CN104277190B - The preparation of a kind of core-shell type Ultraluminescence molecular engram material and material application in sulfanilamide detects - Google Patents
The preparation of a kind of core-shell type Ultraluminescence molecular engram material and material application in sulfanilamide detects Download PDFInfo
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Abstract
The present invention relates to preparation and the material application in sulfanilamide detects of a kind of core-shell type Ultraluminescence molecular engram material.The method is with the fluorescent functional monomer with lysine as skeleton of band FMOC fluorophor; use surface aggregate method; with sulfamethyldiazine as template molecule; with the fluorescence molecule of synthesized lysine base as function monomer; with ethylene glycol dimethacrylate as cross-linking agent; with azodiisobutyronitrile as initiator, it is polymerized the imprinted layer of last layer about 10 nanometer at the silicon ball surface that the metering system of monodispersed a diameter of 100 nanometers is acylated.With soxhlet extraction, the template molecule of trace in material is washed away, obtain the fluorescence molecule trace sensing material having fluorescent quenching to respond template molecule, there is good dispersibility, faster response speed, stable optical property, and it is applied to the mensuration of sulfamethyldiazine content in mark-on milk, the uv absorption signal of analyte can be changed into sensitive fluorescence signal by this method indirectly, improve detection sensitivity, obtain the preferable response rate.
Description
Technical field
The present invention relates to the preparation of the core-shell type fluorescence molecule imprinted polymer of a kind of Ultraluminescence radical functino
Method, function of dominant technical field of nanometer material preparation.
Background technology
Abuse of antibiotics and the antibiotic remains that causes is undoubtedly " accelerated evolutionary " to pathogenic bacterium medicine resistance ability, " drug resistance
Pathogenic bacteria " it is exactly largely that antibacterials abuse expedites the emergence of out.Go down if this situation continued spreads deterioration, it is likely that
When making facing mankind infect without medicine can condition.At present multiple instrument is set up in the detection of residual antibiotic in food
Analysis method, but all must be by accurate expensive instrument, and operation processes wastes time and energy, and is unsuitable for on-the-spot and quickly analyzes,
Therefore, develop convenient, quick, the method for Sensitive Detection antibiotic remains and material has important realistic meaning.
Molecular engram generally refers to the polymeric material having single-minded absorbability to certain or certain class template molecule, its tradition
Preparation method is that after polymer raw mixing self assembly, template molecule is carried out combined polymerization, and template molecule trace is embedded into polymerization
In substrate, then by after template molecule elution, it is left with functional group in the material and size has matched with template molecule, tool
There is the trace hole of special identification function, absorption template molecule that can be single-minded in complicated matrix environment.It identifies process
It is similar to " antigen-antibody " recognition mechanism of biosystem, and there is good stability (high temperature resistant, acid and alkali-resistance, high pressure resistant, resistance to has
The biological poor environment such as machine solvent), use the features such as time length (renewable recycling), preparation are simple, know at target molecule
Not, sample pre-treatments, catalyze and synthesize, the aspect such as pharmaceutical carrier shows good application prospect (Chem. Soc. Rev.,
2011,40,2922 2942).
In recent years, the sensing unit that the absorption behavior of template molecule can produce signal response is incorporated into molecular engram material
In material, select absorbability, the capacity of resisting disturbance of imprinted material are integrated mutually with the Sensitive Detection ability of sensing unit, be not required to by
The analyte desorption of imprinted material absorption is got off and is detected by complex instrument, it is achieved the enrichment detection integration of template molecule, is
The focus of functional molecular trace research field and difficult point.Detection technique of fluorescence has highly sensitive, detects fireballing advantage.If
In the trace hole of molecular engram, embedding can produce the fluorophor of fluorescence response with template molecule so that material is at absorption mould
The change of fluorescence signal is produced, by the monitoring of this transducing signal being can be achieved with directly target molecule before and after plate molecule
Quantitative detection.
At present, most fluorescence imprinted material is all with the quantum dot of quantum dot or hydridization for fluorescence sense unit, and it was prepared
Journey is the most relatively complicated, and due to the size limitation of quantum effect, prepared quantum dot kernel mostly particle diameter is less, surface
Can be high, when modifying imprinted layer, material is easily reunited, and affects dispersibility and the optical stability (RSC of final gained imprinted material
Adv., 2014, 4, 2764–2771 ) ( Biosensors and Bioelectronics 2014, 57, 310–316
), and then affect the repeatability of testing result.
Summary of the invention
In view of above-mentioned, the purpose of the present invention aims to provide the preparation side of a kind of core-shell type Ultraluminescence molecular engram material
Method.Fluorescence molecule imprinted material prepared by the method has more preferable dispersibility, faster response speed, more stable optical
Matter, is a kind of good dispersion, and imprinted layer is thin, pattern is homogeneous, have the molecular engram material of Smart fluorescent detection function.
Another object of the present invention is to the sulfamethyldiazine detection in mark-on milk of Ultraluminescence molecular engram material
In application.The most outside fluorescence molecule imprinted material trace hole is embedded with and can produce the fluorescence of fluorescence response with template molecule
Group so that imprinted material produces the quencher of fluorescence signal after absorption template molecule, by system fluorescent quenching amount and solution
Linear relationship between middle template molecular concentration realizes the fast qualitative detection by quantitative of sulfamethyldiazine in mark-on milk.
It is an object of the invention to be achieved through the following technical solutions:
Ultraluminescence function monomer N-fluorenes methyl ester formyl-N-methyl acryloyl group-lysine methyl ester (FMOC-Lys-MC)
Preparation
A) N-fluorenes methyl ester formoxyl-N-tert-butyl ester formoxyl-lysine (FMOC-Lys-BOC) is joined 40 ml chlorine
Change in the methanol that hydrogen is saturated, 2 hour after sucking filtration is stirred at room temperature, be dried to obtain the lysine first of FMOC protection with methanol washing
Ester hydrochloride；
B) raw material having taken off BOC is joined (DCM) in anhydrous methylene chloride, then in above-mentioned solution, add methyl-prop
Dripping anhydrous triethylamine (TEA) after alkene acyl chlorides, material molar ratio is followed successively by 3 mmol:3.3 mmol:6 mmol, reacts 1 hour
Terminating, add DCM dilution, the most successively with hydrochloric acid and the saturated aqueous common salt washing of pH=1, organic facies anhydrous sodium sulfate is dried
Rear concentrated pillar (ethyl acetate: petroleum ether=1:2), is spin-dried for obtaining N-fluorenes methyl ester formyl-N-methyl acryloyl group-bad ammonia
Acid methyl ester (FMOC-Lys-MC) white crystal；
The preparation of fluorescence molecule trace FMIP
C) the silicon ball carrier that metering system is acylated is prepared: mixed by ethanol, water, ammonia 80 ml:6 ml:3 ml by volume
After even, dripping tetraethyl orthosilicate (TEOS) under stirring, room temperature reaction is ethanol centrifuge washing 2 times after 8 hours, then by gained
Naked silicon ball is distributed in ethanol, adds water and ammonia, lower dropping methyclyloxypropyl trimethoxy silane (MPS) of stirring,
Ethanol centrifuge washing is used 3 times after reacting 12 hours, 50 DEG C of vacuum drying, obtain the silicon ball that metering system is acylated, cold preservation is stand-by；
D) by polymer raw template molecule sulfamethyldiazine (SMZ), fluorescent functional monomer FMOC-Lys-MC, cross-linking agent
Ethylene glycol dimethacrylate (EGDMA), initiator azodiisobutyronitrile (AIBN) 0.15 mmol:0.15 in molar ratio
Mmol:1.5 mmol:0.06 mmol is dividedly in some parts in acetonitrile, drum nitrogen after be stirred at room temperature 5 hours ultrasonic uniformly, add
The acetonitrile solution of methacryl SiClx ball, drum nitrogen is 60 DEG C of stirring reactions 20 hours after 5 minutes, at the silicon that metering system is acylated
Ball surface aggregate last layer imprinted layer；
E) d) step resulting materials is used ethanol, acetonitrile centrifuge washing 1 time respectively, then with methanol: acetic acid=8:1) mixed
Closing liquid and carry out Soxhlet washing, until supernatant does not has the uv absorption of template molecule SMZ, be vacuum dried at 60 DEG C, it is right to have obtained
Template molecule has fluorescence molecule trace sensing material (FMIP) that fluorescent quenching responds.
Advantages of the present invention and the beneficial effect of generation:
The present invention overcomes when preparing fluorescence imprinted material with quantum dot for sensing unit that preparation flow is loaded down with trivial details, material is easily reunited
Shortcoming, with simple amino acid derivativges as sensing unit, with the slightly larger single dispersing silicon ball of size as carrier, by molecular engram
Technology and fluorescence detection method combine, and are prepared for sulfamethyldiazine antibiosis in solution is have fluorescent quenching power of test
Sensing imprinted material.By transmission electron microscope, scanning electron microscope, Fourier transform infrared spectroscopy, fluorescence spectrum, thermogravimetric
Analysis etc. demonstrates the success of this fluorescence molecule imprinted material and prepares, and the material scatter of gained is good, pattern is homogeneous, response is fast
Degree fast (up to response balance in 30 minutes), optical property are stable, have easily preparation, the feature of low cost.Pass through with this material
The linear relationship of Stern Volmer quencher establishing equation system fluorescence intensity and sulfamethyldiazine concentration, by this material
Preliminary Applications, to the detection of sulfamethyldiazine antibiotic in actual sample, is applied to sulfamethyldiazine content in mark-on milk
Mensuration.Indirectly the ultraviolet of analyte can be inhaled in the sulfamethyldiazine of about 267nm, this method for uv absorption
The collection of letters number is changed into sensitive fluorescence signal, produces fluorescent quenching response, improves detection sensitivity.In milk substrate, this inspection
The range of linearity of survey method fluorescent quenching standard curve is 0.5 μM 20 μMs, and lowest detection is limited to 98.4 nM (26.0 ng/
Ml) there is the ability of rapid and convenient detection sulfanilamide antibiotic.
Accompanying drawing explanation
Fig. 1 is the reactions steps schematic diagram of FMOC-Lys-MC fluorescent functional monomer.
Fig. 2 a, b are respectively the H of prepared fluorescent functional monomer FMOC-Lys-MC1Spectrum and C13Spectrogram.
Fig. 3 is the mass spectrum of prepared fluorescent functional monomer.In spectrogram, 451.17 is fluorescent functional monomer FMOC-Lys-MC
+ H the molecular ion peak of (molecular weight is 450.22).
Fig. 4 is the Electronic Speculum phenogram of prepared material.Wherein a, b, c are respectively 15000 times of transmission electron microscopes of silicon ball carrier
Figure, 200000 times of transmission electron microscope pictures, scanning electron microscope (SEM) photographs.D, e, f are respectively obtained nucleocapsid structure fluorescence molecule trace
(FMIP) 15000 times of transmission electron microscope pictures, 200000 times of transmission electron microscope pictures, scanning electron microscope (SEM) photographs.G, h, i are respectively obtained conduct
15000 times of transmission electron microscope pictures of the non-imprinted material of fluorescence (FNIP) of comparison, 200000 times of transmission electron microscope pictures, scanning electron microscope (SEM) photographs.
Fig. 5 a, b, c represent silicon ball carrier, the infrared spectrum of FMIP, FNIP respectively.
Fig. 6 a, b, c are respectively naked silicon ball, FMIP, FNIP thermogravimetric curve figure in the range of 0 ~ 700 DEG C.
Fig. 7 is material excitation-emission spectrogram in acetonitrile solution, and wherein a, b represent FMIP swashing in acetonitrile respectively
Send out spectral line (Em=304), the FMIP spectral line of emission (Ex=267) in acetonitrile；C, d represent FNIP excitation spectrum in acetonitrile respectively
Line (Em=304), the FNIP spectral line of emission (Ex=267) in acetonitrile；E is template molecule SMZ ultraviolet absorption spectrum in acetonitrile
Line.
Fig. 8 is the acetonitrile solution fluorescent stability of material, and wherein a represents the acetonitrile solution fluorescence intensity of FMIP and FNIP
(excitation wavelength Ex=267 nm launches wavelength Em=304nm, a width of 1.5-3 of excitation-emission slit), b generation over time
The pure water solution fluorescence intensity of table FMIP and FNIP (Ex=267, Em=304, slit width 1.5-3) over time.
Fig. 9 a is the static adsorbance contrasts in the ACN solution of SMZ of 5 batches of block polymeric materials, and Fig. 9 b is 5 batches of block polymerizations
Material static adsorbance contrast in the DMF solution of SMZ.
After Figure 10 is FMIP fluorescence imprinted material and analyte SMZ mixes in the solution, system fluorescent quenching degree is at any time
Between change schematic diagram.
Figure 11 b is the fluorescence intensity curves of variable concentrations FMIP material acetonitrile solution.
Figure 12 a be the FMIP acetonitrile solution of 100 μ g/ml to the SMZ response balance of variable concentrations after, the fluorescence of each system
The spectral line of emission (Ex=267, Em=304, slit width 1.5-3).
Figure 13 is the fluoroscopic examination selectivity schematic diagram of FMIP and FNIP.
Figure 14 a be the FMIP milk substrate solution of 100 μ g/ml with the SMZ response balance of variable concentrations after, each system
Fluorescent line (Ex=267, Em=318, slit width 3-5).
Detailed description of the invention
Below in conjunction with specific embodiment and experimental example, the present invention is described further again:
Embodiment 1
Prepare fluorescent functional monomer: by the N-fluorenes methyl ester formoxyl-N-tert-butyl ester formoxyl-lysine (FMOC-of 2.5g
Lys-BOC) join in the methanol that 40 ml hydrogen chloride gas are saturated, sucking filtration after 2 hours is stirred at room temperature, wash with 25 ml methanol
After washing 3 times, it is dried to obtain the lysine methyl ester hydrochlorate of FMOC protection.Take 3 mmol taken off the raw material of BOC join 25ml without
In water dichloromethane (DCM), after adding 3.3 mmol methacrylic chlorides, drip 6 mmol anhydrous triethylamines (TEA), react 1
Hour terminate, add dilution in 25ml anhydrous methylene chloride (DCM), the most successively with hydrochloric acid and the saturated common salt washing of pH=1
Washing, the dried concentrated pillar purification of organic facies anhydrous sodium sulfate (ethyl acetate: petroleum ether=1:2), product is spin-dried for obtaining N-
Fluorenes methyl ester formyl-N-methyl acryloyl group-lysine methyl ester (FMOC-Lys-MC) white crystal.Polymer raw template is divided
Sub-sulfamethyldiazine, fluorescent functional monomer FMOC-Lys-MC, cross-linking agent ethylene glycol dimethacrylate, initiator azo
Bis-isobutyronitrile 0.15 mmol:0.15 mmol:1.5 mmol:0.06 mmol in molar ratio be dividedly in some parts in acetonitrile ultrasonic uniformly,
Adding the acetonitrile solution of methacryl SiClx ball, drum nitrogen is 60 DEG C of stirring reactions 20 hours after 5 minutes, at methacryl
The silicon ball surface polymerization last layer imprinted layer changed.
Synthetic schemes as it is shown in figure 1, product nuclear-magnetism and mass spectral characteristi as shown in Figure 2,3, it was demonstrated that fluorescent monomer FMOC-
The successful synthesis of Lys-MC.Product hydrogen spectrum, carbon spectrum, mass spectrometric data are as follows:
1H NMR (400 MHz, CDCl3 ) δ= 7.72 (d, J = 7.5 Hz, 2H), 7.63 – 7.51 (m,
2H), 7.36 (t, J = 7.3 Hz, 2H), 7.28 (dd, J = 10.6, 4.3 Hz, 2H), 6.30 (s, 1H),
5.85 (d, J = 8.1 Hz, 1H), 5.66 (s, 1H), 5.24 (d, J = 1.6 Hz, 1H), 4.49 – 4.28
(m, 3H), 4.18 (t, J = 7.1 Hz, 1H), 3.70 (s, 3H), 3.27 (dd, J = 13.0, 6.6 Hz,
2H), 1.91 (s, 3H), 1.88 – 1.63 (m, 2H), 1.53 (dt, J = 13.4, 6.8 Hz, 2H), 1.44
– 1.28 (m, 2H)。
13C NMR (101 MHz, CDCl3 ) δ=173.12, 168.83, 156.34, 143.82, 141.33,
140.14, 127.82, 127.17, 125.21, 120.08, 119.54, 67.08, 53.84, 52.51, 47.18,
39.13, 31.86, 29.04, 22.62, 18.83。
ESI-MS m/z (M + H+) = 451.17。
Embodiment 2
The fluorescence imprinted material preparing 5 batches of doping difference in functionality monomers by the method for polymerisation in bulk (therefrom filters out optimum
Proportioning raw materials): as a example by the fluorescence imprinted material of the methacrylic acid that adulterates (MAA) function monomer, by 0.15 mmol template molecule
The MAA of SMZ and 0.45 mmol joins in 3 ml bottles with cover, is subsequently adding 1.5 ml DMFs (DMF)
Dissolve, add fluorescent monomer FMOC-Lys-MC after magnetic agitation 1 h, be stirred at room temperature 1 hour after drum nitrogen, stand overnight.Then
It is sequentially added into 1.5 mmol cross-linking agent ethylene glycol dimethacrylate (EGDMA), 6 mg initiator azodiisobutyronitriles
(AIBN), after drum nitrogen, 60 DEG C are stirred 20 hours, grind Soxhlet after sieving and wash (methanol: acetic acid=8:1) until supernatant does not has mould
The uv absorption of plate molecule, is vacuum dried stand-by at 60 DEG C.This material number is M2.The preparation process of M1 compared with M2, except
Being not added with outside MAA, remaining is identical；The preparation process of M3, M4, M5 is compared with M2, except the MAA of doping is changed to propylene successively
Outward, remaining is identical for amide (AM), 2-acrylamide-2-methylpro panesulfonic acid (AMPS), 4 vinylpyridines (4-VP)；Make
For the preparation method of each group of non-imprinted material N1, N2, N3, N4, N5 compareed, except being not added with template molecule SMZ, remaining is with corresponding
The preparation method of M1, M2, M3, M4, M5 is identical, and filtering out M1, N1 group material by Staticadsorption experiment is optimum feed stock
Proportioning, i.e. template molecule sulfamethyldiazine, fluorescent functional monomer FMOC-Lys-MC, cross-linking agent ethyleneglycol dimethacrylate
Ester, the mol ratio of initiator azodiisobutyronitrile are 0.15 mmol:0.15 mmol:1.5 mmol:0.06 mmol.
Embodiment 3
Prepare the silicon ball carrier that metering system is acylated: after 80 ml ethanol, 6 ml water, 3 ml ammonia mixings, under stirring
Dripping 8 ml tetraethyl orthosilicates (TEOS), room temperature reaction is ethanol centrifuge washing 2 times after 8 hours, then disperseed by gained silicon ball
In 60 ml ethanol, add 0.5 ml water and 0.1 ml ammonia, the lower methyclyloxypropyl three dripping 1 ml of stirring
Methoxy silane (MPS), uses ethanol centrifuge washing 3 times after reacting 12 hours, and 50 DEG C of vacuum drying obtain 2.28 g metering systems
Acylated silicon ball, cold preservation is stand-by.
0.15 mmol template molecule SMZ and 0.15 mmol fluorescent monomer FMOC-Lys-MC is joined 25ml acetonitrile
Middle ultrasonic dissolution, is stirred at room temperature 5 hours after drum nitrogen, is then sequentially added into 1.5 mmol cross-linking agent EGDMA, 300 mg methyl-prop
Alkene is acylated 5 ml acetonitrile solutions of silicon ball, 0.06 mmol initiator azodiisobutyronitrile AIBN, drum nitrogen after 5 minutes 60 DEG C stir
Mix reaction 20 hours, at the silicon ball surface polymerization last layer imprinted layer that metering system is acylated；Resulting materials is respectively with ethanol, acetonitrile
Centrifuge washing 1 time.Then Soxhlet washing (methanol: acetic acid=8:1) is not until supernatant has a uv absorption of template molecule SMZ, and 60
It is vacuum dried stand-by at DEG C.This material number is FMIP, as preparation method and the FMIP phase of the non-imprinted material FNIP compareed
With being simply not added with template molecule SMZ.As shown in Figure 4, wherein a, b, c are respectively the 15000 of silicon ball carrier to resulting materials Electronic Speculum figure
Times transmission electron microscope picture, 200000 times of transmission electron microscope pictures, scanning electron microscope (SEM) photographs.D, e, f are respectively obtained nucleocapsid structure fluorescence and divide
15000 times of transmission electron microscope pictures of sub-trace (FMIP), 200000 times of transmission electron microscope pictures, scanning electron microscope (SEM) photographs.G, h, i are respectively obtained
As comparison 15000 times of transmission electron microscope pictures of the non-imprinted material of fluorescence (FNIP), 200000 times of transmission electron microscope pictures, scanning electron microscopies
Figure.Contrasted by the transmission electron microscope picture of Fig. 4 b and e, h and understand after polymerization reaction on the solid surface, at the single dispersing silicon of diameter 100 nm
The thick imprinted layer of about 10 nm successfully it has been coated with outside ball carrier.
In Fig. 5, a, b, c represent silicon ball carrier, the infrared spectrum of FMIP, FNIP respectively.Fig. 5 shows, FMIP(b) and FNIP
1730 cm occurred in (c) spectrogram-1、1260 cm-1、1450 cm-1Peak is corresponding in turn to carbonyl C=O in cross-linking agent EGDMA
The symmetrical stretching vibration peak of carbon oxygen singly-bound C-O, polymerization carbochain methylene CH in stretching vibration peak, EGDMA2Stretching vibration
Peak；2965 cm-1Peak is the eigen vibration peak of C-H, 690 cm-1It it is the characteristic peak on FMOC group phenyl ring；Meanwhile, compared to silicon
Ball carrier (a), FMIP(b) and FNIP(c) 3420 cm in spectrogram-1、1100 cm-1Silicone hydroxyl Si-OH and Si-O-Si at place is special
Levy peak intensity to weaken, it was demonstrated that polymerization imprinted layer is successfully coated on silicon ball carrier.Additionally, FMIP(b) and FNIP(c) collection of illustrative plates basic
Unanimously, can substantially be eluted totally by the template molecule in indirect proof FMIP.
Fig. 6 a, b, c are respectively naked silicon ball, FMIP, FNIP thermogravimetric curve figure in the range of 0 ~ 700 DEG C.Permissible from Fig. 6
Seeing, between 30 280 DEG C, each material occurs the most weightless, and about 4 % 5 %, this is by physical absorption in material
Water and solvent volatilization cause.Between 280 450 DEG C, material occurs second time weightless, the weightlessness of naked silicon ball (a) be by
The TEOS wherein remained prepares raw material and decomposes and cause, FMIP(b) and weightlessness FNIP(c) be due to its surface coated organic print
Trace layer thermal decomposition causes, and weight-loss ratio is respectively 40.20 % and 42.25 %, it was demonstrated that the successful modification of imprinted layer.Imprinted material is with non-
The difference of the degree of cross linking that the template molecule presence or absence during slightly difference is likely due to polymerization process of imprinted material weight-loss ratio causes
Different, and then cause density of material difference to be caused.According to thermogravimetric curve, prepared fluorescence imprinted material is being less than
Good stability is had under the conditions of 280 DEG C.
Fig. 7 is material excitation-emission spectrogram in acetonitrile solution, and wherein a, b represent FMIP swashing in acetonitrile respectively
Send out spectral line (Em=304), the FMIP spectral line of emission (Ex=267) in acetonitrile；C, d represent FNIP excitation spectrum in acetonitrile respectively
Line (Em=304), the FNIP spectral line of emission (Ex=267) in acetonitrile；E is template molecule SMZ ultraviolet absorption spectrum in acetonitrile
Line.
Be can be seen that the exciting of prepared FMIP and FNIP material, the spectral line of emission are complete by the contrast of Fig. 7 a, b, c, d spectral line
Unanimously, simply intensity slightly difference, it may be possible to the presence or absence in trace hole causes.Additionally, due to the absorption line (e) of SMZ and material
The exciting line (a) of material has the biggest overlapping, just can absorb wherein fluorescent base after template molecule SMZ enters the imprinted cavity of material
The excitation energy of group, thus produce the response signal of fluorescent quenching, the concentration for detection template molecule has established theoretical basis.
Fig. 8 is the acetonitrile solution fluorescent stability of material, and wherein a represents the acetonitrile solution fluorescence intensity of FMIP and FNIP
(excitation wavelength Ex=267 nm launches wavelength Em=304nm, a width of 1.5-3 of excitation-emission slit), b generation over time
The pure water solution fluorescence intensity of table FMIP and FNIP (Ex=267, Em=304, slit width 1.5-3) over time.
Be can be seen that material has good dispersibility in organic solvent by Fig. 8 a, thus in a long time (30
Minute) fluorescence signal is stable.Found out that material increases in aqueous solvent over time by Fig. 7 b and have certain sedimentation, for material
Hydrophobic forces between the organic imprinted layer in surface causes, but system after scattering in 10 minutes fluorescence signal be stable
's.
Fig. 48 illustrates that the core-shell type Ultraluminescence molecular engram material successfully prepared has favorable dispersibility, optical property
Stable feature.
Test example 1
First the proportioning raw materials of fluorescence imprinted material is prepared in optimization, will prepare 5 batch of materials respectively by the method for polymerisation in bulk
In acetonitrile (ACN) solution and DMF solution of the SMZ of 500 μMs, do Staticadsorption experiment, determine that absorbability and imprinting factor are
Excellent one group, prepares the fluorescence molecule imprinted material of nucleocapsid structure by its proportioning raw materials.
Staticadsorption experiment: weigh respectively prepare with bulk polymerization the M1 of 10 mg, N1, M2, N2, M3, N3, M4,
N4, M5, N5 material, is separately added into SMZ acetonitrile solution and DMF solution that 5 ml concentration are 500 μMs, and under room temperature, to adsorb 16 little for shaking table
Centrifuging and taking supernatant time after, calculates the unit adsorbance of every kind of material by UV absorption after diluting 20 times.It is repeated twice, finally
Determine the fluorescence molecule imprinted material preparing nucleocapsid structure by the proportioning raw materials preparing M1, N1.Adsorption experimental results such as Fig. 9 institute
Show.Wherein, Fig. 9 a is the static adsorbance contrasts in the ACN solution of SMZ of 5 batches of block polymeric materials, and Fig. 9 b is 5 batches of block polymerizations
Material static adsorbance contrast in the DMF solution of SMZ.
Being contrasted by Fig. 9 it can be seen that polymeric material advantages of good adsorption effect in acetonitrile solvent, undope general function monomer
First material (MIP1, NIP1) imprinting factor is the highest, and the absorbability difference of M1 and N1 is the most obvious, thus with prepare M1,
The proportioning raw materials of N1 prepares the fluorescence molecule imprinted material of next step core-shell type.
Test example 2
The fluoroscopic examination condition of FMIP is optimized, determine respectively the response time of detection, the material concentration of detection,
The standard curve of detection.
1, the time needed for first research material and analyte arrive fluorescence response balance, joining 2ml concentration is 125 μ g/ml
FMIP acetonitrile solution, add the SMZ acetonitrile solution that 20 μ l concentration are 500 μMs, different time record system fluorescent value become
Change (excitation wavelength Ex=267 nm launches wavelength Em=304nm, a width of 1.5-3 of excitation-emission slit), be repeated 3 times.With the time
For abscissa, fluorescent quenching rate is vertical coordinate, optimal response time (material and when hatching of analyte when finally determining detection
Between) it is 30 minutes, as shown in Figure 10.After Figure 10 is FMIP fluorescence imprinted material and analyte SMZ mixes in the solution, system is glimmering
Optical quenching degree schematic diagram over time.As can be seen from Figure 10: system Quenching of fluorescence speed starts quickly, after gradually
Tending to definite value, when thereby determining that detection, the optimal response time is 30 minutes.
2, then investigating the impact of material concentration equity component analysis thing quencher rate, joining 2 ml concentration is 10～150 μ g/ml
FMIP acetonitrile solution, be separately added into the SMZ solution that 20 μ l concentration are 500 μMs, respective fluorescence surveyed by shaking table after hatching 30 minutes
Quencher situation (Ex=267 nm, Em=304nm, a width of 1.5-3 of slit).It is repeated 3 times, with material concentration as abscissa, fluorescence
Quencher rate is vertical coordinate, obtains the variable concentrations FMIP material quencher situation to same concentrations SMZ.Finally determine during detection optimal
Material concentration is 100 μ g/ml, as shown in figure 11.Figure 11 b is the fluorescence intensity curves of variable concentrations FMIP material acetonitrile solution.
Material concentration and fluorescence intensity are good linear relationship as can be seen from Figure 11.Figure 11 a is variable concentrations FMIP material equity
The quencher rate curve of amount SMZ analyte, it will be seen that the amount of material is the most, and equivalent SMZ is the lowest to its quencher rate.In detection solution
During SMZ concentration, the consumption of FMIP material affects the range of linearity and the sensitivity of detection, so the fluorescence intensity of material should be taken into account
And quenching ability, when finally determining detection, optimal material concentration is 100 μ g/m.
3, then measure the equivalent material quencher rate to variable concentrations analyte, set up fluorescent quenching rate with analyte concentration
Linear relationship, in the FMIP acetonitrile solution of the 100 μ g/ml of 2 ml, add different amounts of SMZ respectively, make SMZ in system dense
Degree is 1 ~ 11 μM, and shaking table records respective fluorescence intensity after hatching 30 minutes (Ex=267 nm, Em=304nm, slit is a width of
1.5-3), with Stern Volmer quencher equation (F0 / F=1+KSV ·[C]) surveyed peak value is processed, F in formula0For body
Being initial fluorescent intensity, F is the fluorescence intensity after system balance, and C is analyte concentration in system (μM/L), KSVFor quenching constant
(L/ μM).With analyte concentration as abscissa, quencher rate (F0 / F-1) it is vertical coordinate, obtain the fluorescent quenching rate pair of FMIP
The standard curve of SMZ concentration, as shown in figure 12.Figure 12 a is that the SMZ of variable concentrations is rung by the FMIP acetonitrile solution of 100 μ g/ml
After should balancing, the fluorescent emission spectral line (Ex=267, Em=304, slit width 1.5-3) of each system, as can be seen from Figure 12 along with
The increase of SMZ concentration, the gradually quencher of the fluorescence of system.Figure 12 b is for processing each spectral line peak with Stern Volmer quencher equation
After value, mapping obtains the fluorescent quenching rate of the material standard curve to SMZ concentration, in studied concentration range (1 9 μMs)
Having good linear relationship, its mark Qu Fangcheng is F0/ F-1=0.0426 C-0.012, correlation coefficient is 0.998, according to sky
Lean type system deviation and slope of standard curve are obtained the detection of the method in acetonitrile solvent and are limited to 463.5 nM (122.5 ng/
ml )。
Test example 3
The fluoroscopic examination selectivity of FMIP is studied, respectively with sulfa drugs sulfamethazine (SDM), sulphur
Amine isoxazole (SIZ) and ribavirin (RBV), diethylstilbestrol (DES), dexamethasone (DEX), ibuprofen (IBU) are for compareing
Thing does the fluorescent quenching selectivity experiment of FMIP in acetonitrile.Join FMIP and the FNIP acetonitrile that several parts of concentration is 100 μ g/ml molten
Liquid 2 ml, is separately added into different amounts of SMZ, SDM, SIZ, RBV, DES, DEX, IBU solution successively, and in making system, each analyte is dense
Degree is followed successively by 1,2,3,5,7 μMs, and shaking table detects respective fluorescent quenching situation (Ex=267 nm, Em=after hatching 30 minutes
304nm, a width of 1.5-3 of slit), calculate FMIP and the FNIP quenching constant Ksv to each analyte respectively, be repeated 3 times.Result
As shown in figure 13.Figure 13 is the fluoroscopic examination selectivity schematic diagram of FMIP and FNIP.Figure shows the fluorescent quenching constant of FMIP
Be all higher than non-imprinted material FNIP, and FMIP the quencher degree of template molecule SMZ is significantly greater than SIZ and DEX, DES,
RBV, IBU, and to SDM, its quencher rate is only somewhat below SMZ, this is owing to SDM is very much like with template molecule SMZ structure, says
Template molecule SMZ is had and preferably selects binding ability by bright prepared imprinted material FMIP, finds out this detection method simultaneously
There is preferable capacity of resisting disturbance.
Test example 4
Actual sample is detected by research FMIP:
1, the FMIP material standard curve to SMZ quencher in milk is first done: take two parts of 20 ml milk, each addition 200 μ l
Hydrochloric acid and 3 ml acetonitriles, after vortex, 6000 leave the heart 5 minutes, take supernatant, place 10 min after being adjusted to neutrality with sodium hydroxide, then
The milk that centrifuging and taking supernatant obtains removing protein is clear, obtains the milk substrate of detection with water constant volume to 50 ml, joins 100 μ g/ with it
The milk clear solution of the FMIP of ml, takes the milk substrate solution number part of FMIP, adds different amounts of SMZ solution and makes SMZ in system
Concentration is 1 μM 15 μMs, and shaking table detects each autofluorescence (Ex=267nm, Em=318nm, a width of 3-5 of slit) after hatching 30 minutes,
Process with Stern Volmer quencher equation and obtain FMIP quenched standard curve in milk substrate, as shown in figure 14.Figure 14
A be the FMIP milk substrate solution of 100 μ g/ml with the SMZ response balance of variable concentrations after, the fluorescent line (Ex=of each system
267, Em=318, slit width 3-5).As can be seen from Figure 14 along with the increase of SMZ concentration, the gradually quencher of the fluorescence of system.Figure 14
After b is for processing each spectral line peak value with Stern Volmer quencher equation, mapping obtains the fluorescent quenching rate of material to SMZ concentration
Standard curve, in studied concentration range, (1 15 μMs) have good linear relationship, quencher mark in milk substrate
Qu Fangcheng is F0/ F-1=0.0868 C+0.0111, correlation coefficient is 0.9982, bent according to blank system fluorescence deviation and standard
Line slope is obtained the detection of the method and is limited to 98.4 nM/L (26.0 ng/ml)
2, the then concentration of SMZ in detection mark-on milk: take 5 parts of 6 ml milk, be separately added into 0 ml, 0.1 ml, 0.2
Ml, 1 ml, 2 ml concentration are to mix after the SMZ acetonitrile solution of 50 μMs, respectively add 100 μ l hydrochloric acid, are then separately added into
Being centrifuged after 2ml, 1.9ml, 1.8ml, 1ml, 0 ml acetonitrile vortex, the sodium hydroxide solution of supernatant pH=12 is put after being adjusted to neutrality
Putting 10 minutes, then be centrifuged removing protein, after taking supernatant, each addition 0.5 ml concentration is the acetonitrile solution of 2000 μ g/ml materials, finally
Being settled to 10ml with water respectively, obtain adding SMZ mark concentration and be respectively 0,0.5,1,5,10 μMs, material concentration is 100 μ g/ml
Milk clear solution, shaking table hatches fluorescence intensity (Ex=267nm, Em=318nm, a width of 3-5 of slit) after 30min, according to
Mark song extrapolates detectable concentration, calculates the response rate, is repeated 3 times.The results are shown in Table 1.
Table 1 is by the result of SMZ concentration in fluorescent quenching method detection mark-on milk
In table: spiked levels is followed successively by 0.5,1,5,10 μMs, obtains the preferable response rate, and the deviation of the response rate is due to cattle
The albumen impact of non-Ex-all in milk substrate causes.Simultaneously for bigger concentration SMZ(10 μM of mark-on) the sample detection response rate inclined
Low, it is owing to SMZ dissolubility in neutral water is relatively low, during centrifugal removing protein, loss is caused.
Claims (2)
1. a preparation method for core-shell type Ultraluminescence molecular engram material, the steps include:
The preparation of Ultraluminescence function monomer N-fluorenes methyl ester formyl-N-methyl acryloyl group-lysine methyl ester
A) N-fluorenes methyl ester formoxyl-N-tert-butyl ester formoxyl-lysine FMOC-Lys-BOC is joined 40 ml hydrogen chloride gas
In the methanol that body is saturated, 2 hour after sucking filtration is stirred at room temperature, is dried to obtain the lysine methyl ester hydrochloric acid of FMOC protection with methanol washing
Salt；
B) raw material having taken off BOC is joined in anhydrous methylene chloride, then drip after addition methacrylic chloride in above-mentioned solution
Adding anhydrous triethylamine, material molar ratio is followed successively by 3 mmol:3.3 mmol:6 mmol, reacts end in 1 hour, adds 25ml bis-
Chloromethanes dilutes, and the most successively with hydrochloric acid and the saturated aqueous common salt washing of pH=1, organic facies anhydrous sodium sulfate concentrates after drying
Crossing pillar, eluent is ethyl acetate: the volume ratio of petroleum ether is 1:2, and 40 DEG C of vacuum are spin-dried for, obtain N-fluorenes methyl ester formoxyl-
N-methylacryloyl-lysine methyl ester white crystal；
The preparation of Ultraluminescence molecular engram FMIP
C) the silicon ball carrier that metering system is acylated is prepared: by the 80 ml:6 ml:3 ml mixing by volume of ethanol, water, ammonia
After, dripping tetraethyl orthosilicate under stirring, room temperature reaction is ethanol centrifuge washing 2 times after 8 hours, then by naked for gained silicon ball point
It is scattered in ethanol, adds and drip methacryloxypropyl trimethoxy silane, after reacting 12 hours under water and ammonia, stirring
With ethanol centrifuge washing 3 times, 50 DEG C of vacuum drying, obtaining the silicon ball that metering system is acylated, cold preservation is stand-by；
D) by polymer raw template molecule sulfamethyldiazine, fluorescent functional monomer N-fluorenes methyl ester formyl-N-methyl acryloyl
Base-lysine methyl ester, cross-linking agent ethylene glycol dimethacrylate, initiator azodiisobutyronitrile 0.15 mmol in molar ratio:
0.15 mmol:1.5 mmol:0.06 mmol is dividedly in some parts in acetonitrile, is stirred at room temperature 5 hours, adds methyl-prop after drum nitrogen
Alkene is acylated the acetonitrile solution of silicon ball, and drum nitrogen is 60 DEG C of stirring reactions 20 hours after 5 minutes, at the silicon ball surface that metering system is acylated
Polymerization last layer imprinted layer；
E) d) step resulting materials is used ethanol, acetonitrile centrifuge washing 1 time respectively, then with methanol: the volume ratio of acetic acid is 8:
1 mixed liquor carries out Soxhlet washing, until supernatant does not has the uv absorption of template molecule sulfamethyldiazine, at 60 DEG C, vacuum is done
Dry；Obtain the fluorescence molecule trace sensing material FMIP having fluorescent quenching to respond template molecule.
2. prepared by claim 1, a kind of core-shell type Ultraluminescence molecular engram material sulfamethyldiazine in mark-on milk contains
Measure the application in determining.
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