Yuan et al. | Metal nanoparticles for diagnosis and therapy of bacterial infection | |
| Liu et al. | Development of a fluorescence aptasensor for rapid and sensitive detection of Listeria monocytogenes in food | |
| Luo et al. | Nanotechnology in the detection and control of microorganisms | |
| Ray et al. | Nanomaterials for targeted detection and photothermal killing of bacteria | |
| Xu et al. | Dual-mode of magnetic assisted Au@ Ag SERS tags and cationic conjugated UCNPs for qualitative and quantitative analysis of multiple foodborne pathogens | |
| Tang et al. | Preparation of functional magnetic nanoparticles mediated with PEG-4000 and application in Pseudomonas aeruginosa rapid detection | |
| Bahari et al. | Ratiometric fluorescence resonance energy transfer aptasensor for highly sensitive and selective detection of Acinetobacter baumannii bacteria in urine sample using carbon dots as optical nanoprobes | |
| Imai et al. | Dark-field microscopic detection of bacteria using bacteriophage-immobilized SiO2@ AuNP core–shell nanoparticles | |
| Sun et al. | An optical and rapid sandwich immunoassay method for detection of Salmonella pullorum and Salmonella gallinarum based on immune blue silica nanoparticles and magnetic nanoparticles | |
| Ali et al. | Detection of E. coli O157: H7 in feed samples using gold nanoparticles sensor | |
| Liu et al. | Graphene-DNAzyme-based fluorescent biosensor for Escherichia coli detection | |
| Basso et al. | A new immunoassay of hybrid nanomater conjugated to aptamers for the detection of dengue virus | |
| Chang et al. | Fluorescent-magnetic Janus nanorods for selective capture and rapid identification of foodborne bacteria | |
| CN107941779A (en) | The method of gold shell magnetic nanoparticles and nanometer label surface enhancing Raman scattering detection bacterium based on vancomycin modification | |
| Sadsri et al. | Simple colorimetric assay for Vibrio parahaemolyticus detection using aptamer-functionalized nanoparticles | |
| Li et al. | A novel low-field NMR biosensor based on dendritic superparamagnetic iron oxide nanoparticles for the rapid detection of Salmonella in milk | |
| CN109283333A (en) | A method of chiral dimer is above converted based on golden shell-to Drug Resistance of E. coli quantitative analysis | |
| Kumar et al. | Biofunctional magnetic nanotube probe for recognition and separation of specific bacteria from a mixed culture | |
| Carvalho et al. | Advances in screening, detection and enumeration of Escherichia coli using nanotechnology-based methods: A review | |
| Bahavarnia et al. | Bio-assay of Acintobacter baumannii using DNA conjugated with gold nano-star: A new platform for microorganism analysis | |
| Deng et al. | Poly-l-lysine-functionalized magnetic beads combined with polymerase chain reaction for the detection of Staphylococcus aureus and Escherichia coli O157: H7 in milk | |
| Liu et al. | In-taken labeling and in vivo tracing foodborne probiotics via DNA-encapsulated persistent luminescence nanoprobe assisted autofluorescence-free bioimaging | |
| Bhattacharya et al. | Detection of total count of Staphylococcus aureus using anti-toxin antibody labelled gold magnetite nanocomposites: a novel tool for capture, detection and bacterial separation | |
| Lee et al. | Rapid detection of Escherichia coli O157: H7 in fresh lettuce based on Localized Surface Plasmon Resonance combined with immunomagnetic separation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |