CN103411816B - A kind of nano material and preparation method thereof for enriching and purifying pathogenic microorganism - Google Patents
A kind of nano material and preparation method thereof for enriching and purifying pathogenic microorganism Download PDFInfo
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Abstract
The invention discloses a kind of nano materials and preparation method thereof for the purifying of pathogenic microorganism fast enriching, and the chemical structure of the nano material is expressed as SiO2@Fe3O4@SiO2@CdTe@CMCS, the nano material are the core-shell type multifunctional magnetic Nano materials for having both targeting transport, fluorescent marker and specific bond ability, can fast and efficiently be enriched with pathogenic microorganism when carrying out live Diagnosis of Infectious Diseases.
Description
Technical field
The invention belongs to nano biological medical material fields, are related to a kind of receiving for the purifying of pathogenic microorganism fast enriching
Rice material and preparation method thereof.
Background technique
The means of biological detection are many kinds of at present, but the pre-processing of sample is to determine detection efficiency and qualification result
It is crucial.Although the quick detection of infectious disease pathogens is greatly facilitated in appearance and application based on modern technologies such as biosensors,
But still the demand of field quick detection cannot be fully met.Complex system where the infectious disease pathogens such as respiratory tract and enteron aisle, such as
Expiratory air, throat swab and excrement etc., the interference to sensor detection performance are a more universal phenomenons, are mainly manifested in:
First is that the non-targeted object content such as inorganic or organic present in infectious disease pathogens medium is typically much deeper than object, severe jamming
The combination of object and sensor sensing interface, influence detection specificity and sensitivity, and to the pre-treatment of sample require compared with
Height, and extend entire detection time, the medium of infectious disease pathogens are the mixed systems such as soil, blood, urine, need by from
The tedious processing step such as the heart, extraction could be used for biosensor detection, this process at least needs 2-3 hours；Second is that more
Dependent on labelling technique, usually enzyme or fluorescent marker, and infectious disease pathogens usually have autofluorescence, are easy to bring background dry
It disturbs, affects detection sensitivity.
Quantum dot (quantum dots, ODs) is also known as semiconductor nanocrystals, is by II-VI group or iii-v element
Composition can emit the nano particle of fluorescence after the excitation of particular excitation wave.The size of three dimensions of quantum dot all 100nm with
Under, appearance just like a very small pointing object, all limited to by movement of the internal electron in all directions.Quantum dot it is special
Structure causes it with skin effect, quantum size effect, Dielectric confinement effect and macro quanta tunnel effect, to show
Many is different from the physicochemical properties and the unique characteristics of luminescence of macroscopical block materials, makes it in biological fluorescent labelling, biology
The fields such as imaging have broad application prospects.On the other hand since poor biocompatibility, the toxicity of quantum dot are big, make it in life
Object field is still difficult to effectively be applied.
Nuclear shell type nano meter particle is in nanoscale spheric granules using a size as core, coats several layers uniformly on its surface
Nano thin-film and a kind of compound heterogeneous structure formed are connected with each other between core and shell by physically or chemically effect.With spy
It is multi-functional compound can to assign nucleocapsid by the composition and component for changing increase Shell Materials for the nanocomposite of different core-shell structure
Characteristic.
, background interference more demanding for Sample pretreatment present in current infectious disease pathogens on-site test is big and relies on
The problems such as labelling technique, the present invention are intended to provide before can be applied to pathogenic microorganism by nuclear shell type nano meter particle technology
The multifunctional nanocomposites of the fast enriching purifying of sample in treatment process.
Summary of the invention
The object of the present invention is to provide a kind of core-shell type multifunctional magnetism for the purifying of pathogenic microorganism fast enriching to receive
Rice material and preparation method thereof.
To achieve the above object, the present invention discloses a kind of for being enriched with the nano material of pathogenic microorganism first, special
Sign is: the entitled carboxymethyl chitosan cladding cadmium telluride coated silica coated ferriferrous oxide cladding two of nano material
The hud typed composite Nano ball of silica, the representation of the nano material are SiO2@Fe3O4@SiO2@CdTe@CMCS, wherein@
It is the meaning of cladding, such as SiO2@Fe3O4It indicates to coat one layer of nano ferriferrous oxide outside silica nanosphere.
The invention also discloses a kind of preparation methods of above-mentioned nano material, and described method includes following steps:
(1) with the monodispersed silica nanosphere of ferroso-ferric oxide cladding, (monodisperse refers to particle single variety and grain
Diameter distribution is very narrow)；
(2) product obtained again with monodispersed coated with silica step (1)；
(3) product obtained again with cadmium telluride quantum dot encapsulation steps (2)；
(4) product finally obtained with carboxymethyl chitosan encapsulation steps (3).
In a preferred technical solution of the invention, monodispersed silica nanosphere described in step (1) is
WithMethod preparation, and step (1) is carried out with heat sink area method.
In a preferred technical solution of the invention, step (2) be withWhat method carried out.
In a preferred technical solution of the invention, step (3) is carried out with chemical synthesis.
In a preferred technical solution of the invention, step (4) is the hydroxyl and carboxymethyl by silica surface
What the carboxyl on chitosan surface was realized by condensation reaction.
In a preferred technical solution of the invention, the reaction solution of heat sink area method described in step (1) is volume ratio
For 3: 1 deionized water and the mixed solution of ethylene glycol, reaction temperature is 40-100 DEG C, and the reaction time is 1-10 hours.
In a preferred technical solution of the invention, the reacting solution pH value of chemical synthesis described in step (3) is
5-14, reaction temperature are 50-300 DEG C, and the reaction time is 1-10 hours.
The present invention also provides application of the nano material in the purifying of pathogenic microorganism fast enriching.
Preferably, the nano material is used for piezoelectricity electrochemical immunosensor.
Compared with prior art, the beneficial effects of the present invention are: core-shell type multifunctional magnetic Nano disclosed by the invention
Material has superparamagnetism, fluorescence and pathoklisis, specifically, the partial size of magnetic particle of the invention is small, specific surface
Product is big, convenient for being efficiently coupled with target product；Nano ferriferrous oxide has superparamagnetism, divides under the action of externally-applied magnetic field
From very simple, it is made to be highly convenient for separating；Cadmium telluride quantum dot have it is good it is water-soluble, special fluoresce characteristic and
Photochemical stability has fluorescence；Outermost carboxymethyl chitosan has nontoxicity, good water-soluble, good biology
Compatibility is convenient for conjugated biological molecules with heavy metal there are chelation and surface is rich in a large amount of functional groups, both can be with
Protection cadmium telluride quantum dot will not reveal, and make resulting materials have good biocompatibility, can its surface modification its
His group assigns core-shell type multifunctional material pathoklisis.Therefore nano material of the invention can by infectious disease pathogens from
It separates, be enriched with and purify in complex samples medium, realize the quick pretreatment of cause of disease sample, entire pretreatment process takes around
0.5 hour, and its separation and concentration efficiency is up to 70-80%.
Detailed description of the invention
Fig. 1 core-shell type multifunctional magnetic Nano material prepares schematic diagram；
Fig. 2 core-shell type multifunctional magnetic Nano material is used for the schematic diagram of pathogenic microorganism fast enriching purifying；
Fig. 3 is sample to be tested real-time fluorescence quantitative PCR result figure；
Fig. 4 is the standard curve drawn according to Fig. 3.
Specific embodiment
The invention will now be further described with reference to specific embodiments, the advantages and features of the present invention will be with description and
It is apparent.But examples are merely exemplary for these, does not constitute to the scope of the claims in the present invention further
Restriction.
Embodiment 1: preparation embodiment
(1) it usesMethod prepares monodispersed silica nanosphere (bibliography: W.,
A.Fink.Controlled growth of monodisperse silica spheres in the micron size
Range [J] .J.Colloid Interface Sci., 1968,26 (1): 62-69)；
(2) ferroso-ferric oxide coated silica nanosphere is prepared using heat deposition method, specific preparation process is as follows: will
0.25gSilica nanosphere made from method is added into the mixed solution containing 50ml ethylene glycol and 150ml water,
Reaction solution is transferred in three-necked flask after ultrasonic disperse is uniform, in 45 DEG C of water-bath, mechanical stirring.15 minutes backward anti-
Answer addition 0.25g frerrous chloride, mechanical stirring 5 hours in 45 DEG C of water-bath in system.After completion of the reaction, by gained black
Solution centrifugation, and cleaned 3 times with deionized water and ethyl alcohol according to this, hud typed ferroso-ferric oxide coated silica nanometer is made
Ball, products therefrom is stand-by in 60 DEG C of oven dryings；
(3) it usesMethod prepares monodispersed coated with silica ferroso-ferric oxide coated silica nanosphere；
(4) the 0.1g coated with silica ferroso-ferric oxide coated silica nanosphere ultrasonic disperse prepared is existed
In 100ml deionized water, 5ml cadmium chloride solution, 5ml mercaptopropionic acid is added, and with the sodium hydrate regulator solution pH of 1mol/L
Value is 8.0.Then be added 0.8g sodium tellurite, and 60 DEG C mechanical stirring 2 hours, hud typed cadmium telluride cladding dioxy is made
SiClx coated ferriferrous oxide coated silica nanosphere, products therefrom acetone, water are cleaned, and are waited in 60 DEG C of oven dryings
With；
(5) it is dissolved firstly, taking 0.025g carboxymethyl chitosan sodium salt that 1.5mol/L, 5ml hydrochloric acid to the sodium salt is added dropwise, acidification
0.5 hour, dehydrated alcohol is then added dropwise into solution, is precipitated out carboxymethyl chitosan after standing 12 hours, 15000rpm
Centrifugation 10 minutes, with dehydrated alcohol centrifuge washing 7-8 times, every time 10 minutes, obtains product carboxymethyl chitosan CMCS.Take 0.1g system
Hud typed cadmium telluride coated silica coated ferriferrous oxide coated silica nanosphere is obtained, is added dropwise contains thereto
PB (phosphate buffer, 0.2mol/L, pH=6) 4ml ultrasound of 0.025gEDC 10 minutes, is then added 1ml shell containing carboxymethyl
The PB (0.2mol/L, pH=6,0.025g/ml) of glycan, ultrasound 1 hour remove surplus solution after being adsorbed with magnet, gained produces
Object is cleaned 3 times with deionized water and ethyl alcohol according to this, is obtained carboxymethyl chitosan cladding cadmium telluride coated silica and is coated four oxygen
Change the hud typed composite Nano ball of three-iron coated silica.
Fig. 1 is that core-shell type multifunctional magnetic Nano material disclosed in this invention prepares schematic diagram；Wherein 1 is titanium dioxide
Silicon；2 be ferroso-ferric oxide coated silica；3 be coated with silica ferroso-ferric oxide coated silica；4 be cadmium telluride
Coated silica coated ferriferrous oxide coated silica；5 coat cadmium telluride coated silica for carboxymethyl chitosan
Coated ferriferrous oxide coated silica.
2 Application Example of embodiment
Fig. 2 is the schematic diagram that nano material prepared by the present invention is used for the purifying of pathogenic microorganism fast enriching.It is shown in figure,
Nanometer magnetic bead can be specifically bound after specific antibody is modified with target antigen, and the antigen-antibody obtained after enriched separation is exempted from
Epidemic disease compound can be used for the detection of next step.
100ml small beaker is taken, the analog sample of pre-fabricated known pathogenic microorganism concentration and complex dielectrics are diluted
Liquid pours into beaker for use together, and the core-shell type multifunctional magnetic Nano material (hereinafter referred to as magnetic bead) of above-mentioned preparation is modified
Can with the antibody of specific recognition target pathogenic microorganism, then by after modification magnetic bead investment beaker in sample to be tested at room temperature
It is common to be incubated for 0.5 hour, make the respiratory infectious diseases such as influenza virus or adenovirus cause of disease and magnetic bead surfaces anti-current in complex dielectrics
The polyclonal antibody of the respiratory infectious diseases cause of disease such as Influenza Virus or adenovirus sufficiently combines, then will be in beaker under magnetic fields
Magnetic bead be totally separated out, so far complete a separation and concentration pathogenic microorganism sample from complex dielectrics pre-treatment
Journey.The magnetic bead for being combined with antigen is transferred in 100ml acidic buffer, release surface antigen simultaneously measures its concentration, with simulation
The original concentration of sample cause of disease is compared, and obtains magnetic bead to the separation and concentration efficiency of the cause of disease.
Separation and concentration efficiency passes through the method validation of Real Time-PCR:
1. the foundation of standard curve: preparing standard sample, after doubling dilution, produce standard curve.
2. evaluation separation and concentration efficiency: the magnetic bead that piezoelectric transducer crystal-vibration-chip surface is combined is eluted down with PBS solution
Come, measure the content of wherein pathogenic microorganism, is then this with the ratio of the total amount of pathogenic microorganism known in analog sample
Invent the separation and concentration efficiency of the material.
Standard sample and sample to be tested are measured using U.S.'s BIO-RAD company IQ5 real-time fluorescence quantitative PCR instrument.
Fig. 3 be gradient dilution sample to be tested real-time fluorescence quantitative PCR result figure (totally 8 gradient concentrations, each concentration do 3 it is parallel
Experiment)；Fig. 4 is the standard curve drawn according to Fig. 3 Ct threshold value and gradient concentration relationship.This is calculated by the ratio of two concentration
It invents the material and this hair is illustrated by separation and concentration efficiency is calculated up to 70-80% to the separation and concentration efficiency of sample
The bright material realizes the micro- life of separation and concentration cause of disease from complex dielectrics on the basis of guaranteeing has compared with high score from bioaccumulation efficiency
The quick pretreatment process of object sample.
Claims (3)
1. a kind of for being enriched with the nano material of pathogenic microorganism, it is characterised in that: the chemical structure of the nano material indicates
For SiO2@Fe3O4@SiO2@CdTe@CMCS；
The preparation method of the nano material specifically:
(1) it usesMethod prepares monodispersed silica nanosphere；
(2) ferroso-ferric oxide coated silica nanosphere is prepared using heat deposition method, specific preparation process is as follows: by 0.25g
The silica nanosphere is added into the mixed solution containing 50ml ethylene glycol and 150ml water, after ultrasonic disperse is uniform
Reaction solution is transferred in three-necked flask, in 45 DEG C of water-bath, mechanical stirring；It is added in 15 minutes backward reaction systems
0.25g frerrous chloride, mechanical stirring 5 hours in 45 DEG C of water-bath；After completion of the reaction, gained dark solution is centrifuged, and according to
This is cleaned 3 times with deionized water and ethyl alcohol, hud typed ferroso-ferric oxide coated silica nanosphere is made, by products therefrom
It is stand-by in 60 DEG C of oven dryings；
(3) it usesMethod prepares monodispersed coated with silica ferroso-ferric oxide coated silica nanosphere；
(4) the 0.1g coated with silica ferroso-ferric oxide coated silica nanosphere ultrasonic disperse prepared is gone in 100ml
In ionized water, 5ml cadmium chloride solution, 5ml mercaptopropionic acid is added, and is 8.0 with the sodium hydrate regulator solution pH value of 1mol/L；
Then be added 0.8g sodium tellurite, and 60 DEG C mechanical stirring 2 hours, hud typed cadmium telluride coated silica cladding is made
Ferroso-ferric oxide coated silica nanosphere cleans products therefrom acetone, water, stand-by in 60 DEG C of oven dryings；
(5) it is dissolved firstly, taking 0.025g carboxymethyl chitosan sodium salt that 1.5mol/L, 5ml hydrochloric acid to the sodium salt is added dropwise, acidification 0.5
Hour, dehydrated alcohol is then added dropwise into solution, is precipitated out carboxymethyl chitosan after standing 12 hours, 15000rpm centrifugation
10 minutes, with dehydrated alcohol centrifuge washing 7~8 times, every time 10 minutes, obtain product carboxymethyl chitosan CMCS；0.1g is taken to be made
Hud typed cadmium telluride coated silica coated ferriferrous oxide coated silica nanosphere is added dropwise contains thereto
Then the pH=6 of 0.025gEDC, 0.2mol/L phosphate buffer 4ml, ultrasound 10 minutes are added 1ml and contain carboxymethyl chitosan
PH=6,0.2mol/L phosphate buffer containing the carboxymethyl chitosan is 0.025g/ in the phosphate buffer
Ml, it is l hours ultrasonic, remove surplus solution after being adsorbed with magnet, products therefrom is cleaned 3 times with deionized water and ethyl alcohol according to this, obtained
To SiO2@Fe3O4@SiO2@CdTe@CMCS nano material.
2. application of the nano material in the purifying of pathogenic microorganism fast enriching according to claim 1.
3. application according to claim 2, which is characterized in that the nano material is used for piezoelectricity electrochemistry immuno-sensing
Device.
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| CN103614143A (en) * | 2013-11-28 | 2014-03-05 | 天津大学 | Preparation method of ferroferric oxide@silicon dioxide@zinc selenide fluorescent magnetic nanometer particles |
| CN103730223B (en) * | 2013-12-31 | 2017-10-13 | 深圳先进技术研究院 | Super-paramagnetism nano ball and preparation method thereof |
| CN111176003B (en) * | 2020-02-07 | 2022-12-06 | 武汉华星光电半导体显示技术有限公司 | Color developing material and display panel thereof |
| CN112881510A (en) * | 2021-01-15 | 2021-06-01 | 传鸣(宁波)化学科技有限公司 | Mixture, method and application for enriching microorganisms |
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