CN107099293A - Streptomysin molecular engram quanta polymer and preparation method for detecting streptomysin - Google Patents
Streptomysin molecular engram quanta polymer and preparation method for detecting streptomysin Download PDFInfo
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- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
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- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/08—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials
- C09K11/88—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials containing selenium, tellurium or unspecified chalcogen elements
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- C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
- C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
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- C09K11/025—Use of particular materials as binders, particle coatings or suspension media therefor non-luminescent particle coatings or suspension media
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6432—Quenching
Abstract
The present invention discloses a kind of streptomysin molecular engram quanta polymer and preparation method for being used to detect streptomysin, comprise the following steps, first silica shell quantum dot is obtained in quantum dot surface modification silica shell, then quantum dot compound is obtained in the surface modification silanization phenyl boric acid and streptomysin of silica shell quantum dot, quantum dot compound streptomycin is finally eluted to the streptomysin molecular engram quanta polymer obtained with streptomysin space identity site and phenyl boric acid glycosyl affine combination site.The streptomysin molecular engram quanta polymer can it is special, quick, sensitive, detect content of streptomycin with high throughput.
Description
Technical field
The present invention relates to molecular engram preparation field, more particularly to a kind of streptomysin molecule print for being used to detect streptomysin
Mark-quanta polymer and preparation method.
Background technology
Aminoglycoside antibiotics is cheap, broad-spectrum antiseptic, animal can be prevented to fall ill, and with the work for promoting growth of animal
With, be widely used in China in animal husbandry and aquaculture, most-often used aminoglycoside veterinary drug includes, gentamicin,
Streptomysin, dihydrostreptomycin etc..Its streptomycin has the toxicity such as ototoxicity, renal toxicity, neuromuscular blockade, largely using chain
Mycin is also easy to produce drug resistance.European Union's clear stipulaties prohibit the use of streptomysin as the growth promoter of domestic animal.To ensure people
Group's health, China, the U.S., European Union are determined to the maximum allowable residue limits of animal derived food streptomycin veterinary drug.Therefore,
How the residual of food streptomycin veterinary drug is rapidly and accurately determined, to ensureing that it is important that food security, guarantee population health have
Meaning.
There are microbial method, enzyme linked immunological (ELISA) method, chromatogram currently used for the method for food streptomycin residue of veterinary drug
Method, mass spectrography etc..The characteristics of microbial method can be with bacteria growing inhibiting using antibiotic, according to bacterial growth situation judgement sample
In whether there is antibiotic, simply, without complicated equipment, but cannot distinguish between specific Antibiotics.ELISA method belongs to
Immunoassay method, it is simple to operate using certain antibiotic of specific TPPA, quickly, high specificity, but streptomysin is small
Molecular compound, its Antibody preparation is difficult, and Antibody stability is poor, has strict demand, food samples to heat, pH, salinity etc.
Need, by pretreatment, to remove after chaff interference, just can guarantee that the sensitivity and specificity of ELISA detections, should not be used in live straight
Connect quick detection.Chromatography and mass spectrography can determine the species and content of specific streptomysin, but need to locate before complicated sample
Reason process and expensive instrument and equipment, it is impossible to for Site Detection.The structure of particularly streptomysin lacks chromophore, in chromatogram
Need to use EISD in detection, or be measured using fluorescence after being derived.EISD
Complex and expensive, common laboratory is not equipped with, and detection sensitivity is low.Derivatization method sensitivity is high, but influence factor is numerous, accuracy
It is relatively low.It can be seen that, at present in the urgent need to can rapidly and accurately determine the new method of streptomysin, effectively to monitor Determination of Streptomycin Residues, protect
Hinder population health.
Fluoroscopic examination is conventional quick determination method, and sensitivity is high, and equipment is simple, but Streptomyces is in unstressed configuration base
The compound of group, it is impossible to directly detected using fluorescence.In addition, fluoremetry specificity is relatively low, it is many dry into branch in food
Disturb the accuracy of fluoroscopic examination.Accordingly, it would be desirable to which one kind can specifically bind target antibiotic, done while effectively reducing background fluorescence
Disturb, and the new method of aminoglycoside antibiotics of the fluoremetry without fluorescence can be used.
Artificial antibody's (also known as molecularly imprinted polymer, MIP) is the high molecular polymer of chemical synthesis, by specific empty
Between structure and chemical bond (including covalent bond, ionic bond, hydrogen bond etc.) specific recognition and binding target molecule, its absorption specificity with
Natural antibody is similar, but tolerance organic solvent, ion, soda acid, high temperature and high pressure.The wherein MIP containing fluorescence is fluorescence MIP, no
Template molecule can be only specifically bound, while the combination of template molecule, which may further result in fluorescence MIP, occurs changing for fluorescence signal
Become (such as wavelength of fluorescence changes, or fluorescence intensity changes), according to the change of fluorescence signal, you can determine special with fluorescence MIP
Property combine template molecule content.It can be seen that, fluorescence MIP can determine non-blooming compound using fluorescence signal, and MIP with
The specific binding of template molecule ensure that the specificity of fluorescence MIP detections, be very suitable for being used to quickly determine non-fluorescence chemical combination
Thing, such as bibliography " Y.Wu, et al.Monitoring bisphenol A and its biodegradation in
water using a fluorescent molecularly imprinted chemosensor.Chemosphere,2015,
119:515-523”。
Typically contain two or more amino in streptomysin structure, and be attached by glycosidic bond.Phenyl boric acid and
Its derivative can be with saccharide compound (including aminoglycoside antibiotics) 1,2- or 1,3- glycol group and many alcoholic extract hydroxyl groups
Mutual covalent bond, this reaction has higher specificity.Quantum dot is semiconductor fluorescence nano material, and its fluorescing frequencies has sharp
Luminous spectrum width, emission spectrum is narrow, and fluorescence intensity is high, the advantages of being not susceptible to fluorescent bleach.Studies have found that, in quantum dot table
Face synthesizes glycoprotein MIP gels by the use of phenyl boronic acid derivative as function monomer, can simultaneously using MIP space identity power and
Phenyl boric acid-glycosyl affine combination power, improves binding specificity such as document " W Zhang, et al.As of the MIP to target molecule
Fluorescence Nanosensor for Glycoproteins with Activity Based on the
MolecularlyImprinted Spatial Structure of the Target and Boronate
Affinity.Angew.Chem.Int.Ed.2014,53,12489–12493”.But MIP prepared by this method can only determine dilute
The target protein in 100 times of urine is released, display this method Detection results in the case where there is high concentration chaff interference are not good,
The detection of complex sample is not suitable for.
The content of the invention
In order to solve problem above, it is an object of the invention to provide a kind of streptomysin molecule print for being used to detect streptomysin
Mark-quanta polymer and preparation method, the streptomysin molecular engram-quanta polymer energy can special, quick, sensitive, height
Content of streptomycin is detected flux, and the kit for detecting streptomysin is provided.
To achieve the above object, the designed technical scheme of the present invention includes：
For the preparation method for the streptomysin molecular engram-quanta polymer for detecting streptomysin, comprise the following steps, it is first
Silica shell quantum dot first is obtained in quantum dot surface modification silica shell, then in silica shell quantum dot
Surface modification silanization phenyl boric acid and streptomysin obtain quantum dot compound, finally by quantum dot compound streptomycin elute
Get off and obtain streptomysin molecular engram-quantum with streptomysin space identity site and phenyl boric acid-glycosyl affine combination site
Point polymer.
Preferably, for the preparation method for the streptomysin molecular engram-quanta polymer for detecting streptomysin, tool
Body comprises the following steps：
1) silica shell quantum dot is prepared：
In mass ratio, CdTe-GSH quantum dots (QDs), water, absolute ethyl alcohol, tetraethoxysilane (TEOS) are with ammoniacal liquor
0.1~1.0:4~10:35~45:0.6~1.0:1 is well mixed, and particle is collected after stirring 16~24h under oxygen free condition, through washing
Wash and obtain silica shell quantum dot (QDs@SiO after drying2)；
2) quantum dot compound is prepared：
In molar ratio, streptomysin and silanization phenyl boric acid are 8~12:1 is dissolved in pure water, then adds QDs@SiO2, table
Under face activating agent, crosslinking agent and ammoniacal liquor, oxygen free condition, it is stirred at room temperature after 20~28h and obtains quantum dot compound；
3) streptomysin is eluted：
Quantum dot compound is eluted for 4.0~5.0 hydrochloric acid solution 6~12 times, 30~50min is eluted every time using pH,
Elution uses pure water after finishing, and last centrifugal drying produces streptomysin molecular engram-quanta polymer (streptomysin MIP- amounts
Sub- point).
Preferably, the surfactant is cetyl trimethylammonium bromide (CTAB), and the crosslinking agent is
Tetraethyl orthosilicate (TEOS).
Streptomysin molecular engram-quanta polymer for detecting streptomysin, the streptomysin molecular engram-quantum dot
Polymer is what is prepared according to the preparation method of above streptomysin molecular engram-quanta polymer.
Streptomysin detection kit, it include streptomysin molecular engram-quanta polymer, streptomysin standard liquid and
Boric acid borax buffer solution.
Streptomysin detection kit application method is：
Step one：Streptomysin molecular engram-quanta polymer is dissolved in detection liquid and obtains streptomysin MIP- quantum
Point solution (0.025g/L), is added in 96 hole elisa Plates, determines fluorescence intensity (I at 620nm on multi-function microplate reader at once
0)。
Step 2：Add after 10 μ L streptomysins standard liquids or sample, reaction 3h and surveyed on multi-function microplate reader per hole
Determine fluorescence intensity at 620nm (I).
Step 3：Using I0/I as ordinate, streptomysin concentration is that abscissa sets up standard curve, according to I0/I and streptomysin
Linear relationship between concentration, you can determine the concentration of streptomysin.
Application of the streptomysin molecular engram-quanta polymer in detection sample streptomycin content.Especially in detection
The application of food streptomycin content.
The principle that the present invention prepares streptomysin molecular engram-quanta polymer is：
Transparent Bio-sil shell is modified in quantum dot surface, and molecular engram is carried out thereon, Bio-sil is obtained
The network structure of formation, can play the function of similar molecular sieve, not only can effectively protect quantum dot in complex system
Fluorescent stability (Youngdo Kim et al.CdSe quantum dot-encapsulated molecularly
imprinted mesoporous silica particles for fluorescent sensing of bisphenol
A.J.Mater.Chem., the materials such as the protein of macromolecular 2012,22,24075-24080), can also be prevented to enter.Therefore,
The preparation scheme that the present invention is used for：Silica shell is modified in quantum dot surface first, silanization phenyl boric acid is then utilized
For function monomer, prepared in silica shell layer surface has space identity site and phenyl boric acid-glycosyl affine combination position simultaneously
Streptomysin molecular engram-quanta polymer of point, the streptomysin molecular engram-quanta polymer recognizes streptomysin simultaneously
The glycosidic bond on space structure and its surface, and combined with streptomysin molecular engram-quanta polymer according to streptomysin front and rear
Fluorescence changes, the content of streptomycin in Accurate Determining sample.
The advantage of the invention is that：
This method is simultaneously using a variety of with streptomysin space identity site and phenyl boric acid-glycosyl affine combination site etc.
Adhesion improves the specificity of fluoroscopic examination, and quantum dot fluorescence ensure that the sensitivity of detection, and utilize silica shell
Molecular sieving effect, effectively reduce biologic single molecular and enter, so as to improve the inspection of fluorescence streptomysin molecular engram-quanta polymer
Survey the stability and accuracy of the streptomysin of trace in the complex matrices such as food, effectively overcome current streptomysin detection sensitivity and
Stability is low, complex operation the shortcomings of, realize and determine food streptomycin content quick, sensitive and accurately, agricultural, food,
There to be very big application prospect in the subjects such as environment.
Brief description of the drawings
Fig. 1 synthesizes the flow chart of streptomysin molecular engram-quanta polymer for the present invention；
Fig. 2A is the scanning electron microscope (SEM) photograph of quantum dot (QDs), and Fig. 2 B are quantum dot (the QDs@SiO for being coated with silica2)
Scanning electron microscope (SEM) photograph, Fig. 2 C are the scanning electron microscope (SEM) photograph of streptomysin MIP- quantum dots, and Fig. 2 D is NIP- quantum dots (negative control)
Scanning electron microscope (SEM) photograph；
Fig. 3 is Fourier transform infrared spectroscopy (FT-IR) figure of streptomysin MIP- quantum dots.Wherein A lines are quantum dot, B
Line is QDs@SiO2, C lines are streptomysin MIP- quantum dots, and D lines are NIP- quantum dots.Its ordinate is light transmittance, and abscissa is
Wave number (centimetre-1)；
Fig. 4 is absorption specificity figure of the streptomysin MIP- quantum dots to different aminoglycoside antibiotics；Ordinate is
The change in fluorescence (I0/I) of MIP- quantum dots, abscissa is the species for adding analyte；
Fig. 5 is detection specificity of the streptomysin MIP- quantum dots in chaff interference containing space to streptomysin；
Fig. 6 is that detection specificity of the streptomysin MIP- quantum dots in the chaff interference of site containing glycosyl to streptomysin is schemed；
The standard curve that Fig. 7 is obtained when being streptomysin detection kit examination criteria solution；
Fig. 8 be a variety of chaff interferences (gentamicin, amikacin, streptomysin and D-Glucose) while under existence condition, chain
Mycin detection kit detects the standard curve of streptomysin.
Embodiment
To more fully understand the present invention, invention is described in detail below with reference to accompanying drawing and instantiation.
Embodiment 1
With reference to shown in Fig. 1, the preparation method of streptomysin molecular engram-quanta polymer is：
Silica shell quantum dot is obtained in quantum dot surface modification silica shell first, then in silica
The surface modification silanization phenyl boric acid and streptomysin of shell quantum dot obtain quantum dot compound, finally by quantum dot compound
Streptomysin elutes the streptomysin molecule obtained with streptomysin space identity site and phenyl boric acid-glycosyl affine combination site
Trace-quanta polymer.Specific method comprises the following steps：
1) silica shell quantum dot is prepared：
Weigh 0.4gCdTe-GSH quantum dots (QDs) to be dissolved in 16ml pure water, then add 80g absolute ethyl alcohols, 1.6g tetra-
Ethoxysilane is well mixed with 2g ammoniacal liquor, collects particle after stirring 20h under oxygen free condition, titanium dioxide is obtained after scrubbed drying
Silicon shell quantum dot (QDs@SiO2)；
2) quantum dot compound is prepared：
10mol streptomysins and 1mol silanization phenyl boric acids are taken, is dissolved in after being well mixed in pure water, 20mg QDs@are added
SiO2, 0.2933g CTAB, 20 μ L TEOS and 300 μ L ammoniacal liquor, under oxygen free condition, be stirred at room temperature after 24h and obtain quantum dot and be combined
Thing；
3) streptomysin is eluted：
Quantum dot compound is eluted for 4.5 hydrochloric acid solution 10 times, 40min is eluted every time, elution is used after finishing using pH
Pure water 6 times, last centrifugal drying produces streptomysin molecular engram-quanta polymer (streptomysin MIP- quantum dots).
Prepare comparative example：The method of blank imprinted polymer (NIP- quantum dots) and the system of above streptomysin MIP- quantum dots
Preparation Method is identical, and difference is step 2) in be added without streptomysin.
Use hydro-thermal method synthesis hydrophilic CdTe-GSH quantum dots (QDs)：By CdCl2、GSH、Na2TeO3In molar ratio 5:
6:1 ratio, adds excess NaBH4, in pH=10.5, after temperature is stirring 1h in 110 DEG C of distilled water, terminating reaction, plus
Enter two volumes absolute ethyl alcohol, 10min (4500rpm) is centrifuged after mixing, supernatant discarding is placed in vacuum drying chamber after being repeated 3 times
Middle drying, it is CdTe-GSH quantum dots (QDs) to obtain powder
Embodiment 2
Following streptomysin molecular engram-quanta polymer writes a Chinese character in simplified form streptomysin MIP- quantum dots.
The performance evaluation of streptomysin MIP- quantum dots
1) morphological assessment：As shown in Fig. 2A~2C, transmission electron microscope analysis shows that the diameter of quantum dot is about 3nm, QDs
SiO2Diameter be about 9nm, the diameter of streptomysin MIP- quantum dots is about 25nm, and MIP layers of thickness is about 8nm.Such as A in Fig. 3
Shown in~D lines, FT-IR shows QDs@SiO2In 1068cm-1, 795cm-1And 463cm-1There is obvious absworption peak at place, respectively generation
Table Si-O-Si asymmetric stretching vibration, midplane extrusion vibration and flexural vibrations, illustrate to successfully synthesize in quantum dot surface
SiO2 shells.Streptomysin MIP- quantum dots are in 1402cm-1And 1602cm (B-O)-1(C=C) absworption peak prompting silanization benzene
The presence of boric acid function monomer, it was demonstrated that the present invention is in QDs@SiO2Surface be successfully prepared MIP layers.
2) streptomysin MIP- quantum dots adsorption capacity is evaluated：Using Scatchard slope of a curves as equilibrium dissociation constant Kd,
With formula：[Bond]/[Free]=- ([Bond]/Kd)+(Bmax/Kd), calculates maximum adsorption capacity Bmax.
3) streptomysin MIP- quantum dots binding specificity is evaluated：Using IPB (imprinting-induced promotion
Of binding) value evaluation：IPB=(Cmip-Cnip)/Cnip × 100%.Wherein, Cmip is and streptomysin MIP- quantum dots
With reference to target antibiotic amount, Cnip is the target antibiotic amount that is combined with NIP- quantum dots.As a result it see the table below 1:
The characterization of adsorption of the streptomysin MIP- quantum dots of table 1
As seen from Table 1, streptomysin MIP- quantum dots can show that this method can be used for preparing with specific recognition streptomysin
Determine the MIP- quantum dots of streptomysin.
Embodiment 3
Detection specificity of the streptomysin MIP- quantum dots to streptomysin
Various aminoglycoside antibiotics structures are similar, it is possible to can disturb the binding specificity of MIP and template molecule.For
This, carries out following test：
Detection specificity of the streptomysin MIP- quantum dots to different antibiotic：First by 190 μ L streptomysin MIP- quantum dots
Solution (0.025g/L) is added in 96 hole elisa Plates, determines fluorescence intensity (I0) at 620nm on multi-function microplate reader at once.Often
Hole adds 10 μ L streptomysins or gentamicin+amikacin+neomycin mixed solution, in multi-function microplate reader after reacting 3 hours
Fluorescence intensity (I) of the upper measure at 620nm.Calculate the fluorescence change (I0/I) before and after sample-adding.As shown in figure 4, result table
Bright streptomysin has most strong fluorescent quenching ability to streptomysin MIP- quantum dots, and MIP- quantum dots caused by other analogs
Fluorescence change (I0/I) and NIP- quantum dots without marked difference, meanwhile, the fluorescence changes (I0/I) of MIP- quantum dots compared with
NIP- quantum dots are big, and less than the change in fluorescence caused by streptomysin, illustrate streptomysin MIP- quantum dots can specificity streptomysin,
The antibiotic absorption affinity similar to other structures is very low.
Embodiment 4
Streptomysin MIP- quantum dots detect the specificity of streptomysin in when containing analogue
Embodiment 3 analyze streptomysin MIP- quantum dots individually to certain antibiotic binding specificity on the basis of, point
Streptomysin MIP- quantum dots are analysed at the similar antibiotic of various structures (i.e. during Existential Space structure chaff interference), MIP- quantum dots and
The binding specificity of streptomysin.
Detection specificity of the streptomysin MIP- quantum dots in chaff interference containing space to streptomysin：By 190 μ L streptomysins
MIP- quantum dot solutions (0.025g/L) are added in 96 hole elisa Plates, determine fluorescence at 620nm on multi-function microplate reader at once
Intensity (I0).The μ L of antibiotic mixed liquor 10 of various combination are added per hole, and (its streptomycin 100 μ g/L, gentamicin is respectively
0,10,100,500 μ g/L), reaction determines the fluorescence intensity (I) at 620nm on multi-function microplate reader after 3 hours.Such as Fig. 5
It is shown, as a result show under various concentrations gentamicin existence condition, the fluorescence of streptomysin MIP- quantum dots changes (I0/I) nothing
Notable difference, illustrating the presence of gentamicin does not influence streptomysin MIP- quantum dots to the accurate quantitative analysis of streptomysin.
Embodiment 5
Streptomysin MIP- quantum dots detect the spy of aminoglycoside antibiotics in the site of glycosyl containing various concentrations chaff interference
The opposite sex.
Saccharide compound may interfere with phenyl boric acid on streptomysin MIP-glycosyl affine combination power, therefore, analysis has sugar
During base chaff interference, the binding specificity of streptomysin MIP- quantum dots and template molecule.
Detection specificity of the streptomysin MIP- quantum dots in the chaff interference of site containing glycosyl to streptomysin：By 190 μ L strepto-s
Plain MIP- quantum dot solutions (0.025g/L) are added in 96 hole elisa Plates, determine glimmering at 620nm on multi-function microplate reader at once
Luminous intensity (I0).Then be separately added into per hole 10 μ L containing streptomysin and (100 μ g/L) and various concentrations D- glucose solution (0,
10,100,500 μ g/L) mixed liquor, reaction determines fluorescence intensity at 620nm on multi-function microplate reader after 3 hours
(I).As shown in fig. 6, comparing the fluorescence change (I0/I) added before and after mixed liquor.As a result the presence containing D-Glucose is shown not
Influence specific binding of the streptomysin MIP- quantum dots to streptomysin.
Embodiment 6
The sensitivity analysis of streptomysin detection kit
(1) sensitivity that streptomysin detection kit is detected to streptomysin
MIP- quantum dots are diluted to 0.025g/L with boric acid borax buffer solution (pH=9.0,0.02mol/L), 190 μ L are taken
MIP- quantum dot solutions are added in 96 hole elisa Plates, determine fluorescence intensity (I0) at 620nm on multi-function microplate reader at once.Often
Hole is added after 10 μ L streptomysins standard liquids (0,2,10,50,100,250,500,1000 μ g/L), reaction 3h in multifunctional enzyme
Mark and fluorescence intensity (I) at 620nm is determined on instrument.As shown in fig. 7, I0/I and streptomysin concentration are in 2-1000 μ g/L scope memory lines
Sexual intercourse, detection sensitivity is 0.76 μ g/L, illustrates that the streptomysin detection kit prepared using the present invention detects the range of linearity
Extensively, sensitivity is high, is expected to be directly used in the detection and analysis of low concentration content of streptomycin in the actual samples such as food, environment and biology
(Fig. 7).
(2) influence of the chaff interference to streptomysin detection kit detection sensitivity
190 μ L MIP- quantum dot solutions (0.025g/L) are added in 96 hole elisa Plates, at once on multi-function microplate reader
Determine fluorescence intensity (I0) at 620nm.10 μ L various concentrations streptomysins (0,2,10,50,100,250,500,1000 are added per hole
μ g/L) mixed liquor with chaff interference (neomycin, amikacin and gentamicin are 100 μ g/L, and D-Glucose is 25 μ g/L),
Reaction determines the fluorescence intensity (I) at 620nm on multi-function microplate reader after 3 hours.As shown in figure 8, comparing addition mixing
Fluorescence change (I0/I) before and after liquid.As a result show that chaff interference does not influence detection spirit of the streptomysin MIP- quantum dots to streptomysin
Sensitivity (Fig. 8).
Embodiment 7
The stability analysis of streptomysin detection kit
Various concentrations streptomysin mycin titer, the recovery of standard addition of streptomysin are detected using streptomysin MIP- quantum dots method
In 93.6.2-102.9%, in a few days deviation is 6.2-7.8% (n=6), and deviation is 3.8-7.6% (n=5), group difference in the daytime
For 4.8-8.1%.
Embodiment 8
The streptomysin in various samples is determined using streptomysin detection kit.
Milk sample：
Method 1：10% trichloroacetic acid (TCA) effect 3h, after centrifugation, the drying of part supernatant nitrogen are added in milk sample
Afterwards, with after boric acid borax buffer solution (pH=9.0,0.02mol/L) constant volume, contained with streptomysin MIP- quantum dots method detection streptomysin
Amount.Part supernatant is carried out after Solid phase extraction sample using commercialization SPE (SPE), is surveyed after deriving with HPLC-FLD
Determine content of streptomycin.As shown in table 2, streptomysin MIP- quantum dots method can determine 20 μ g/kg-400 μ g/kg streptomysin and
HPLC-FLD is only capable of determining 100 μ g/kg-400 μ g/kg streptomysin.It can be seen that, although sample is without Solid phase extraction, chain
Mycin MIP- quantum dots method still can be higher than HPLC-FLD methods with the streptomysin of low concentration in determination sample, its detection sensitivity.
Streptomysin MIP- quantum dots can rapidly and accurately determine the streptomysin of various concentrations.(table 2)
Muscle class sample (chicken, pork, the flesh of fish)
Contain muscle glycogen in musculature, the affine site of phenyl boric acid-glycosyl on MIP can be disturbed to resist with aminoglycoside
The combination of raw cellulose content.In chicken, pork, flesh of fish homogenised sample, 10% trichloroacetic acid (TCA) effect 3h, centrifugation are separately added into
Afterwards, after the drying of part supernatant nitrogen, with boric acid borax buffer solution (pH=9.0,0.02mol/L) constant volume, MIP- quantum dots are used
Method detects content of streptomycin.Part supernatant, sample is purified using commercialization solid-phase extraction column (Agilent PPL solid-phase extraction columns)
Afterwards, content of streptomycin is determined after deriving with HPLC-FLD.As shown in table 2, streptomysin MIP- quantum dots method can determine 20 μ g/kg-
400 μ g/kg streptomysin, and HPLC-FLD is only capable of determining 100 μ g/kg-400 μ g/kg streptomysin.It can be seen that, although sample is not
By Solid phase extraction, streptomysin MIP- quantum dots method still can determine low in muscle samples in the presence of a variety of chaff interferences
The aminoglycoside antibiotics content of concentration, its detection sensitivity is higher than HPLC-FLD methods.MIP- quantum dots can be quick and precisely
Ground determines the streptomysin of various concentrations in musculature.(table 2)
Animal's liver sample
Contain substantial amounts of hepatic glycogen in animal's liver, the affine site of phenyl boric acid-glycosyl in MIP- quantum dots can be disturbed
And the combination of streptomysin.Therefore, in pork liver, the homogenised sample of fish liver, 10% trichloroacetic acid (TCA) effect 3h is separately added into,
After centrifugation, after the drying of part supernatant nitrogen, with boric acid borax buffer solution (pH=9.0,0.02mol/L) constant volume, with MIP- amounts
Sub- point method detects aminoglycoside antibiotics content.Part supernatant, utilizes commercialization solid-phase extraction column (Agilent PPL solid phases
Extraction column) purify after sample, determine aminoglycoside antibiotics content with HPLC-FLD after deriving.As shown in table 2, streptomysin
MIP- quantum dots method can determine 20 μ g/kg-400 μ g/kg streptomysin, and HPLC-FLD is only capable of determining 100 μ g/kg-400 μ g/
Kg streptomysin.It can be seen that, although sample still can be determined in liver specimens without Solid phase extraction, MIP- quantum dots method
The content of streptomycin of low concentration, its detection sensitivity is higher than HPLC-FLD methods (table 2)
Animal kidney sample
In pig kidney homogenised sample, 10% trichloroacetic acid (TCA) effect 3h, after centrifugation, part supernatant liquid nitrogen are separately added into
After air-blowing is dry, with boric acid borax buffer solution (pH=9.0,0.02mol/L) constant volume, aminoglycoside is detected with MIP- quantum dots method
Antibiotic content.Part supernatant, is purified after sample using commercialization solid-phase extraction column (Agilent PPL solid-phase extraction columns), spread out
After life aminoglycoside antibiotics content is determined with HPLC-FLD.As a result show, MIP- quantum dots method can determine 20 μ g/kg-400
μ g/kg content of streptomycin, and HPLC-FLD is only capable of determining 100 μ g/kg-400 μ g/kg content of streptomycin.It can be seen that, although sample
Product still can determine the aminoglycoside antibiosis of low concentration in kidney samples A without Solid phase extraction, MIP- quantum dots method
Cellulose content, its detection sensitivity is higher than HPLC-FLD methods.MIP- quantum dots can rapidly and accurately determine various concentrations in pig kidney
Content of streptomycin (table 2)
Concrete outcome see the table below 2.
The distinct methods of table 2 determine the streptomysin in various samples
The scope of the present invention is not limited by the specific embodiments described, and the embodiment is only used as illustrating of the invention each
Also include the method and component of functional equivalent in the single example of individual aspect, the scope of the invention.In fact, except as described herein
Outside content, those skilled in the art can easily grasp a variety of improvement to the present invention with reference to described above and accompanying drawing.Institute
Improvement is stated to also fall within the scope of the appended claims.Every bibliography mentioned above is all included in conduct herein in full
With reference to.
Claims (7)
1. a kind of preparation method for being used to detect streptomysin molecular engram-quanta polymer of streptomysin, it is characterised in that：Bag
Following steps are included, silica shell quantum dot are obtained in quantum dot surface modification silica shell first, then in dioxy
The surface modification silanization phenyl boric acid and streptomysin of SiClx shell quantum dot obtain quantum dot compound, are finally combined quantum dot
Thing streptomycin elutes the streptomysin obtained with streptomysin space identity site and phenyl boric acid-glycosyl affine combination site
Molecular engram-quanta polymer.
2. the preparation side according to claim 1 for being used to detect streptomysin molecular engram-quanta polymer of streptomysin
Method, it is characterised in that：Specifically include following steps：
1) silica shell quantum dot is prepared：
In mass ratio, CdTe-GSH quantum dots, water, absolute ethyl alcohol, tetraethoxysilane and ammoniacal liquor are 0.1~1.0:4~10:35
~45:0.6~1.0:1 is well mixed, and collects particle after stirring 16~24h under oxygen free condition, dioxy is obtained after scrubbed drying
SiClx shell quantum dot；
2) quantum dot compound is prepared：
In molar ratio, streptomysin and silanization phenyl boric acid are 8~12:1 is dissolved in pure water, then adds silica shell quantum
Under point, surfactant, crosslinking agent and ammoniacal liquor, oxygen free condition, it is stirred at room temperature after 20~28h and obtains quantum dot compound；
3) streptomysin is eluted：
Quantum dot compound is eluted for 4.0~5.0 hydrochloric acid solution 6~12 times, 30~50min, elution are eluted every time using pH
Pure water is used after finishing, last centrifugal drying produces streptomysin molecular engram-quanta polymer.
3. the preparation side according to claim 2 for being used to detect streptomysin molecular engram-quanta polymer of streptomysin
Method, it is characterised in that：The surfactant is cetyl trimethylammonium bromide, and the crosslinking agent is tetraethyl orthosilicate.
4. a kind of streptomysin molecular engram-quanta polymer for being used to detect streptomysin, it is characterised in that：The streptomysin point
Sub- trace-quanta polymer is to be polymerize according to streptomysin molecular engram according to any one of claims 1 to 3-quantum dot
What the preparation method of thing was prepared.
5. a kind of streptomysin detection kit, it is characterised in that：It include streptomysin molecular engram as claimed in claim 4-
Quanta polymer, streptomysin standard liquid and boric acid borax buffer solution.
6. a kind of streptomysin molecular engram-quanta polymer as claimed in claim 4 is in detection sample streptomycin content
Application.
7. streptomysin molecular engram-quanta polymer according to claim 6 is in detection food streptomycin content
Using.
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| CN113304708A (en) * | 2021-06-11 | 2021-08-27 | 天津医科大学 | Preparation method of glycoprotein microreactor with boron affinity surface imprinting of mesoporous molecular sieve |
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