CN109536571A - A kind of nanometer bio probe and preparation method thereof detecting pathogenic bacteria - Google Patents
A kind of nanometer bio probe and preparation method thereof detecting pathogenic bacteria Download PDFInfo
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/18—Testing for antimicrobial activity of a material
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- 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
- G01N33/532—Production of labelled immunochemicals
- G01N33/535—Production of labelled immunochemicals with enzyme label or co-enzymes, co-factors, enzyme inhibitors or enzyme substrates
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- 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
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- 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
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- 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
- G01N33/56938—Staphylococcus
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- G01N2333/245—Escherichia (G)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/305—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Micrococcaceae (F)
- G01N2333/31—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Micrococcaceae (F) from Staphylococcus (G)
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The present invention provides a kind of nanometer bio probe and preparation method thereof for detecting pathogenic bacteria, the nanometer bio probe is formed by magnetic core-shell nanostructure and for the specific recognition molecules coupling of pathogenic bacteria, the magnetic core-shell nanostructure is shell by core, the silicon dioxide layer embedded with Pt nanoparticle of ferriferrous oxide particles, it is whole negatively charged, there is Mimetic enzyme activity.Nanometer bio probe provided by the invention has Mimetic enzyme activity, and can use chromatmetry and be used for quickly detecting pathogenic bacteria whether there is.Preparation method simple process provided by the invention, strong operability and environmental-friendly, nanometer bio probe obtained can reach the purpose and effect of quickly detection pathogenic bacteria, have a good application prospect.
Description
Technical field
The present invention relates to novel nano-material preparation field more particularly to it is a kind of detect pathogenic bacteria nanometer bio probe and
Preparation method.
Background technique
Pathogenic bacteria are the microorganism that can cause disease, also referred to as pathogenic microorganism comprising bacterium, virus, conveyor screw,
Rickettsia, Chlamydia, mycoplasma, fungi and actinomyces etc..
Pathogenic bacteria not only seriously damages the health of the mankind, also causes significant impact to economy, it has also become in the world
One of public health problem most outstanding.Traditional technique in measuring pathogenic bacteria (such as Escherichia coli, staphylococcus aureus etc.) are first
It acquires sample and carries out enrichment culture, the quantity for being detected bacterium is made to reach detectable level in the sample, it then could basis
Morphological feature observation and a series of identification of physio-biochemical characteristics, the process take a long time, generally require 3~5 days.Therefore, urgently
Ground needs quick, specific and sensitive method to detect pathogenic bacteria, monitors especially online, in real time.
Currently, quickly the method for detection pathogenic bacteria includes small-sized biological test chemical, immunology test, based on nucleic acid probe
Method, polymerase chain reaction etc..But the above method is there is also the device is complicated, the problems such as false positive, and needs bacterium
The operations such as enrichment early period, can't fully meet the demand in market.
Summary of the invention
In view of the problems of the existing technology, the present invention provide it is a kind of detect pathogenic bacteria nanometer bio probe and its preparation
Method.
The present invention provides a kind of nanometer bio probe for detecting pathogenic bacteria, by magnetic core-shell nanostructure and is directed to pathogenic bacteria
Specific recognition molecules be coupled composition, the magnetic core-shell nanostructure using ferriferrous oxide particles as core, be embedded with platinum
The silicon dioxide layer of grain is shell, whole negatively charged, has Mimetic enzyme activity.
In above-mentioned technical proposal, the magnetic core-shell nanostructure in nanometer bio probe has superparamagnetism and simulation peroxide
Compound enzymatic activity, nanometer bio probe first pass through superparamagnetism and are enriched with pathogenic bacteria to be measured, then simulate peroxide using it
Hydrogen peroxide and TMB can be added in compound enzymatic activity into system to be measured, based on ELISA method according to the depth of final color whether there is or not
The qualitative or quantitative analysis for carrying out pathogenic bacteria has the advantages that quick and high sensitivity, especially suitable for the online of pathogenic bacteria
Real-time monitoring has a good application prospect.
Preferably, the size of the magnetic core-shell nanostructure is 30~50nm, and wherein ferriferrous oxide particles size is
10~20nm, silicon dioxide layer thickness are 5~10nm, and platinum grain size is 2~3nm.
Preferably, the specific recognition molecules be one of antibody, target polypeptide, aptamer and antibiotic or
It is a variety of.
The present invention also provides the preparation methods of above-mentioned nanometer bio probe, comprising the following steps:
(1) ferroso-ferric oxide in organic phase is subjected to coated with silica using reverse micelle of microemulsion, four oxygen is prepared
Change three-iron-silica dioxide granule；
(2) amination modification is carried out to the ferroso-ferric oxide-silica dioxide granule, keeps its surface positively charged；
(3) platinum grain is made to be supported on the positively charged ferroso-ferric oxide-silica dioxide granule by electrostatic adsorption
Magnetic core-shell nanostructure is prepared in surface；
(4) it is coupled by the magnetic core-shell nanostructure and for the specific recognition molecules of pathogenic bacteria.
Above-mentioned preparation method simple process, strong operability and environmental-friendly, nanometer bio probe obtained can reach
The quickly purpose and effect of detection pathogenic bacteria.
Preferably, the step (1) specifically includes: the ferroso-ferric oxide that will be dispersed in organic phase is dispersed in polyethylene glycol
In octyl phenyl ether/n-hexyl alcohol/water mixed solution, Reverse microemulsion is formed, ammonium hydroxide and ethyl orthosilicate is then added, makes just
Silester hydrolyzes to form coated with silica layer under alkaline environment.
Preferably, the ferroso-ferric oxide solvent-applied thermal method preparation, being dispersed in concentration in hexamethylene is 1~10mg/mL,
More preferably 1mg/mL.
Preferably, Triton X-100 in the Triton X-100/n-hexyl alcohol/water mixed solution,
The molar ratio of n-hexyl alcohol and water is 1:2:4~8, preferably 1:2:6.
Preferably, the ethyl orthosilicate, the ferroso-ferric oxide, the ammonium hydroxide mass ratio be 1:5:1.
Preferably, the step (2) specifically includes: by the ferroso-ferric oxide-silica dioxide granule and 3- aminopropyl three
The mixing of methoxy radical siloxane, carries out amination modification, and the two mass ratio is 1:10~1:100, preferably 1:40, reaction time 4
~for 24 hours, preferably 14h.
Preferably, ferroso-ferric oxide-silica dioxide granule positively charged described in the step (3) and the platinum grain
Mass ratio be 100:1~1:1, preferably 10:1, the reaction time be 4~for 24 hours, preferably 14h.
Preferably, coupling method is glutaraldehyde spacerarm method or EDC/NHS method, more preferable glutaraldehyde in the step (4)
Spacerarm method.
Specific embodiment as one preferred, the preparation method comprises the following steps:
(1) ferroso-ferric oxide that will be dispersed in organic phase is dispersed in Triton X-100/n-hexyl alcohol/water mixing
In solution, Reverse microemulsion is formed, ammonium hydroxide and ethyl orthosilicate is then added, hydrolyzes ethyl orthosilicate under alkaline environment
Coated with silica layer is formed, ferroso-ferric oxide-silica dioxide granule is prepared；
(2) ferroso-ferric oxide-silica dioxide granule and 3- aminopropyl trimethoxy siloxane are mixed, carries out amino
Change modification, keeps its surface positively charged；
(3) platinum grain is made to be supported on the positively charged ferroso-ferric oxide-silica dioxide granule by electrostatic adsorption
Surface, the two mass ratio are 1:1~1:100, and magnetic core-shell nanostructure is prepared；
(4) glutaraldehyde spacerarm is used by the magnetic core-shell nanostructure and for the specific recognition molecules of pathogenic bacteria
Method is coupled.
Another object of the present invention is provide application of the above-mentioned nanometer bio probe in preparation pathogenic bacteria detection kit.
Nanometer bio probe provided by the invention has Mimetic enzyme activity, can use chromatmetry and carries out quickly
Detection pathogenic bacteria whether there is.Preparation method simple process provided by the invention, strong operability and environmental-friendly, can reach
The quickly purpose and effect of detection pathogenic bacteria, have a good application prospect.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is the present invention
Some embodiments for those of ordinary skill in the art without creative efforts, can also basis
These attached drawings obtain other attached drawings.
Fig. 1 is the Fe that embodiment 1 is prepared3O4The TEM of@Si-Pt schemes；
Fig. 2 is the Fe that embodiment 1 is prepared3O4The Mimetic enzyme activity analysis result of@Si-Pt；
Fig. 3 is the Fe that embodiment 2 is prepared3O4The TEM of@Si-Pt schemes；
Fig. 4 is the Fe that embodiment 3 is prepared3O4The TEM of@Si-Pt schemes；
Fig. 5 is the Fe that embodiment 4 is prepared3O4The TEM of@Si schemes；
Fig. 6 is that the TMB of various concentration staphylococcus aureus in application examples 1 absorbs spectrogram；
Fig. 7 is the linear relationship fitted figure of TMB colour developing and staphylococcus aureus number in application examples 1.
Specific embodiment
In order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below in conjunction with the embodiment of the present invention
In attached drawing, technical scheme in the embodiment of the invention is clearly and completely described, it is clear that described embodiment is
A part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, those of ordinary skill in the art
Every other embodiment obtained without creative efforts, shall fall within the protection scope of the present invention.
Embodiment 1
The present embodiment provides a kind of nanometer bio probes for detecting staphylococcus aureus, by magnetic core-shell nanostructure and
It is coupled composition for the specific antibody of staphylococcus aureus, wherein magnetic core-shell nanostructure is with ferriferrous oxide particles
Core, the silicon dioxide layer embedded with platinum grain are shell, whole negatively charged, have Mimetic enzyme activity.Its preparation side
Method the following steps are included:
(1) ferroferric oxide nano granules (1mg/mL, 10mL) that will be dispersed in hexamethylene are dispersed by mechanical stirring
In 10mL Triton X-100 (Triton X-100)/n-hexyl alcohol/water mixed solution (Triton X-100, just oneself
Alcohol, water molar ratio are 1:2:6), Reverse microemulsion is formed, 200 μ L (mass fraction 28%) of ammonium hydroxide and positive silicon are then added
200 μ L of acetoacetic ester, makes ethyl orthosilicate hydrolyze to form coated with silica layer under alkaline environment, and four oxidations three are prepared
Iron-silica dioxide granule；
(2) by ferroso-ferric oxide-silica dioxide granule (1mg/mL, 5mL) and 3- aminopropyl trimethoxy siloxane
(APTMS, 200 μ L) mixing carries out amination and modifies 14h, keeps its surface positively charged；
(3) by positively charged ferroso-ferric oxide-silica dioxide granule (1mg/mL, 5mL) and electronegative nano platinum particle
(1mg/mL, 0.5mL) hybrid reaction 14h makes nano platinum particle be supported on ferroso-ferric oxide-titanium dioxide by electrostatic adsorption
Silicon particle surface obtains the magnetic core-shell using ferriferrous oxide particles as core, the silicon dioxide layer embedded with platinum grain for shell
Nanostructure is denoted as Fe3O4@Si-Pt；
(4) use glutaraldehyde spacerarm method by Fe3O4@Si-Pt and for staphylococcus aureus specific antibody carry out
Coupling is to get target product.
Fig. 1 is the Fe that the present embodiment is prepared3O4The TEM of@Si-Pt schemes.It can be seen from the figure that in the present embodiment
Fe3O4@Si-Pt grain diameter size is in 30~50nm, and wherein ferriferrous oxide particles size is 10~20nm, silicon dioxide layer
Thickness is 5~10nm, and nano platinum particle size is 2~3nm, Fe3O4@Si-Pt particle is in using ferriferrous oxide particles as unitary core
Core-shell structure, size uniformity, dispersibility very well.
The Fe that analysis the present embodiment is prepared3O4The Mimetic enzyme activity of@Si-Pt, makes a concrete analysis of operating process
It is as follows: to select sodium-acetate buffer (pH=4), prepare reaction system 1mL, wherein 940 μ L of buffer, 20 μ L of hydrogen peroxide is (initial
Concentration 50mM), TMB 20 μ L (initial concentration 50mM), Fe3O420 μ L (initial concentration 1mg/mL) of@Si-Pt reacts 30 minutes,
200 μ L are taken to be placed in 96 orifice plates respectively, microplate reader measures the absorption spectra at 652nm.
As a result as shown in Fig. 2, it can be seen from the figure that compared to hydrogen peroxide and Fe3O4@Si, Fe3O4The mould of@Si-Pt
Quasi- peroxidase activity is remarkably reinforced, and TMB has obvious absorption at 652nm, and after having modified specific antibody, mould
Quasi- peroxidase activity is not significantly affected.
Embodiment 2
The present embodiment provides a kind of nanometer bio probes for detecting staphylococcus aureus, the difference from embodiment 1 is that:
It is positively charged ferroso-ferric oxide-silica dioxide granule (1mg/mL, 2mL) and electronegative nano platinum particle in step (3)
(1mg/mL, 2mL) carries out hybrid reaction 14h.
Fig. 3 is the Fe that the present embodiment is prepared3O4The TEM of@Si-Pt schemes.It can be seen from the figure that prepared by the present embodiment
Obtained Fe3O4@Si-Pt granular size is more uniform, and surface platinum particles are more, but clustering phenomena has occurred in part, causes entirety
Granule-morphology becomes no longer uniform, is unfavorable for the application of later period in biosystem.
Embodiment 3
The present embodiment provides a kind of nanometer bio probes for detecting staphylococcus aureus, the difference from embodiment 1 is that:
Triton X-100 in Triton X-100 (Triton X-100)/n-hexyl alcohol/water mixed solution in step (1), just
The molar ratio of own alcohol and water is 1:2:3.
Fig. 4 is the Fe that the present embodiment is prepared3O4The TEM of@Si-Pt schemes.It can be seen from the figure that shape in the present embodiment
At Reverse microemulsion it is less desirable, cause coated with silica effect poor, formed multi-core.
Embodiment 4
The present embodiment provides a kind of ferroso-ferric oxide-silica dioxide granule (Fe3O4@Si) preparation method, comprising: will point
It is pungent that the ferroferric oxide nano granules (1mg/mL, 10mL) being dispersed in hexamethylene by mechanical stirring are dispersed in 10mL polyethylene glycol
In base phenyl ether (Triton X-100)/n-hexyl alcohol/water mixed solution (Triton X-100, n-hexyl alcohol, water molar ratio are 1:2:
6) Reverse microemulsion, is formed, 200 μ L of ammonium hydroxide and 250 μ L of ethyl orthosilicate is then added, makes ethyl orthosilicate in alkaline environment
Under hydrolyze to form coated with silica layer.
Fig. 5 is the Fe that the present embodiment is prepared3O4The TEM of@Si schemes.It can be seen from the figure that ethyl orthosilicate is excessively led
Coated with silica layer is caused to thicken, ferroso-ferric oxide core is relatively smaller, and there are free silica spheres to occur.
Embodiment 5
The present embodiment provides a kind of nanometer bio probes for detecting Escherichia coli, by magnetic core-shell nanostructure and for big
The specific antibody of enterobacteria is coupled composition, wherein magnetic core-shell nanostructure using ferriferrous oxide particles as core, be embedded with platinum
The silicon dioxide layer of particle is shell, whole negatively charged, has Mimetic enzyme activity.Preparation method includes following step
It is rapid:
(1)~(3) are the same as embodiment 1；
(4) use glutaraldehyde spacerarm method by Fe3O4It@Si-Pt and is coupled for the specific antibody of Escherichia coli,
Up to target product.
Application examples 1
The nanometer bio probe that Application Example 1 provides is based on ELISA method fast detecting Staphylococcus aureus, including
Following steps:
(1) vancomycin of 2% BSA and 50mg/mL is coupled using EDC/NHS method, as capturing probe, packet
By in 96 orifice plate bottoms；
(2) staphylococcus aureus concentration gradient is designed as negative control, 10,102、103、104With 105cfu/mL；
(3) 150 μ L of staphylococcus aureus is added in every hole, and 37 DEG C of shaking tables shake 1 hour；
(4) bacterium solution is sucked out, PBS is washed three times；
(5) nanometer bio probe that the embodiment 1 that 100 μ L PBS are added in every hole and 20 μ L concentration are 1mg/mL provides, is incubated
It educates 1 hour；
(6) supernatant is siphoned away, PBS is washed three times；
(7) every hole adds 100 μ L acetate buffer solutions, 2 μ L hydrogen peroxide and TMB (50mM), and absorbance at 652nm is surveyed after 1 hour.
As a result as shown in fig. 6, the TMB for various concentration staphylococcus aureus absorbs spectrogram.It is aobvious that TMB is obtained according to Fig. 6
The linear relationship fitted figure of color and staphylococcus aureus number, as shown in fig. 7, it can be seen from the figure that TMB colour developing and bacterial population
With good linear relationship, with the increase of staphylococcus aureus quantity, colour developing is more obvious.
Application examples 2
The nanometer bio probe that Application Example 3 provides, it is golden yellow using being detected with detection method identical in application examples 1
Staphylococcus, as a result TMB colour developing and the linear relationship of staphylococcus aureus number are worse than application examples 1.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention, rather than its limitations；Although
Present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that: it still may be used
To modify the technical solutions described in the foregoing embodiments or equivalent replacement of some of the technical features；
And these are modified or replaceed, technical solution of various embodiments of the present invention that it does not separate the essence of the corresponding technical solution spirit and
Range.
Claims (10)
1. a kind of nanometer bio probe for detecting pathogenic bacteria, which is characterized in that by magnetic core-shell nanostructure and be directed to pathogenic bacteria
Specific recognition molecules be coupled composition, the magnetic core-shell nanostructure using ferriferrous oxide particles as core, be embedded with platinum
The silicon dioxide layer of grain is shell, whole negatively charged, has Mimetic enzyme activity.
2. nanometer bio probe according to claim 1, which is characterized in that the size of the magnetic core-shell nanostructure is
30~50nm, wherein ferriferrous oxide particles size is 10~20nm, and silicon dioxide layer thickness is 5~10nm, platinum grain size
For 2~3nm.
3. nanometer bio probe according to claim 1 or 2, which is characterized in that the specific recognition molecules be antibody,
One of target polypeptide, aptamer and antibiotic are a variety of.
4. the preparation method of the described in any item nanometer bio probes of claims 1 to 3, which comprises the following steps:
(1) ferroso-ferric oxide in organic phase is subjected to coated with silica using reverse micelle of microemulsion, four oxidations three is prepared
Iron-silica dioxide granule；
(2) amination modification is carried out to the ferroso-ferric oxide-silica dioxide granule, keeps its surface positively charged；
(3) platinum grain is made to be supported on the positively charged ferroso-ferric oxide-silica particles by electrostatic adsorption,
Magnetic core-shell nanostructure is prepared；
(4) it is coupled by the magnetic core-shell nanostructure and for the specific recognition molecules of pathogenic bacteria.
5. the preparation method according to claim 4, which is characterized in that the step (1) specifically includes: will be dispersed in organic
Ferroso-ferric oxide in phase is dispersed in Triton X-100/n-hexyl alcohol/water mixed solution, forms Microemulsions body
Then system adds ammonium hydroxide and ethyl orthosilicate, ethyl orthosilicate is made to hydrolyze to form coated with silica layer under alkaline environment.
6. preparation method according to claim 5, which is characterized in that the ferroso-ferric oxide being dispersed in organic phase is dense
Degree is 1~10mg/mL；
And/or Triton X-100, n-hexyl alcohol in the Triton X-100/n-hexyl alcohol/water mixed solution
Molar ratio with water is 1:2:4~8, preferably 1:2:6；
And/or the mass ratio of the ethyl orthosilicate, the ferroso-ferric oxide, the ammonium hydroxide is 1:5:1.
7. the preparation method according to claim 4, which is characterized in that the step (2) specifically includes: described four are aoxidized
Three-iron-silica dioxide granule and the mixing of 3- aminopropyl trimethoxy siloxane, carry out amination modification, and the two mass ratio is 1:10
~1:100, preferably 1:40, the reaction time be 4~for 24 hours, preferably 14h.
8. the preparation method according to claim 4, which is characterized in that four positively charged oxidations described in the step (3)
The mass ratio of three-iron-silica dioxide granule and the platinum grain is 100:1~1:1, preferably 10:1；
And/or coupling method is glutaraldehyde spacerarm method or EDC/NHS method, preferably glutaraldehyde spacerarm in the step (4)
Method.
9. the preparation method according to claim 4, which comprises the following steps:
(1) ferroso-ferric oxide that will be dispersed in organic phase is dispersed in Triton X-100/n-hexyl alcohol/water mixed solution
In, Reverse microemulsion is formed, ammonium hydroxide and ethyl orthosilicate is then added, hydrolyzes to form ethyl orthosilicate under alkaline environment
Ferroso-ferric oxide-silica dioxide granule is prepared in coated with silica layer；
(2) ferroso-ferric oxide-silica dioxide granule and 3- aminopropyl trimethoxy siloxane are mixed, carries out amination and repairs
Decorations, keep its surface positively charged；
(3) platinum grain is made to be supported on the positively charged ferroso-ferric oxide-silica particles by electrostatic adsorption,
The two mass ratio is 1:1~1:100, and magnetic core-shell nanostructure is prepared；
(4) by the magnetic core-shell nanostructure and for pathogenic bacteria specific recognition molecules using glutaraldehyde spacerarm method into
Row coupling.
10. application of the described in any item nanometer bio probes of claims 1 to 3 in preparation pathogenic bacteria detection kit.
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| WO2022225193A1 (en) * | 2021-04-23 | 2022-10-27 | 재단법인대구경북과학기술원 | Microstructure for actively sampling microbe and method for actively sampling microbe by using same |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0599926A (en) * | 1991-10-11 | 1993-04-23 | Tdk Corp | Method for measuring bacterium, cell and virus |
| CN101672771A (en) * | 2009-09-23 | 2010-03-17 | 东南大学 | Method for applying magnetic gamma-Fe2O3 nano-particle mimetic enzyme to biological detection |
| CN102645536A (en) * | 2012-04-19 | 2012-08-22 | 沈鹤柏 | Method for detecting staphylococcus aureus |
| CN104749365A (en) * | 2013-12-31 | 2015-07-01 | 中国科学院上海微***与信息技术研究所 | Difunctional composite nanosphere and method for rapidly detecting food-borne pathogenic bacteria |
| CN106248933A (en) * | 2016-08-19 | 2016-12-21 | 浙江工商大学 | The method quickly detecting the rugged Cronobacter sakazakii of slope based on PGM and magnetic Nano microsphere |
| CN108187741A (en) * | 2018-01-09 | 2018-06-22 | 天津大学 | More carboxyl magnetic Nano silicon ball solid-borne platinum catalysts and its preparation method and application |
-
2018
- 2018-10-18 CN CN201811217049.7A patent/CN109536571B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0599926A (en) * | 1991-10-11 | 1993-04-23 | Tdk Corp | Method for measuring bacterium, cell and virus |
| CN101672771A (en) * | 2009-09-23 | 2010-03-17 | 东南大学 | Method for applying magnetic gamma-Fe2O3 nano-particle mimetic enzyme to biological detection |
| CN102645536A (en) * | 2012-04-19 | 2012-08-22 | 沈鹤柏 | Method for detecting staphylococcus aureus |
| CN104749365A (en) * | 2013-12-31 | 2015-07-01 | 中国科学院上海微***与信息技术研究所 | Difunctional composite nanosphere and method for rapidly detecting food-borne pathogenic bacteria |
| CN106248933A (en) * | 2016-08-19 | 2016-12-21 | 浙江工商大学 | The method quickly detecting the rugged Cronobacter sakazakii of slope based on PGM and magnetic Nano microsphere |
| CN108187741A (en) * | 2018-01-09 | 2018-06-22 | 天津大学 | More carboxyl magnetic Nano silicon ball solid-borne platinum catalysts and its preparation method and application |
Non-Patent Citations (2)
| Title |
|---|
| HUACHAO ZAI ET,AL.: "A novel hierachically-nanostructured Pt/SiO2/Fe3O4 catalyst with high activity and recyclability towards hydrosilylation", 《RSC ADVANCES》 * |
| LANLIU ET,AL.: "Construction of surface charge-controlled reduced graphene oxide-loaded Fe3O4 and Pt nanohybrid for peroxidase mimic with enhanced catalytic activity", 《ANALYTICA CHIMICA ACTA》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2022225193A1 (en) * | 2021-04-23 | 2022-10-27 | 재단법인대구경북과학기술원 | Microstructure for actively sampling microbe and method for actively sampling microbe by using same |
| KR20220146116A (en) * | 2021-04-23 | 2022-11-01 | 재단법인대구경북과학기술원 | Microstructure for active collection of microorganisms and active collection method of microorganisms using the same |
| KR102537287B1 (en) | 2021-04-23 | 2023-05-26 | 재단법인대구경북과학기술원 | Microstructure for active collection of microorganisms and active collection method of microorganisms using the same |
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