CN104592446B - The preparation method and application of the nanofiber molecularly imprinted polymer of tetracycline antibiotics - Google Patents
The preparation method and application of the nanofiber molecularly imprinted polymer of tetracycline antibiotics Download PDFInfo
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Abstract
The invention discloses the preparation method and application of a kind of nanofiber molecularly imprinted polymer, feature is that nanofiber is carried out silanization treatment degeneration, again with doxycycline as template molecule, acetonitrile/methanol is porogen, methacrylic acid is function monomer, trimethylol propane trimethyl acrylic acid acid esters is cross-linking agent, azodiisobutyronitrile is initiator, synthesis nano-cellulose molecularly imprinted polymer, characterize with to synthetic polymeric material, it is filled in solid phase extraction column, and combine high performance liquid chromatography, the concentration of tetracycline antibiotics in testing sample is determined according to standard curve, advantage be have highly sensitive, detection speed is fast, detection limit is low and efficient, inexpensively.
Description
Technical field
The present invention relates to field of detection of food safety, especially relate to a kind of for detecting tetracycline antibiotics in food
The nanofiber molecularly imprinted polymer preparation method and application of residual.
Background technology
Tetracycline antibiotics (Tetracycline Antibiotic) is the antimicrobial drug of spectral type, be usually used in prevention and
Treatment livestock and poultry process, but due to the unreasonable use of medicine, animal-derived food is commonly present the residual of tetracycline medication
Problem, endangers human health.Conventional Fourth Ring four class antibiotic mainly has tetracycline (Tetracycline, TC), chlortetracycline
(Chlortetracycline, CTC), oxytetracycline (Oxytetracycline, OTC) etc., part antibiotic also can be at animal body
The interior epitetracycline. that is converted into through metabolism, does not only result in drug effect and reduces, and its toxic and side effects increases.U.S. food medicine pipe
Reason office specifies that above-mentioned three kinds of tetracycline antibiotics maximum residue limits are 2 μ g/g, the Ministry of Agriculture's regulation antibiotic maximum residual
Amount is 100ng/g.Therefore, in food, the detection of tetracycline antibiotics is extremely urgent.Microbial method is most common method, but
It is that the method is complicated, time-consuming, accuracy is the highest.High performance liquid chromatography combine the detection technique of additive method have high sensitivity,
High selectivity and accuracy, can meet the qualitative and quantitative analysis to trace amount tetracycline antibiotics, but operation is complicated, pre-treatment
Requiring height, sample introduction requires strict.Therefore, sample is enriched with, purification, step indispensable during becoming trace analysis
Suddenly.Solid phase extraction techniques, because its equipment is simple, automaticity advantages of higher, is widely used in sample pre-treatments.General sample
Complicated component, solid phase extraction procedure specificity is the highest, and molecular engram solid phase extraction (Molecularly imprinted
Solid-phase extraction, MISPE) it is the high selective cleaning and enrichment new technique that fast development is got up in recent years,
Molecularly imprinted polymer is filled in solid phase extraction column, sample is carried out pre-treatment, improve detection specificity, accuracy.
Nano-cellulose (Nanocrystalline Cellulose, NCC), also referred to as Cellulose nanocrystal body, is a kind of
Diameter at the rod-like fibre element of tens to hundreds of nanometer, has high-purity, high-crystallinity, high intensity etc. excellent in 1-100nm, length
Point.Cellulose is natural polymers simultaneously, and NCC is because of the particularity on aggregated structure and physical property so that it is in high-performance
Polymer demonstrates huge application prospect.At present, any receiving about tetracycline antibiotics is not the most disclosed
The correlational study report of the preparation method and application of rice fiber molecule imprinted polymer.
Summary of the invention
The technical problem to be solved be to provide one have highly sensitive, detection speed is fast, detection limit is low and
Efficiently, the preparation method and application of the nanofiber molecularly imprinted polymer of cheap tetracycline antibiotics.
The present invention solves the technical scheme that above-mentioned technical problem used: the nanofiber of a kind of tetracycline antibiotics
The preparation method of molecularly imprinted polymer, using nanofiber molecule as supporting carrier, carries out silanization to its surface, with strength
Mycin be template molecule, acetonitrile/methanol be porogen, methacrylic acid (MAA) be function monomer, trimethylol propane trimethyl
Acrylic acid acid esters (TRIM) be cross-linking agent, azodiisobutyronitrile (AIBN) be initiator, the nanometer of synthesis tetracycline antibiotics
Fiber molecule imprinted polymer.
Specifically include following steps:
(1) preparation of nanofiber
Take 10g alpha-cellulose powder to be dissolved in the anhydrous N,N-dimethylacetamide of 100mL (DMAC), after ultrasonic 20min,
Activating 2h under the conditions of 160 DEG C, rotation is steamed 3-4h(and is steamed part N,N-dimethylacetamide), add 1mmol anhydrous Lithium chloride, turn
Speed 200rpm, is sufficiently stirred for 10h at 60 DEG C, and the milkiness that 3-4h removing part N,N-dimethylacetamide obtains is steamed in rotation the most again
Liquid, by emulsion under 8000～12000rmp rotating speeds, centrifugal 15min, takes off layer gelinite and is repeatedly washed to cleanout fluid pH and be
6.5-7.5, then dialysed overnight in distilled water, finally under the conditions of-40 DEG C, lyophilization 24h, process the fibre obtaining loosening
Dimension element nanometer；
(2) preparation of silanization nanofiber
Take 0.9g nano-cellulose to be scattered in 300mL ethanol/water mixed liquor, sonic oscillation 15min, be subsequently adding 15mL
Ammonia, 2.1mL tetraethyl orthosilicate (TEOS) in reaction system, under the conditions of 40 DEG C, stirring reaction 12h, by distilled water and second
Alcohol drip washing repeatedly, under 8000～12000rmp rotating speeds, centrifugal 15min, takes off layer gelinite under the conditions of-40 DEG C, lyophilization
24h, obtains silanization nanofiber particle；Wherein ethanol/water mixed liquor is mixed with water 4:1 by volume by ethanol；
(3) degenerative treatments of silanization nanofiber
Take 0.25mg silanization nanofiber particle join 30mL containing 3-mercaptopropyl trimethoxysilane (MPTS)
Volume fraction is in the toluene solution of 1/6, then in nitrogen protection, under the conditions of 120 DEG C, after back flow reaction 24h, use toluene successively
Wash 3～5 times with distilled water, then under 8000～12000rmp rotating speeds, centrifugal 15min, takes off layer gelinite at-40 DEG C of bars
Under part, lyophilization 24h, obtain the silanization nano-fiber material of degeneration；
(4) precipitation polymerization method synthesis nanofiber molecularly imprinted polymer
Take round-bottomed flask, be sequentially added into the silanization nano-fiber material of degeneration in 30mg step (3), 3.0mmol strength
Mycin, 60mL acetonitrile/methanol mixed liquor (35:30, V/V), the pretreated methacrylic acid of 0.68mL (MAA), ultrasonic 15min
After, add 10mmol pretreated trimethylol propane trimethyl acrylic acid acid esters (TRIM), ultrasonic 10min；Protect at nitrogen
Protecting down, add the pretreated azodiisobutyronitrile of 45mg (AIBN), be polymerized 24h in 60 DEG C of magnetic agitation, polyreaction is tied
Shu Hou, reduce pressure sucking filtration, is washed 3～5 times by filter cake methanol/acetic acid mixture (7:1, V/V), again with methanol/acetic acid mixture
(7:1, V/V) surname extraction 24h, after having extracted, sample under the conditions of-40 DEG C, lyophilization 24h, obtain Tetracyclines antibiosis
The nanofiber molecularly imprinted polymer of element.
Acetonitrile/methanol mixed liquor described in step (5) is mixed with methanol 35:30 by volume by acetonitrile, described
Methanol/acetic acid mixture mixed with acetic acid 7:1 by volume by methanol.
The preprocessing process of the methacrylic acid described in step (5) is by methacrylic acid decompression distillation, collects 69 DEG C
Fraction ,-30 DEG C of Refrigerator stores are standby；The preprocessing process of described trihydroxy methyl propane trimethyl acrylic acid acid esters is for using
Activated carbon adsorption remove impurity, filtration, collect filtrate, and-30 DEG C of Refrigerator stores are standby；The preprocessing process of described azodiisobutyronitrile
After mixing with methanol 1:10 in mass ratio for azodiisobutyronitrile, 50 DEG C of water-baths, while hot sucking filtration, filtrate stands 30min, again takes out
Filter, collects crystallized product, seals, and-40 DEG C save backup.
Described tetracycline antibiotics is tetracycline, chlortetracycline and oxytetracycline.
The method of tetracycline antibiotics concentration in a kind of nanofiber molecularly imprinted polymer detection sample, including following
Step:
(1) sample treatment
Rubbing uniformly by testing sample tissue mashing machine, the Mcllvaine buffering adding 4 times of volumes of testing sample is molten
Liquid, ultrasonic extraction 20min, centrifugal 15min take supernatant, and residue adds the Mcllvaine buffering of 4 times of volumes of testing sample
Solution, ultrasonic extraction 20min, centrifugal 15min take supernatant, merge two times centrifugal gained supernatant, supernatant is used 0.45 μm
Filtering with microporous membrane obtain analyte sample fluid, cold preservation is standby；
(2) prepared by molecular imprinted solid phase extraction cartridge
Accurately weigh 150mg nanofiber molecularly imprinted polymer, fill post with methanol wet, after loading well, use vacuum pump
Solid phase extraction column is taken out tightly, obtains molecular imprinted solid phase extraction cartridge；
(3) tetracycline antibiotics concentration measures
By molecular imprinted solid phase extraction cartridge 3mL methanol solution under conditions of flow velocity is 2mL/min, repeatedly drip washing is lived
After change, pure water cleans, and reloads 2mL analyte sample fluid, and after absorption terminates, pure water cleans 3-4 time, again with methanol eluant solution 3
～5 times, collect eluent, by eluent high-performance liquid chromatogram determination, record data, mould according to tetracycline, chlortetracycline and soil
3 kinds of tetracycline antibiotics liquid chromatograph peak areas of element and the quantitative relationship of corresponding tetracycline antibiotics concentration, determine to be measured
Tetracycline, chlortetracycline and the concentration of 3 kinds of tetracycline antibiotics of oxytetracycline in sample.
The compound method of described Mcllvaine buffer solution is as follows: 7.10g disodium hydrogen phosphate (Na2HPO4·
12H2O, 12.9g ten citrate dihydrate (C6H8O7·12H2O), 3.72g disodiumedetate, with diluting after water dissolution and fixed
Hold to 1000mL.
High-efficient liquid phase chromatogram condition: chromatographic column: ZORBAX Eclipse XDB-C18,4.6 × 150mm, 5 μm；Flowing phase:
The sodium dihydrogen phosphate of 0.01mol/L pH 2.8: acetonitrile=79:21；Detection wavelength: 355nm；Flow velocity: 0.8mL/min；Sample size:
20μL；Column temperature: 30 DEG C.
Principle: using nanofiber molecule as supporting carrier, its surface is carried out silanization, divides for template with doxycycline
Son, acetonitrile/methanol be porogen, methacrylic acid (MAA) be function monomer, trimethylol propane trimethyl acrylic acid acid esters
(TRIM) be cross-linking agent, azodiisobutyronitrile (AIBN) be initiator, template molecule and function monomer are by covalent bond or non-co-
Valence link combines and produces functional group and space structure complementary action formation coordination compound, then in the presence of cross-linking agent, initiation template molecule-
Function monomer coordination compound generation polyreaction, forms highly cross-linked rigid macromolecule polymer, then removes template molecule,
To functional group, space structure and template molecule complementation, and tetracycline antibiotics is had the molecule of specific adsorption ability
Imprinted polymer.Molecularly imprinted polymer is filled in solid-phase extraction column, makes solid phase extraction column, through overbalance, loading,
Drip washing and these steps of eluting, target molecule (tetracycline antibiotics) and the hole knot in molecularly imprinted polymer in sample
Close combined highly effective liquid chromatography and measure the trace residue of three kinds of tetracycline antibiotics of meat kind simultaneously.
Compared with prior art, it is an advantage of the current invention that: the nanofiber molecule print of tetracycline antibiotics of the present invention
The preparation method and application of mark polymer, make use of nano-cellulose to have a polyhydroxy and surface is prone to the feature of chemical modification,
To its surface silanization, with doxycycline as template molecule, acetonitrile/methanol as porogen, methacrylic acid (MAA) be function list
Body, trimethylol propane trimethyl acrylic acid acid esters (TRIM) be cross-linking agent, azodiisobutyronitrile (AIBN) be initiator, synthesis
Nano-cellulose molecularly imprinted polymer, is filled in solid phase extraction column, and combines high performance liquid chromatography, measures in sample four
The trace residue of ring element class antibiotic.It utilizes molecularly imprinted polymer to have specific recognition and adsorptivity to target molecule
Can, it is achieved 3 kinds of tetracycline antibiotics in sample are detected simultaneously.Nanofiber surface molecular engram is gathered by the present invention
Laminate material is as solid extracting agent, and combines the analysis method of high performance liquid chromatography, and utilizes nanofiber surface molecule to print
The specific adsorption characteristic of mark polymeric material, reaches to improve detection specificity, the purpose of sensitivity, and the present invention has special
Property, the multiple advantage such as quick, cheap.
Accompanying drawing explanation
Fig. 1 is scanning (SEM) the Electronic Speculum figure of nanofiber molecularly imprinted polymer；
Fig. 2 is transmission (TEM) the Electronic Speculum figure of nanofiber molecularly imprinted polymer；
Fig. 3 is infrared chromatography analysis (FT-IR) figure of nanofiber and nanofiber molecularly imprinted polymer；
Fig. 4 is the liquid chromatogram of tetracycline antibiotics standard specimen；
Fig. 5 is the absorption to three kinds of tetracycline antibiotics of the nanofiber molecularly imprinted polymer；
Fig. 6 is nanofiber molecularly imprinted polymer and the non-molecularly imprinted polymer adsorption curve to oxytetracycline；
Fig. 7 is the Scatchard analysis chart that oxytetracycline is adsorbed by nanofiber molecularly imprinted polymer；
Fig. 8 is the Scatchard analysis chart that oxytetracycline is adsorbed by the non-molecularly imprinted polymer of nanofiber.
Detailed description of the invention
Below in conjunction with accompanying drawing embodiment, the present invention is described in further detail.
Specific embodiment one
A kind of using nanofiber molecule as supporting carrier, its surface is carried out silanization, divides for template with doxycycline
Son, acetonitrile/methanol be porogen, methacrylic acid (MAA) be function monomer, trimethylol propane trimethyl acrylic acid acid esters
(TRIM) be cross-linking agent, azodiisobutyronitrile (AIBN) be initiator, synthesize nano-cellulose molecularly imprinted polymer, gather with this
Compound is adsorbent, uses Solid-Phase Extraction-liquid chromatograph United Technologies, the residual of tetracycline antibiotics in detection food.
Specific embodiment two
The concrete preparation method of the nano-cellulose molecularly imprinted polymer of the tetracycline antibiotics of above-described embodiment one,
Experiment material is as follows:
Doxycycline (Aladdin reagent company limited)；Tetracycline (Aladdin reagent company limited)；Oxytetracycline (Aladdin
Reagent company limited)；Chlortetracycline (Aladdin reagent company limited), alpha-cellulose powder (Aladdin reagent company limited)；Anhydrous
Lithium chloride (the upper smooth Science and Technology Co., Ltd. of Haitai)；N,N-dimethylacetamide (DMAC, Aladdin reagent company limited)；
3-mercaptopropyl trimethoxysilane (MPTS, Chemical Reagent Co., Ltd., Sinopharm Group)；Methacrylic acid (MAA, traditional Chinese medicines grouping of the world economy
Learn reagent company limited)；Trimethylol propane trimethyl acrylic acid acid esters (TRIM, Mayan reagent company limited)；Azo two is different
Butyronitrile (AIBN, Tianjin recovery fine chemistry industry institute)；Tetraethyl orthosilicate (TEOS, the upper Haitai smooth science and technology limited public affairs of share
Department)；Sodium dihydrogen phosphate (NaH2PO4·2H2O, Yong great chemical reagent company limited)；Disodium hydrogen phosphate (Na2HPO4·12H2O, forever
Big chemical reagent company limited)；Disodiumedetate (Chemical Reagent Co., Ltd., Sinopharm Group)；Citric acid (C6H8O7·
12H2O, Yong great chemical reagent company limited)；Acetonitrile (chromatographically pure, Di Ma Science and Technology Ltd.)；Methanol (chromatographically pure, enlightening equine
Skill company limited).Below the reagent of non-grade labeling is analytical pure, and deionized water is prepared from laboratory.
Specifically include following steps:
(1) preparation of nanofiber
Take 10g alpha-cellulose powder to be dissolved in the anhydrous N,N-dimethylacetamide of 100mL (DMAC), after ultrasonic 20min,
Activating 2h under the conditions of 160 DEG C, rotation is steamed 3-4h(and is steamed part N,N-dimethylacetamide), add 1mmol anhydrous Lithium chloride, turn
Speed 200rpm, is sufficiently stirred for 10h at 60 DEG C, and rotation steaming 3-4h removing part N,N-dimethylacetamide obtains milkiness the most again
Liquid, by emulsion under 8000～12000rmp rotating speeds, centrifugal 15min, takes off layer gelinite and is repeatedly washed to cleanout fluid pH and be
6.5-7.5, then dialysed overnight in distilled water, finally under the conditions of-40 DEG C, lyophilization 24h, process the fibre obtaining loosening
Dimension element nanometer；
(2) preparation of silanization nanofiber
Take 0.9g nano-cellulose to be scattered in 300mL ethanol/water mixed liquor (4:1, V/V), sonic oscillation 15min, so
Rear addition 15mL ammonia, 2.1mL tetraethyl orthosilicate (TEOS) in reaction system, under the conditions of 40 DEG C, stirring reaction 12h, use
The drip washing repeatedly of distilled water and ethanol, under 8000～12000rmp rotating speeds, centrifugal 15min, takes off layer gelinite-40 DEG C of conditions
Under, lyophilization 24h, obtain silanization nanofiber particle；
(3) degenerative treatments of silanization nanofiber
Take 0.25mg silanization nanofiber particle join 30mL containing 3-mercaptopropyl trimethoxysilane (MPTS)
Volume fraction is in the toluene solution of 1/6, then in nitrogen protection, under the conditions of 120 DEG C, after back flow reaction 24h, use toluene successively
Wash 3～5 times with distilled water, then under 8000～12000rmp rotating speeds, centrifugal 15min, takes off layer gelinite at-40 DEG C of bars
Under part, lyophilization 24h, obtain the silanization nano-fiber material of degeneration；
(4) agent treated: 1. by methacrylic acid decompression distillation, collecting the fraction of 69 DEG C ,-30 DEG C of Refrigerator stores are standby；
2. trihydroxy methyl propane trimethyl acrylic ester activated carbon adsorption remove impurity, filtration, collect filtrate, and-30 DEG C of Refrigerator stores are standby
With；3. azodiisobutyronitrile mixes with methanol (mass ratio is 1:10), 50 DEG C of water-baths, while hot sucking filtration, and filtrate stands 30min, then
Secondary sucking filtration, collects crystallized product, seals, and-40 DEG C save backup；
(5) precipitation polymerization method synthesis nanofiber molecularly imprinted polymer
Take round-bottomed flask, be sequentially added into the silanization nano-fiber material of degeneration in 30mg step (3), 3.0mmol strength
Mycin, 60mL acetonitrile/methanol mixed liquor (35:30, V/V), the pretreated methacrylic acid of 0.68mL (MAA), ultrasonic 15min
After, add 10mmol pretreated trimethylol propane trimethyl acrylic acid acid esters (TRIM), ultrasonic 10min；Protect at nitrogen
Protecting down, add the pretreated azodiisobutyronitrile of 45mg (AIBN), be polymerized 24h in 60 DEG C of magnetic agitation, polyreaction is tied
Shu Hou, reduce pressure sucking filtration, is washed 3～5 times by filter cake methanol/acetic acid mixture (7:1, V/V), again with methanol/acetic acid mixture
(7:1, V/V) surname extraction 24h, after having extracted, sample under the conditions of-40 DEG C, lyophilization 24h, obtain Tetracyclines antibiosis
The nanofiber molecularly imprinted polymer of element, wherein tetracycline antibiotics is tetracycline, chlortetracycline and oxytetracycline 3 kinds.According to
Above method, is not added with doxycycline, synthesizes matched group non-molecularly imprinted polymer NIP；
(6) sign of nanofiber molecularly imprinted polymer: scanning electron microscope (SEM), transmission electron microscope
(TEM) the microscopic appearance structure of sample, the chemical constituent of Fourier transform infrared spectrometer (FT-IR) test sample are observed.Institute
Having sign sample all to pulverize, pressing potassium bromide troche, wave-length coverage is 400～4000cm-1, resolution is 4 cm-1。
By scanning (SEM) Electronic Speculum figure and the polymerization of Fig. 2 nanofiber molecular engram of Fig. 1 nanofiber molecularly imprinted polymer
Transmission (TEM) the Electronic Speculum figure of thing is found out, the caliber of nanofiber molecularly imprinted polymer about between 500～600nm, Nanowire
Dimension molecularly imprinted polymer is slightly reunited, nanofiber molecular engram rough surface, uneven, with the presence of a large amount of apertures, little
Molecular compound can penetrate into the inside of polymer by hole.
Infrared chromatography analysis (FT-IR) figure at Fig. 3 nanofiber molecularly imprinted polymer Yu non-molecularly imprinted polymer
In, 1632cm-1For the vibration performance peak of carbon-carbon double bond (C=C), 3428 cm-1It is the characteristic absorption peak of H-O, in nanofiber
Content the highest.806 cm-1It is Si-O-Si symmetric vibration absworption peak, 1096 cm-1It is the characteristic absorption peak of Si-O, and
Now the activity of oxygen atom is relatively strong, it could therefore be concluded that nanofiber surface success silanization.At molecular engram nanofiber
In characteristic absorption peak, Si-O absorption intensity reduces, and can release more Si-O key and participate in the formation in molecular engram hole.
Specific embodiment three
The adsorption test of nanofiber molecularly imprinted polymer, mark song and detection limit, specifically comprise the following steps that
(1) high-efficient liquid phase chromatogram condition: chromatographic column: ZORBAX Eclipse XDB-C18,4.6 × 150mm, 5 μm,
Agilent；Flowing phase: 0.01mol/L sodium dihydrogen phosphate (pH 2.8): acetonitrile=79:21；Detection wavelength: 355nm；Flow velocity:
0.8mL/min；Sample size: 20 μ L；Column temperature: 30 DEG C, such as the liquid chromatogram that Fig. 4 is tetracycline antibiotics standard specimen, three kind four
Ring element class antibiotic, under above-mentioned liquid chromatograph experiment condition, can be analyzed within 15min and terminate, and tetracycline and oxytetracycline are front
Can go out peak within 7min, chlortetracycline went out peak before 12min；
(2) make standard curve: make solvent with methanol, compound concentration is 2.5 μ g/mL, 5.0 μ g/mL, 10 μ g/mL, 25 μ
G/mL, 50 μ g/mL, the tetracycline of 100 μ g/mL and oxytetracycline, concentration is 5.0 μ g/mL, 10 μ g/mL, 20 μ g/mL, 50 μ g/mL,
100 μ g/mL, the mixed standard solution of 200 μ g/mL chlortetracycline, by above-mentioned liquid phase chromatogram condition, make linear regression analysis, table 1
Linear equation, relative coefficient and detection limit for tetracycline antibiotics standard specimen.Result shows, three kinds of tetracycline antibiotics
Good (R is all higher than 0.999) in this concentration range internal linear relation；
(3) mensuration of method detection limit: (concentration ＜ 100 μ g/L) tests 5 concentration and one under low concentration scope
Blank group, each concentration is tested 10 times respectively, calculates standard deviation, draws the regression line with standard deviation-concentration, calculates the regression line
Being exactly the detection limit of the method with the intersection point (i.e. concentration is standard deviation when zero) of vertical coordinate, table 1 is tetracycline antibiotics
The linear equation of standard specimen, relative coefficient and detection limit, wherein X represents concentration, and Y represents liquid chromatograph peak area.Found out by table 1
In invention method for designing for tetracycline, oxytetracycline, the detection limit of chlortetracycline be respectively 9.61 μ g/Kg, 7.84 μ g/Kg, 11.93
μg/Kg；
Table 1
| Composition | The range of linearity (μ g/mL) | Detection limit (μ g/Kg) | Linear equation | Correlation coefficient (R) |
| Tetracycline (TC) | 0.05～1.1 | 9.61 | Y=19.995X-18.046 | 0.9991 |
| Oxytetracycline (OTC) | 0.05～1.1 | 7.84 | Y=222.35X-0.3748 | 0.9992 |
| Chlortetracycline (CTC) | 0.1～2.2 | 11.93 | Y=869.82X-13.32 | 0.9995 |
(4) adsorption test of molecularly imprinted polymer: take 10 10mL centrifuge tubes, the nanofiber being separately added into 50mg divides
Sub-imprinted polymer, then it is dense containing tetracycline, oxytetracycline, 3 kinds of tetracycline antibiotics of chlortetracycline to be sequentially added into 5mL in 1-10 pipe
Degree is respectively 0.2 mg mL-1、0.4 mg·mL-1、0.6 mg·mL-1、0.8 mg·mL-1、1.0 mg·mL-1、1.2
mg·mL-1、1.4 mg·mL-1、1.6 mg·mL-1、1.8 mg·mL-1、2.0 mg·mL-1Methanol/water (95:5, V/V)
Solution (i.e. 3 kinds tetracycline antibiotics all do above-mentioned 10 variable concentrations), after mixing vibration 8h, 8000rmp is centrifuged 10min,
And collect supernatant, and measuring by high performance liquid chromatography (HPLC), each concentration arranges three groups of repetitions.According to adsorption equilibrium formula
1., calculated equilibrium adsorbance Q(mg g-1):
In formula: V represents liquor capacity (mL)；M represents nanofiber molecularly imprinted polymer quality (g)；C0Represent solution
Initial concentration (mg mL-1)；CeRepresent solution adsorption equilibrium concentration (mg mL-1)；
The molecularly imprinted polymer absorption to target compound, mainly by two kinds of factor impacts, i.e. hole size and target
Compound structure fitness, function monomer and the matching degree of target compound.By Fig. 5 nanofiber molecularly imprinted polymer to four
The absorption of ring element class antibiotic understands, and molecularly imprinted polymer is respectively provided with good absorption to tetracycline, oxytetracycline and chlortetracycline
Property.Because employing doxycycline as template molecule, although it is in the same size with tetracycline molecule amount, but recognition site or group
Nature difference is bigger, therefore the most lower slightly to the adsorption of tetracycline, and to the adsorption of chlortetracycline and oxytetracycline relatively
Strong；
(5) static adsorption and the Scatchard of oxytetracycline are analyzed by nanofiber molecularly imprinted polymer: take 10 10mL
Centrifuge tube, is separately added into the nanofiber molecularly imprinted polymer of 50mg, then is sequentially added into 5mL oxytetracycline concentration in 1-10 pipe
It is respectively 0.2 mg mL-1、0.4 mg·mL-1、0.6 mg·mL-1、0.8 mg·mL-1、1.0 mg·mL-1、1.2 mg·
mL-1、1.4 mg·mL-1、1.6 mg·mL-1、1.8 mg·mL-1、2.0 mg·mL-1Methanol/water (95:5, V/V) solution,
After mixing vibration 8h, 8000rmp is centrifuged 15min, and collects supernatant, measures by high performance liquid chromatography (HPLC), each concentration
Three groups of repetitions are set.According to above-mentioned adsorption equilibrium formula 1., calculated equilibrium adsorbance Q(mg g-1), obtain table 2 nanofiber and divide
The Scatchard of sub-imprinted polymer and non-molecularly imprinted polymer (shown in preparation method as specific embodiment two) analyzes knot
Really,
Table 2
。
Fig. 6 is nanofiber molecularly imprinted polymer and the non-molecularly imprinted polymer adsorption curve to oxytetracycline.It is visible,
The nanofiber molecularly imprinted polymer adsorption to oxytetracycline, adsorbance is all as the increase of initial concentration and increases,
And the adsorption of nanofiber molecularly imprinted polymer is significantly greater than non-molecularly imprinted polymer.Illustrate that the present invention has more
Good selective absorption effect.
The Scatchard analysis chart adsorbed oxytetracycline by Fig. 7 nanofiber molecularly imprinted polymer and upper table, can see
Go out, nanofiber molecularly imprinted polymer equilibrium dissociation constant KdAt 0.17429～0.92533mg mL-1Between, maximum adsorption
Amount QmaxAt 44.886～91.635mg g-1Between, and by Fig. 8 nanofiber non-molecularly imprinted polymer, oxytetracycline maximum is inhaled
Attached amount is 24.41mg g-1.Therefore, the absorption of nanofiber molecularly imprinted polymer has two adsorption sites, absorbability
It is significantly greater than non-molecularly imprinted polymer.
Specific embodiment four
Application nanofiber molecularly imprinted polymer detects the concentration of tetracycline antibiotics in sample simultaneously.
In the DABAI Carnis Anseris domestica matter of Xiangshan, the extraction of tetracycline antibiotics uses Mcllvaine buffer solution dilution method.By group
Knit bruisher to be rubbed uniformly by DABAI Carnis Anseris domestica, be added into the above-mentioned buffer solution of 4 times of volumes, ultrasonic extraction 20min, be centrifuged
15min, residue same procedure repeats to extract 1 time, merges lixiviating solution, and with the filtering with microporous membrane of 0.45 μm, cold preservation is to be measured.
In mark-on process of the test, a certain amount of tetracycline antibiotics need to be dissolved in Mcllvaine buffer solution, make oxytetracycline,
Tetracycline concentration is respectively 0.2 μ g/mL, 0.4 μ g/mL, 0.8 μ g/mL, and chlortetracycline concentration is 0.4 μ g/mL, 0.8 μ g/mL, 1.6 μ
G/mL, the most each concentration does 5 repetitions.The complex prepared by the investigation recovery of standard addition to 3 kinds of tetracycline antibiotics.
Accurately weigh 150mg nanofiber molecularly imprinted polymer, fill post with methanol wet, after loading well, use vacuum pump
Solid phase extraction column is taken out tightly, obtains molecular imprinted solid phase extraction cartridge.Pillar first uses the repeatedly drip washing of 3mL methanol solution before using
Activation, flow velocity 2mL/min.Activation terminates, and pure water cleans, then is loaded into the sample mark-on lixiviating solution of the above-mentioned preparation of 2mL, absorption respectively
After end, pure water cleans, and uses methanol solution eluting, collects eluent, and HPLC is to be measured.Record data, according to corresponding liquid chromatograph
Peak area and the linear equation of mixed mark, draw the concentration of 3 kinds of tetracycline antibiotics in mark-on sample respectively, and calculate every kind of antibiosis
The recovery of standard addition of element, such as table 3,
Table 3
As can be seen from Table 3, the non-molecularly imprinted polymer of nanofiber is to oxytetracycline, Fourth Ring in the DABAI goose sample of Xiangshan
Element, the recovery of standard addition of chlortetracycline medicine are respectively 88.8%, 92.43%, 94.56%；Nanofiber molecularly imprinted polymer object
In the big white goose sample of mountain, oxytetracycline, tetracycline, the recovery of standard addition of chlortetracycline medicine are respectively 91.05%, 95.35%, 95.63%,
Visible, nanofiber molecularly imprinted polymer slightly increases as response rate during adsorbent.The coefficient of variation 1.53～7.80 it
Between, showing that the method for detecting residue of design is reliable, preci-sion and accuracy all can reach teracycline antibiotic residues analysis
Requirement.
The residual of 3 kinds of tetracycline antibiotics in the big white goose brisket of detection Xiangshan, testing result shows, in goose brisket, soil is mould
Cellulose content is 0.027mg/kg, tetracycline content is 0.21 mg/kg, chlortetracycline is not detected by.Illustrate in the big white goose brisket of Xiangshan
The residual quantity of 3 kinds of tetracycline antibiotics is below national standard requirement.
Certainly, described above not limitation of the present invention, the present invention is also not limited to the example above.The art
Those of ordinary skill, in the essential scope of the present invention, the change made, is retrofited, adds or is replaced, and also should belong to the present invention's
Protection domain.
Claims (8)
1. the preparation method of the nanofiber molecularly imprinted polymer of a tetracycline antibiotics, it is characterised in that: with nanometer
Cellulosic molecule as support carrier, its surface is carried out silanization, with doxycycline as template molecule, acetonitrile/methanol be cause
Hole agent, methacrylic acid be function monomer, trihydroxy methyl propane trimethyl acrylic ester be that cross-linking agent, azodiisobutyronitrile are
Initiator, the nanofiber molecularly imprinted polymer of synthesis tetracycline antibiotics.
The preparation method of the nanofiber molecularly imprinted polymer of tetracycline antibiotics the most according to claim 1, its
It is characterised by specifically including following steps:
(1) preparation of nanofiber
Take 10g alpha-cellulose powder to be dissolved in the anhydrous N,N-dimethylacetamide of 100mL, after ultrasonic 20min, live under the conditions of 160 DEG C
Changing 2h, after 3-4h is steamed in rotation, add 1mmol anhydrous Lithium chloride, rotating speed 200rpm, be sufficiently stirred for 10h at 60 DEG C, 3-is steamed in rotation the most again
4h obtains emulsion, by emulsion under 8000～12000rpm rotating speeds, centrifugal 15min, takes off a layer gelinite and is repeatedly washed to
Cleanout fluid pH is 6.5-7.5, then dialysed overnight in distilled water, finally under the conditions of-40 DEG C, and lyophilization 24h, process
To loose nano-cellulose；
(2) preparation of silanization nanofiber
Take 0.9g nano-cellulose to be scattered in 300mL ethanol/water mixed liquor, sonic oscillation 15min, be subsequently adding 15mL ammonia
Water, 2.1mL tetraethyl orthosilicate in reaction system, under the conditions of 40 DEG C, stirring reaction 12h, repeatedly drench with distilled water and ethanol
Wash, under 8000～12000rpm rotating speeds, centrifugal 15min, take off layer gelinite under the conditions of-40 DEG C, lyophilization 24h, obtain silicon
Alkanisation nanofiber particle；Wherein ethanol/water mixed liquor is mixed with water 4:1 by volume by ethanol；
(3) degenerative treatments of silanization nanofiber
Take that 0.25mg silanization nanofiber particle joins 30mL containing 3-mercaptopropyl trimethoxysilane volume fraction is
In the toluene solution of 1/6, then in nitrogen protection, under the conditions of 120 DEG C, after back flow reaction 24h, wash with toluene and distilled water successively
Wash 3～5 times, then under 8000～12000rpm rotating speeds, centrifugal 15min, take off layer gelinite under the conditions of-40 DEG C, freezing
It is dried 24h, obtains the silanization nano-fiber material of degeneration；
(4) precipitation polymerization method synthesis nanofiber molecularly imprinted polymer
Take round-bottomed flask be sequentially added into the silanization nano-fiber material of 30mg degeneration, 3.0mmol doxycycline, 60mL acetonitrile/
The pretreated methacrylic acid of methyl alcohol mixed liquor, 0.68mL, after ultrasonic 15min, adds the pretreated trihydroxy of 10mmol
Trimethacrylate, ultrasonic 10min；Under nitrogen protection, add the pretreated azodiisobutyronitrile of 45mg,
Being polymerized 24h in 60 DEG C of magnetic agitation, after polyreaction terminates, reduce pressure sucking filtration, by filter cake methanol/acetic acid mixture washing 3～5
Secondary, again with methanol/acetic acid mixture surname extraction 24h, after having extracted, sample under the conditions of-40 DEG C, lyophilization 24h,
The nanofiber molecularly imprinted polymer of tetracycline antibiotics.
The preparation method of the nanofiber molecularly imprinted polymer of tetracycline antibiotics the most according to claim 2, its
It is characterised by: the acetonitrile/methanol mixed liquor described in step (4) is mixed with methanol 35:30 by volume by acetonitrile, described
Methanol/acetic acid mixture mixed with acetic acid 7:1 by volume by methanol.
The preparation method of the nanofiber molecularly imprinted polymer of tetracycline antibiotics the most according to claim 2, its
It is characterised by: the preprocessing process of the methacrylic acid described in step (4) is for by methacrylic acid decompression distillation, collecting 69 DEG C
Fraction ,-30 DEG C of Refrigerator stores are standby；The preprocessing process of described trihydroxy methyl propane trimethyl acrylic ester is for living
Property charcoal adsorption-edulcoration, filtration, collect filtrate ,-30 DEG C of Refrigerator stores are standby；The preprocessing process of described azodiisobutyronitrile is
After azodiisobutyronitrile mixes with methanol 1:10 in mass ratio, 50 DEG C of water-baths, while hot sucking filtration, filtrate stands 30min, again takes out
Filter, collects crystallized product, seals, and-40 DEG C save backup.
The preparation method of the nanofiber molecularly imprinted polymer of tetracycline antibiotics the most according to claim 2, its
The tetracycline antibiotics being characterised by described is tetracycline, chlortetracycline and oxytetracycline.
6. tetracycline in the nanofiber molecularly imprinted polymer detection sample that a kind is applied according to any one of claim 1-5
The method of class antibiotic concentration, it is characterised in that comprise the following steps:
(1) sample treatment
Testing sample tissue mashing machine is rubbed uniformly, adds the Mcllvaine buffer solution of 4 times of volumes of testing sample, super
Sound extraction 20min, centrifugal 15min take supernatant, and residue adds the Mcllvaine buffer solution of 4 times of volumes of testing sample,
Ultrasonic extraction 20min, centrifugal 15min take supernatant, merge two times centrifugal gained supernatant, by the supernatant micropore of 0.45 μm
Membrane filtration obtains analyte sample fluid, and cold preservation is standby；
(2) prepared by molecular imprinted solid phase extraction cartridge
Accurately weigh 150mg nanofiber molecularly imprinted polymer, fill post with methanol wet, after loading well, will be solid with vacuum pump
Extraction pillar is taken out tightly mutually, obtains molecular imprinted solid phase extraction cartridge；
(3) tetracycline antibiotics concentration measures
By molecular imprinted solid phase extraction cartridge 3mL methanol solution under conditions of flow velocity is 2mL/min, repeatedly drip washing activation
After, pure water cleans, and reloads 2mL analyte sample fluid, and after absorption terminates, pure water cleans 3-4 time, again with methanol eluant solution 3～5
Secondary, collect eluent, by eluent high-performance liquid chromatogram determination, record data, according to tetracycline, chlortetracycline and oxytetracycline 3
Plant the quantitative relationship of tetracycline antibiotics liquid chromatograph peak area and corresponding tetracycline antibiotics concentration, determine testing sample
Middle tetracycline, chlortetracycline and the concentration of 3 kinds of tetracycline antibiotics of oxytetracycline.
Tetracycline antibiotics concentration in nanofiber molecularly imprinted polymer the most according to claim 6 detection sample
Method, it is characterised in that the compound method of described Mcllvaine buffer solution is as follows: 7.10g disodium hydrogen phosphate,
12.9g ten citrate dihydrate, 3.72g disodiumedetate, with diluting after water dissolution and being settled to 1000mL.
Tetracycline antibiotics concentration in nanofiber molecularly imprinted polymer the most according to claim 6 detection sample
Method, it is characterised in that high-efficient liquid phase chromatogram condition: chromatographic column: ZORBAX Eclipse XDB-C18,4.6 × 150mm, 5 μm；
Flowing phase: the sodium dihydrogen phosphate of 0.01mol/L pH 2.8: acetonitrile=79:21；Detection wavelength: 355nm；Flow velocity: 0.8mL/
min；Sample size: 20 μ L；Column temperature: 30 DEG C.
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