CN104910326B - Multi-walled carbon nano-tube surface molecular imprinting polyionic liquid for detecting 17 beta-estradiol as well as preparation method and application thereof - Google Patents

Abstract

The invention discloses a multi-walled carbon nano-tube surface molecular imprinting polyionic liquid for detecting 17 beta-estradiol as well as a preparation method and an application thereof. The polyionic liquid is a composite material formed by polymerizing multi-walled carbon nano-tubes, a functional monomer, cross-linking agents and template molecules under the action of an initiator and removing the template molecules, wherein the functional monomer is 1-(1'-hydroxylbutane)-3-vinylimidazolium hexafluorophosphate; the cross-linking agents are ethylene glycol dimethacrylate and 3, 3'-(1',4'-butane)-bis-1-vinylimidazole hexafluorophosphate; the template molecules are 17 beta-estradiol. The PIL/MWCNTs-MIP surface molecular imprinting composite material synthesized by the preparation method disclosed by the invention has good electrochemical behaviours on 17 beta-estradiol, as well as is specific in adsorption, high in sensitivity and good in selectivity for 17 beta-estradiol.

Description

For detecting the surface molecule print polyion of the multi-walled carbon nano-tubes of 17 beta estradiols Liquid and its preparation method and application

Technical field

The present invention relates to a kind of molecular imprinted polymer on surface, more particularly, to a kind of many walls for detecting 17 beta estradiols Surface molecule print poly ion liquid of CNT and its preparation method and application.

Background technology

Environmental security is paid close attention to by all circles increasingly, and realizing Environmental security quick detection is national implementation environment protection Basis, however as the fast development of national economy, environmental security is on the rise, and therefore develops quick, accurate, sensitive Detection environment in noxious pollutant new technique very necessary.

Estradiol (estradiol, E₂), it is a kind of typical environmental estrogens in estrogen, be also in estrogen simultaneously A kind of biological activity material the strongest, due to its anabolic impact, has been widely used for fattening of animal.Wherein 17 β-female Glycol (17 β-estradiol) be a kind of this may result at low concentrations being poisoned and carcinogenic natural estrogen, it can be from outer Face enter body, disturb normal physiological process, humoral immunization and the imbalance of cellular immunization can be caused, lead to reproduction, immunity, Many pathological changes such as cardiovascular and nervous system.Therefore realization is efficient, quick, reliable, detect trace in water environment in real time The estradiol of level, has very important practical significance to Homeland Security and health protection.The detection of current 17 beta estradiols Method mainly has HPLC, GC-MS, LC-MS etc..Although these methods can detect the estrogen of low concentration effectively, it Sample pretreatment process complicated, apparatus expensive, sensitivity is low, be not suitable for large-scale application.Radioimmunology (RIA) and enzyme Linked immunoassay method (ELISA) can improve the sensitivity of detection, but its substrate instability limit its extensive application. The features such as electrochemical method has production that is succinct, direct, quick, being conducive to portable and handheld device, the method is applied to The detection of 17 beta estradiols in environmental sample.

Multi-walled carbon nano-tubes (MWCNTs), of great interest due to its unique pattern and chemical property. Polymer/carbon nano-tube (CNTs) nano composite material is because it is compared with straight polymer in recent years, in physics, thermal technology, mechanics Significantly improving of aspect and cause extensive research interest.Although many researchs are carried out, to improve CNT and polymer Between the compatibility.However, CNT is dispersed in polymeric matrix still suffering from significantly challenging, especially for Large-scale commercial Application.Non-covalent functionalized nano pipe after proper treatment is an attractive technology, because It does not destroy extension π-pi-conjugated structure, will not destroy CNTs mechanical performance and electrochemical properties.Recently, find ionic liquid (ILs) be there is strong " π-cation " being interacted with CNT, this is preparation dispersion non-covalent modification in the polymer CNT provides new probability.However, seldom have at present studying CNT (CNTs) dispersion successfully modified ILs In polymeric matrix.In our current research, we are by modifying multiwall carbon nano-tube (MWCNTs) and wide variety of room-temperature ion liquid Body combines prepares molecular imprinted polymer on surface.

Ionic liquid receives much concern as the liquid of specificity property because of it, because ionic liquid only has ion, and table Reveal very high ionic conductivity, not volatile and incombustibility, also have and excellent solvability, heat stability The features such as with being easily recycled.It has been widely used in radical polymerization, ring-opening polymerisation, the ionic conductance polymerization of vinyl monomer Etc. in various polymerization systems, ionic liquid be also used for electrochemistry and enzymatic polymerization during polycondensation and addition polymerization polymerization.For ion The high ionic strength of liquid, the problem that aggregation rate can be increased and mitigate bed contraction or expansion, and then improve imprinted material Selectivity and adsorptivity.Research with regard to ionic liquid being applied to imprinted material be still in the starting stage it is therefore desirable to do into The research of one step.Therefore functionalized carbon nano-tube is combined with ILs and is expected to improve electric conductivity, compatibility and stability, and fully CNT is used for sensor aspect.

Molecularly imprinted polymer (MIP) is the material of synthetic, has specificity and captures the manually generated of target molecule Recognition site.In the presence of molecularly imprinted polymer (MIP) is template molecule, it is prepared from by function monomer and cross-linking agent copolymerization. After template removes, the complementary recognition site with template molecule size and dimension, functionalization ground forms three-dimensional network polymer.Molecule Imprinted polymer (MIP) has many advantages, such as high specific recognition, mechanical stability, motility, and preparation is easier to and material Material low cost.Therefore, molecularly imprinted polymer has been applied to many fields, such as controls drug delivery system, sensor and solid phase The fields such as extraction.However, traditional molecularly imprinted polymer has, template molecule removes hardly possible, imprinted sites are few and mass transfer velocity is slow The shortcomings of, limit it in very many applications.

In order to overcome this problem, researchers are devoted to surface molecule print technology, and preparation is divided based on the surface of carrier Sub- imprinted polymer material, the imprinted sites due to this material are located at material surface, can improve the response speed to template molecule, Reduce non-specific adsorption, thus increasing the adsorbance to target molecule for this material, this material is used for the phase of electrochemical sensor Close the sensitivity that research can improve method, shorten response time.

In surface molecule print method, can need to select different carriers according to different, at present, oxidation reported by document Graphene, Graphene, CdSe quantum dot, silicon dioxide etc. is the molecularly imprinted material on surface of carrier.CNTs and ionic liquid tool There are preferable electro catalytic activity, electric conductivity, compatibility etc..Surface imprinted material with CNTs and novel ion liquid as carrier can Can have preferable electro catalytic activity, electric conductivity etc., therefore, surface imprinted material is prepared for carrier with CNTs and novel ion liquid Material should be a good application prospect.

Content of the invention

The invention discloses a kind of have good electrochemical behavior to 17 beta estradiols, 17 beta estradiol specificitys are inhaled Attached, sensitivity is high, the surface molecule print poly ion liquid of the CNT of good selectivity.

A kind of surface molecule print poly ion liquid of multi-walled carbon nano-tubes, it is by functionalized multi-wall carbonnanotubes, work( The polymerization under initiator effect of energy monomer, cross-linking agent and template molecule forms and removes the composite of template molecule, described work( Can monomer be 1- (1 '-hydroxybutane) -3- vinyl imidazole hexafluorophosphoric acid；Described cross-linking agent is ethyleneglycol dimethacrylate Ester and 3,3 '-(1 ', 4 '-butane)-bis- -1- vinyl imidazole hexafluorophosphoric acids；Described template molecule is 17 beta estradiols.

Specifically, described initiator is azodiisobutyronitrile.

Preferably, described 1- (1 '-hydroxybutane) -3- vinyl imidazole hexafluorophosphoric acid, ethyleneglycol dimethacrylate The mol ratio of ester, 3,3 '-(1 ', 4 '-butane)-bis- -1- vinyl imidazole hexafluorophosphoric acids is 0.3～0.6: 1: 1；It is further preferred that rubbing That ratio is 0.4: 1: 1.

Preferably, the molal of described functionalized multi-wall carbonnanotubes and template molecule is than for 0.08～0.1g: 0.1mmol.

Preferably, the molal weight of described function monomer and initiator is than for 0.1mmol: 3～6mg；It is further preferred that mole weight Amount ratio is for 0.1mmol: 5mg.

Present invention also offers the preparation method of the surface molecule print poly ion liquid of described multi-walled carbon nano-tubes, including Following steps：Template molecule is added in solvent, adds function monomer, cross-linking agent, initiator and carboxylated multi-wall carbon nano-tube Pipe, mix homogeneously carries out polyreaction, collects product, the template molecule in removing product, be finally dried and be obtained after the completion of reaction The surface molecule print poly ion liquid of described multi-walled carbon nano-tubes.

Preferably, the temperature of described polyreaction is 60～80 DEG C, and the time is 12～36 hours；It is further preferred that temperature is 60 DEG C, the time is 24h.

Present invention also offers what a kind of surface molecule print poly ion liquid using described multi-walled carbon nano-tubes was made Electrochemical sensor.

Present invention also offers application in detection 17 beta estradiols for the described electrochemical sensor.

The present invention is with good conductivity, good, good stability the novel ion liquid of compatibility and the good CNTs of electro catalytic activity In conjunction with, with 17 beta estradiols as template molecule, 1- (1 '-hydroxybutane) -3- vinyl imidazole hexafluorophosphoric acid is function monomer, Ethylene glycol dimethacrylate EGDMA/3,3 '-(1 ', 4 '-butane)-bis- -1- vinyl imidazole hexafluorophosphoric acid (1: 1, M/M) For double crosslinker, azodiisobutyronitrile is initiator, has successfully synthesized PIL/MWCNTs-MIP table by non-covalent bond effect Face molecular imprinting composite material, is had the strong π-cation that can be reacted with CNTs based on poly ion liquid, is for entering The synthesis of the non-covalent modification multi-walled carbon nano-tubes that one step is scattered in polymer provides new probability.

Brief description

Fig. 1 is the preparation of PIL/MWCNTs-MIP imprinted material of the present invention and its structure of sensor.

Fig. 2A is [C₄(OH)mim][Cl](a)、[C₄(OH)mim][PF₆](b)、[C₄mim₂] [Br] (c), [C₄mim₂] [PF₆] (d) infrared spectrogram；Fig. 2 B CNTs-COOH (a), PIL/MWCNTs-NIP (b), PIL/MWCNTs-NIP (c) Infrared spectrogram.

Fig. 3 is the TGA mass loss curve of MWCNTs-COOH (a) and PIL/MWCNTs-MIP (b).

Fig. 4 is the SEM phenogram of MWCNTs-COOH (A), PIL-MIP (B) and PIL/MWCNTs-MIP (C).

Fig. 5 is PIL/MWCNTs-MIP/GCE (a) and PIL/MWCNTs-NIP/GCE (b) containing 30 μM of E₂PBS (0.M PH 6.0) cyclic voltammogram (A) in solution and differential pulse voltammetry figure (B) (adsorption time：5min).

Fig. 6 is for PIL/MWCNTs-MIP/GCE (a), Bare GCE (b) and PIL/MWCNTs-NIP/GCE (c) containing 5mM K₃[Fe(CN)₆] and 0.1M KCl mixed solution in cyclic voltammogram (A) and 5mM K₃[Fe(CN)₆], 5mM K₄[Fe(CN)₆] With the impedance spectrum (B) in 0.1M KCl mixed solution.

Fig. 7 A is PIL/MWCNTs-MIP/GCE (a), PIL/MWCNTs-NIP/GCE (b), Bare GCE (c) and tradition MIP/GCE (d) is in 0.5mM K₃[Fe(CN)₆] electricity (Q) in solution and time diagram (Q-t)；Fig. 7 B is PIL/MWCNTs- MIP/GCE (a), PIL/MWCNTs-NIP/GCE (b), Bare GCE (c) and traditional MIP/GCE (d) are in 0.5mM K₃[Fe (CN)₆] electricity (Q) in solution and time root side curve (Q-t^1/2) (test condition：Pulse width 0.25s, sampling interval 0.25ms, time of repose 2s).

Fig. 8 is the concentration (A) of PIL/MWCNTs-MIP, adsorption time (B), pH (C) to PIL/MWCNTs-MIP/GCE's DPV current-responsive affects；PIL/MWCNTs-MIP/GCE is in different pH (a to e is respectively 4.5,5.0,6.0,7.0,8.0) (condition determination is 30 μM of E of 0.1M PBS to DPV figure (D)₂).

Fig. 9 is sensor PIL/MWCNTs-MIP/GCE and PIL/MWCNTs-NIP/GCE to E₂And three kinds of analog absorption DPV current-responsive after 5min；

Wherein, E₁For a female alcohol；E₂For estradiol；E₃For estriol；4-NP is nonyl phenol.

Figure 10 A is sensor PIL-MWCNTs-MIP to E₂DPV response, E₂Concentration (a～k, μM)：0.005、0.01、 0.05、0.5、1、5、10、20、30、50；Figure 10 B sensor PIL-MWCNTs-MIP (a) and PIL-MWCNTs-NIP (b) measures E₂Standard curve.

Figure 11 is PIL-MWCNTs-MIP sensor, traditional MAA sensor, traditional 4-NP sensor and traditional AM sense Device is to 5 μM of E₂DPV detection.

Specific embodiment

The raw material that the present invention adopts is as shown in table 1：

Table 1

Remarks：Phosphate buffer solution (pH 6.00.1M) is by Na₂HPO₄And NaH₂PO₄Prepare.

(1) purification of 4-vinylpridine

The 4-NP taking 20mL is added in the round-bottomed flask of 50mL, is then placed on and carries out vacuum distillation at 70 DEG C, takes Its fraction, is dissolved in blocker therein to remove.

(2) purification of methacrylic acid

Take the round-bottomed flask of a 50mL, be added thereto to 20mL MAA, this carries out vacuum distillation at 30 DEG C, acquisition evaporates Point, thus removing blocker therein.

1st, the preparation of PIL/MWCNTs-MIP sensor

(1) 1- (1 '-hydroxybutane) -3- vinyl imidazole chlorine [C₄(OH) mim] [Cl] ionic liquid preparation：

[C₄(OH) mim] building-up process of [Cl] is：Under stirring energetically, the chloro- n-butyl alcohol of 11.94g (0.11mol) 4- instills 9.411g (0.1mol) 1- vinyl imidazole (0.1M) (99%Aldrich), is dissolved in the methanol of 30mL, this mixture is at 60 DEG C Obtain light yellow viscous liquid after being stirred overnight 15h, then washed with absolute ether 3 times, fall off and be dried at room temperature Obtain 1- (1 '-hydroxybutane) -3- vinyl imidazole chlorine [C to constant weight₄(OH)mim][Cl].

(2) 1- (1 '-hydroxybutane) -3- vinyl imidazole hexafluorophosphoric acid ([C₄(OH)mim][PF₆]) synthesis：

With BF₄-, PF₆- and Tf₂N- is that the ionic liquid of anionic functional group can pass through above-mentioned [C₄(OH)mim] [Cl] and corresponding inorganic salt, i.e. NaBF₄, NH4PF₆, Li [Tf₂N] between anion metathesis reaction obtain.

Weigh [the C that 10.13g has been obtained₄(OH) mim] [Cl] and 8.965g NH₄PF₆Molten in 50mL secondary deionized water After mix homogeneously in liquid, under room temperature, stir 12h.In order that displacement reaction spot is more thoroughly, NH₄PF₆Slightly excessive.Obtain Off-white color solid washed with ether (3 × 30mL) is to remove unreacted raw material.Product after filtering, is washed repeatedly with distilled water Wash, remove the Cl of residual in solution^-.We are by silver nitrate solution come to based on PF₆ ^-Cl in anion ion liquid^-It is No residual is verified, until no white precipitate (AgCl) produces.After 60 DEG C of vacuum is dried overnight, products therefrom is [C₄(OH) mim][PF₆].After weighing, yield is 10.00g, and yield is 82%.

(3) 3,3 '-(1 ', 4 '-butane)-bis- -1- vinyl imidazole bromine ([C₄mim₂] [Br]) and synthesis：

[C₄mim₂] building-up process of [Br] is：Under stirring energetically, 10.80g (0.05mol) Isosorbide-5-Nitrae-dibromobutane instills 9.411g (0.1mol) 1- vinyl imidazole (0.1M) (99%, Aldrich), is dissolved in the methanol of 30mL, and this mixture is in 60 DEG C Under be stirred overnight 15h after obtain light yellow viscous liquid, so viscous solution is poured in 500mL absolute ether into washing, lower floor is glued Magma fall off and at room temperature dry to constant weight.

(4) 3,3 '-(1 ', 4 '-butane)-bis- -1- vinyl imidazole hexafluorophosphoric acid ([C₄mim₂][PF₆]) synthesis：

With BF₄-, PF₆- and Tf₂N- is that the ionic liquid of anionic functional group can pass through above-mentioned [C₄mim₂][Cl] With corresponding inorganic salt, i.e. NaBF₄, NH₄PF₆, Li [Tf₂N] between anion metathesis reaction obtain.20.21g being obtained [C₄mim₂] [Br] and 8.965g NH₄PF₆After mix homogeneously in 50mL secondary deionized water solution, under room temperature, stir 12h.For Displacement is made to react spot more thoroughly, NH₄PF₆Slightly excessive.The off-white color solid washed with ether (3 × 30mL) obtaining with Remove unreacted raw material.Product after filtering, uses distilled water cyclic washing, removes and remains Br in solution^-.We adopt nitric acid Silver-colored solution, to based on PF₆ ^-Br in anion ion liquid^-Whether residual is verified, until no AgBr yellow mercury oxide produces Raw.After 60 DEG C of vacuum is dried overnight, products therefrom is [C₄mim₂][PF₆].

(5) preparation of functionalized multi-wall carbonnanotubes (MWCNTs)：The carboxylated synthetic method of multi-walled carbon nano-tubes is Weigh 100mg CNTs to be placed in 500mL round-bottomed flask, add 400mL nitration mixture (HNO₃∶H₂SO₄=1: 3, V/V) so that it is disperseed, It is subsequently placed in ultrasonic 10h in water bath sonicator instrument, the uniform dispersion standing stay-over demixion of gained, abandoning supernatant are subsequently used Secondary deionized water is washed, centrifugation, until the pH of resulting solution is in neutrality, is then dried to obtain carboxylated CNTs standby.

(6) preparation of PIL/MWCNTs-MIP：The synthetic method of PIL/MWCNTs-MIP is：Sequentially add 0.1mmol17 Beta estradiol (E₂) and 30ml mixed solvent (V_Acetonitrile∶V_Toluene=1: 1) to 50ml round-bottomed flask, add 0.08g CNTs- COOH and 0.4mmol 1- (1 '-hydroxybutane) -3- vinyl imidazole hexafluorophosphoric acid [C₄(OH)mim][PF₆] ionic liquid list Body, magnetic agitation carries out prepolymerization in 2 hours.It is subsequently adding 2.0mmol EGDMA/3,3 '-(1 ', 4 '-butane)-bis- -1- Vinyl imidazole hexafluorophosphoric acid [C₄mim₂][PF₆] (1: 1, M/M) double crosslinker and 20mg AIBN.Seal after inflated with nitrogen 15min Flask mouth, reacts 24h in 60 DEG C of water-bath.Product solvent (V_Methanol∶V_{Acetic acid}=9: 1) soxhlet extraction removes template molecule, It is placed in 40 DEG C of vacuum drying ovens and 48h is dried, obtain PIL/MWCNTs-MIP.

Using same procedure, it is not added with template molecule and prepares blank polymer (non-trace poly- PIL/MWCNTs-NIP).Tradition Blotting procedures are similar, change function monomer and cross-linking agent, and synthesis is sensed trace material accordingly under standing does not stir Material compares.

2nd, Electrochemical Detection

The preparation of sensor：Bare glassy carbon electrode is with 0.05 μm of Al₂O₃After powder is polished to minute surface on chamois leather, use successively Acetone, HNO₃Ultrasonic 5min in (1: 1, V/V), NaOH (1.0mol/L) and secondary deionized water, dries stand-by under room temperature.3mg PIL/MWCNTs-MIP (or PIL/MWCNTs-NIP) is added to the chitosan solution adding to 1mL 0.5%, and ultrasonic mixing is equal Even, then take this mixed solution 5.0 μ L to apply and drop in the bare glassy carbon electrode surface handled well, overnight dry.

Secondary water cleans modified electrode, by PIL/MWCNTs-MIP (or PIL/MWCNTs-NIP) modified electrode be placed in containing In 10mL 0.1M PBS (pH=6.0) of 17 beta estradiols, after stirring and adsorbing 5min, it is placed in blank PBS (pH=6.0,0.1M) In carry out Electrochemical Detection.

3rd, the preparation of PIL/MWCNTs-MIP and its electrochemical sensor are constructed

The preparation of PIL/MWCNTs-MIP sensor and its electrochemical detection ask for an interview Fig. 1.Carboxylated first carbon nanometer Pipe and template molecule 17 beta estradiol (E₂), function monomer 1- (1 '-hydroxybutane) -3- vinyl imidazole hexafluorophosphoric acid [C₄ (OH)mim][PF₆] prepolymerization, MWCNTs-COOH is to E₂There is Selective recognition and generate amido link, function monomer 1- (1 '-hydroxyl Butane) -3- vinyl imidazole hexafluorophosphoric acid [C₄(OH)mim][PF₆] and template molecule E₂By hydrogen bond action.Then distinguish With double crosslinker EGDMA/3,3 '-(1 ', 4 '-butane)-bis- -1- vinyl imidazole hexafluorophosphoric acid [C₄mim₂][PF₆] it is cross-linking agent It is initiator with AIBN, radical polymerization prepares PIL/MWCNTs-MIP.The ethylene linkage of MWCNTs-COOH in terms of trace mechanism Imprinted polymer is occurred on its surface.PIL/MWCNTs-MIP is dissolved in shitosan, and drops to glassy carbon electrode surface, preparation PIL/MWCNTs-MIP modified electrode；Then PIL/MWCNTs-MIP modified electrode is containing E₂PBS in adsorb 5min, finally Carry out DPV Electrochemical Detection in blank PBS.PIL/MWCNTs-MIP produces and E after eluted template molecule₂Size coupling Hole, it can be to E₂There is specific absorption and respond, its surface is contained carboxylic group and can be specifically bound E simultaneously₂, this material Have to E₂Double identification abilitys of molecule, therefore PIL/MWCNTs-MIP modifies film sensors and can achieve E₂Highly sensitive, specificity Detection.

4th, the infrared spectrum characterization of PIL/MWCNTs-MIP

Fig. 2A is [C₄(OH)mim][Cl]、[C₄(OH)mim][PF₆]、[C₄mim₂] [Br] and [C₄mim₂][PF₆] infrared Spectrogram, Fig. 2 B is the infrared spectrogram (FTIR) of CNTs-COOH, PIL/MWCNTs-NIP and PIL/MWCNTs-MIP.By scheming In 2A, curve a, c are respectively [C₄(OH) mim] [Cl] and [C₄mim₂] [Br] infrared be it can be deduced that 3436cm^-1Absworption peak be- OH peak, 3164cm^-1、1575cm^-1、1177cm^-1Left and right is respectively the vibration absorption peak in imidazole ring cation^[103], 1650cm^-1 The characteristic peak of left and right is C=N absworption peak, 1500cm^-1The characteristic peak of left and right is C=C absworption peak, 1107cm^-1In the face of N=C-H Bending vibration, can be drawn by curve b, d, [C₄(OH)mim][Cl]、[C₄mim₂] [Br] respectively with NH₄PF₆After reacting, Occur in that new strong absworption peak 841cm^-1And 843cm^-1, belong to PF₆ ^-Absworption peak^[104].Result shows cation exchange [C is successfully obtained after reaction₄(OH)mim][PF₆] and [C₄mim₂][PF₆] monomer.

Fig. 2 B can be seen that the absworption peak that very strong-COOH compared with CNTs in CNTs-COOH.Can be seen by curve b and c Occur in that strong 3429cm to PIL/MWCNTs-MIP/NIP^-1、1727cm^-1、1632cm^-1And 843cm^-1Absworption peak, these suction Receive peak and be respectively belonging to-OH, C=N, C=C, PF₆ ^-Absworption peak.Result shows the successful preparation of PIL/MWCNTs-MIP/NIP.

5th, the thermogravimetric analysiss of PIL/MWCNTs-MIP

Fig. 3 is thermogravimetric analysiss (TGA) curve of MWCNTs, MWCNTs-COOH and PIL/MWCNTs-MIP.As Fig. 3 (a) institute Show, MWCNTs-COOH is more stable at 25～800 DEG C, more only weight loss, loss rate reaches 17%, and this is attributable to The decomposition of carboxyl.But the TGA curve of PIL/MWCNTs-MIP has big weight loss at 25～800 DEG C, and (curve b), mainly returns Because in the decomposition of oxygen-containing functional group and carbon skeleton, loss rate has reached 95%.PIL/MWCNTs-MIP is relative to MWCNTs-COOH's Heat stability is deteriorated, and this is due to due to the heat stability difference of PIL/MWCNTs-MIP, therefore also further illustrates PIL/ MWCNTs-MIP has been coated on MWCNTs-COOH surface.

6th, the SEM of PIL/MWCNTs-MIP characterizes

SEM is used for characterizing MWCNTs-COOH (A), PIL-MIP (B) and PIL/MWCNTs-MIP (C).As shown in Fig. 4 (A), The average diameter of MWCNTs-COOH is about 20nm.By Fig. 4 (B) Suo Shi, traditional PI L-MIP presents spherical shape, does not have tubulose, Particle diameter is averagely about 350nm, and particle diameter increases the explanation MWCNTs surface aggregate polymeric film layer of one layer of PIL, and film thickness is about 330nm.As Fig. 4 (C), PIL/MWCNTs-MIP has tubular structure, and uniformly, caliber is averagely about 150-170nm to pipe diameter size, Relatively Fig. 4 (A) caliber substantially increases, and the thickness of increase is about 130-150nm, is indicated above PIL/MWCNTs-MIP and successfully makes Standby.

7th, the electrochemical behavior to 17 beta estradiols for the modified electrode

First, by the PIL/MWCNTs-MIP modified electrode PBS containing 30 μM of 17 beta estradiol for the immersion (pH 6.00.1M) Adsorb 5min in solution, make modified electrode and E₂After molecule fully combines, rinse electrode with secondary deionized water, be then immersed in sky Investigate its cyclic voltammetry (CV) and differential pulse voltammetry (DPV) response in white PBS (pH 6.00.1M), repair as shown in Figure 5A Decorations electrode pair E₂Redox peaks, can be seen that it is an irreversible process from peak type, oxidation peak current response ratio reduction peak Big, therefore select E₂Oxidation peak carried out Electrochemical Detection.Rung by the electric current of trace in Fig. 5 A and non-Nanosilica electrode Should understand, PIL/MWCNTs-MIP is bigger than the CV current-responsive of PIL/MWCNTs-NIP modified electrode, is mainly due to PIL/ MWCNTs-MIP material surface defines and template molecule E₂The imprinted cavity that size, shape, structure function match, so as to Target molecule E in specific adsorption solution₂.As can be seen from Figure 5B, PIL/MWCNTs-MIP and PIL/MWCNTs-NIP modifies Electrode all occurs in that E at 0.612V₂Oxidation peak, and PIL/MWCNTs-MIP modified electrode is to 30 μM of E₂DPV peak current It it is 2.21 times of PIL/MWCNTs-NIP modified electrode, PIL/MWCNTs-MIP is to E for this explanation₂There is good absorbability, This is attributable to PIL/MWCNTs-MIP print identification site to E₂Identification.

For the electrochemical properties of further research material, by PIL/MWCNTs-MIP/GCE, PIL/MWCNTs-NIP/ GCE, bare glassy carbon electrode, as working electrode, immerse the K of 5mM respectively₃[Fe(CN)₆] and the mixed solution of 0.1M KCl in, measure Their cyclic voltammetric collection of illustrative plates, the cyclic voltammetric collection of illustrative plates of scanning in -0.2V to 0.8V, as shown in Figure 6A, PIL/MWCNTs- (curve a), PIL/MWCNTs-NIP/GCE response current substantially reduces (curve to the redox current response maximum of MIP/GCE B), this is attributable to PIL/MWCNTs-MIP and has more imprinted cavity, promotes the electron transmission of electrode surface, PIL/ The poorly conductive of MWCNTs-NIP/GCE, in bare electrode, illustrates that non-imprinted material surface does not have imprinted cavity, hinders electrode table The electron transmission in face.Corresponding impedance spectrum as shown in Figure 6B, by PIL/MWCNTs-MIP/GCE, PIL/MWCNTs-NIP/ GCE, bare glassy carbon electrode, immerse 5mM K respectively₃[Fe(CN)₆]、5mM K₄[Fe(CN)₆] and the mixed solution of 0.5M KCl in, Measure their ac impedance spectroscopy it is commonly known that electrochemical AC impedance method can reaction surface modified electrode effectively spy Property, in electrochemical impedance spectroscopy, half diameter of a circle is equal to electron transfer resistance.For example, Fig. 6 B (b) clearly elaborates naked GCE The electron transfer resistance of electrode is about 200 Ω, and the electron transfer resistance of PIL/MWCNTs-MIP/GCE is about 75 Ω (song Line a), the resistance of trace eluting material substantially reduces, after illustrating that PIL/MWCNTs-MIP modifies naked GCE electrode, because trace is empty The satisfactory electrical conductivity of cave and MWCNTs is conducive to [Fe (CN)₆]^3-/4-Electron transfer.However, when PIL/MWCNTs-NIP modifies To after naked GCE electrode, resistance substantially increase (curve c), this explanation non-imprinted material surface there is no imprinted cavity, hinder [Fe(CN)₆]^3-/4-Electron transfer.

The effective surface area of inspecting electrode material further：Respectively by PIL/MWCNTs-MIP/GCE (a), PIL/ MWCNTs-NIP/GCE (b), Bare GCE (c) and traditional MIP/GCE (d) are placed in 0.5mM K₃[FeCN₆] in solution, by meter When coulomb analytic process tested and analyzed, result such as Fig. 7, working electrode PIL/MWCNTs-MIP/GCE (a), PIL/MWCNTs- The effective surface area of NIP/GCE (b), Bare GCE (c) and traditional MIP/GCE (d) is according to Anson^[63]Formula：

A represents the effective surface area of modified electrode；C represents the concentration of substrate；D represents K₃[Fe(CN)₆] the coefficient of dispersion (7.6×10^-6m²s^-1)；Q_dlIt is that double-deck electric charge is negligible relative to background matrix；Q_adsRepresent Faraday constant.Fig. 7-A Q and t can be drawn^1/2Slope be respectively 7.055 × 10^-5、3.181×10^-5、1.240×10^-5、9.615×10^-6, by generation Enter formula 1 obtain corresponding modified electrode effective surface area be followed successively by 0.470,0.212,0.0827,0.0641cm².From result Working electrode GCE can be described after PIL/MWCNTs-MIP and PIL/MWCNTs-NIP modification, effective surface area significantly increases Greatly, thus improve PIL/MWCNTs-MIP to E₂Absorbability, reduce to E₂Lowest detectable limit.

8th, condition determination optimization

Concentration, adsorption time and the adsorption liquid of PIL/MWCNTs-MIP is investigated by DPV and has measured liquid pH to PIL/ The impact of MWCNTs-MIP modified electrode current-responsive.Fig. 8 A is the concentration of PIL/MWCNTs-MIP to E₂The shadow of size of current Ring, find that DPV current-responsive is maximum in 3mg/ml, when concentration is less than 3mg/ml, because dripping in the PIL/ of electrode surface MWCNTs-MIP is few, so corresponding imprinted sites also reduce, phase induced current also diminishes；And work as drop coating material concentration and be more than 3mg/ During ml, to electrode surface, corresponding electric current also diminishes the too thick obstruction electron transfer of modified membrane of PIL/MWCNTs-MIP；

Therefore we select the PIL/MWCNTs-MIP concentration dripping to electrode surface is 3mg/ml.Enrichment time is big to electric current Little impact, when working electrode adsorbed target molecule reaches saturation, tend to be steady time of state of peak current is exactly Good adsorption time.Fig. 8 B is the impact to size of current for the enrichment time, and modified electrode arrives 5min, E 0.5₂Oxidation peak current Linearly increasing, with the increase of enrichment time, accumulation on PIL/MWCNTs-MIP/GCE for the estradiol also increases, in 5min When electric current reach maximum, show that PIL/MWCNTs-MIP/GCE has shorter response time, owing to PIL/MWCNTs-MIP/ The effect of GCE surface imprinted material surface excellent electron transfer performance and molecular engram recognition site is it is contemplated that sensitivity and work Make efficiency, the optimal enrichment time of selection is 5min.Herein the DPV in different pH is schemed, when 4.5 to 6.0, response is electric for pH value of solution Stream increases with pH and increases, and pH is that when 6.0, current-responsive is maximum, and during more than 6.0, response current is gradually reduced, so we PH is selected to be 6.0.Fig. 8 D is PIL/MWCNTs-MIP/GCE in different pH (a to e is respectively 4.5,5.0,6.0,7.0,8.0) DPV figure, with pH from 4.5 to 6.0, maximum spike potential moves right, and illustrates to there occurs proton translocation in electrochemical process, Therefore, E₂Oxidation on PIL/MWCNTs-MIP/GCE is an electronics-proton process, additionally, I_paReach maximum 6.0, Therefore Optimal pH is chosen as 6.0.

9th, the selectivity of sensor

The selectivity of material is a key factor constructing sensor, therefore investigated PIL/MWCNTs-MIP/GCE, PIL/MWCNTs-NIP/GCE is to 30 μM of E₂And the DPV current-responsive of other three kinds of analog, wherein three kinds analog are respectively Estriol (E₃), n nonylphenol (4-NP), a female alcohol (E₁).PIL/MWCNTs-MIP/GCE is to 30 μM of E as shown in Figure 9₂Electricity Stream response is to estriol (E₃), n nonylphenol (4-NP), a female alcohol (E₁) 2.08,2.76,3.12 times, this be attributable to print The reason mark material print identification site.And sensor PIL/MWCNTs-NIP/GCE is to 30 μM of E₂Current-responsive with to female Triol (E₃), n nonylphenol (4-NP), a female alcohol (E₁) response current difference in size less, this is due to PIL/MWCNTs- NIP material surface does not have imprinted cavity.We use estradiol (E₂), estriol (E₃), n nonylphenol (4-NP), a female alcohol (E₁) Peak current size ratio on PIL/MWCNTs-MIP/GCE and PIL/MWCNTs-NIP/GCE to represent its selectivity respectively Can, herein by the current definition on PIL/MWCNTs-MIP/GCE be I_s, by the current definition on PIL/MWCNTs-MIP/GCE For I_o, E₂、E₃、4-NP、E₁Peak current size ratio (I_s/I_o) it is respectively 2.14,1.01,1.06,1.00.Can from data result Draw, PIL/MWCNTs-MIP/GCE is to template molecule E₂There is bigger adsorption capacity, and to E₃、4-NP、E₁Adsorbance all Smaller, thus illustrating that PIL/MWCNTs-MIP/GCE has good affinity and selectivity, E to template molecule₃、4-NP、E₁ Adsorbance less be chemical constitution and E due to them₂Inconsistent, the imprinted cavity size of formation and functional structure are not Join, therefore to template molecule E₂Produce specific adsorption.

10th, the range of linearity, test limit, repeatability and stability

Under all optimal conditions, this experiment have studied PIL/MWCNTs-MIP/GCE to E by DPV method₂Linear model Enclose and test limit.PIL/MWCNTs-MIP/GCE is immersed the E containing variable concentrations₂PBS (pH 6.0) solution in adsorb 5min, then carries out DPV detection, shown in result such as Figure 10 (A), with E₂The increase of concentration, its oxidation peak electricity at 0.6V Stream is also gradually increased, oxidation peak current size and E as seen from the figure₂Concentration assumes good line in the range of 0.005～50.0 μM Sexual intercourse；If Figure 10 (B) is E₂Canonical plotting, the corresponding linear equation of PIL/MWCNTs-MIP/GCE is：I_p(μ A)= 0.02103C (μM)+0.02585, linearly dependent coefficient is 0.9966, and its lowest detection is limited to 0.0015 μM (S/N=3), therewith The front pertinent literature reported is contrasted, as shown in table 2.PIL/MWCNTs-MIP/GCE sensor is to E₂Detection have relatively The wide range of linearity and relatively low test limit.

We have investigated the PIL/MWCNTs-NIP/GCE and E of traditional PI L-MIP/GCE electrode pair variable concentrations simultaneously₂ Carry out DPV mensure, its linear equation is：I_p(μ A)=0.00464C (μM)+0.04405, linearly dependent coefficient is 0.7667. K_{Sv, MIP}And K_{Sv, NIP}It is the linear gradient of PIL/MWCNTs-MIP/GCE, PIL/MWCNTs-NIP/GCE respectively, its value is respectively 0.02103rd, 0.00464, this K_{Sv, MIP}With K_{Sv, NIP}Ratio be defined as the press back factor (IF), for assessment material specificity know Not^[48,67], this IF (K_{Sv, MIP}/K_{Sv, NIP}) result of calculation be 4.53, illustrate that there are a lot of E on PIL/MWCNTs-MIP surface₂Identification position Point, but PIL/MWCNTs-NIP material surface does not have E₂Print identification site, PIL/MWCNTs-MIP/GCE is efficiently electric Stream response shows it to E₂Special sexual compatibility.

Repeatability and stability are the good key factors of sensor performance, therefore, prepare 5 electricity under the same conditions Pole, every electrode runs parallel 30 μM of E of 3 mensure₂DPV current-responsive, weigh this biography by the meansigma methodss of every electrode detection The repeatability of sensor, it is 3.62% that result obtains relative standard deviation.The GCE that PIL/MWCNTs-MIP modifies is in PBS Preserve in refrigerator at 4 DEG C in (pH6.0 0.1M) buffer solution, after 1 week, this sensor DPV response current value remains initially The 97.1% of electric current, after 4 weeks, current-responsive value reduces 15%.The above results explanation PIL/MWCNTs-MIP sensor performance Go out excellent repeatability and stability.

Table 2 this method and other electrochemical processes measure the electrochemical process contrast of NP.

Document source：

(1) Tao H, Wei W Z, Zeng, X D, et al.Electrocatalytic oxidation and determination of estradiol using an electrode modified with carbon nanotubes And an ionic liquid [J] .Microchimica Acta, 2009,166 (1)：53-59.

(2) Hu S S, Wu K B, Yi H C, et al.Voltammetric behavior and determination of estrogens at Nafion-modified glassy carbon electrode in the presence of Cetyltrimethylammonium bromide [J] .Analytica Chimica Acta, 2002,464 (2)：209-216.

(3) Lin X Q, Li Y X.A sensitive determination of estrogens with a Pt nano-clusters/multi-walled carbon nanotubes modified glassy carbon electrode [J] .Biosensors and Bioelectronics, 2005,20 (11)：2341-2346.

(4) Liu X Q, Wong D K Y.Electrocatalytic detection of estradiol at a carbon nanotube|Ni(Cyclam)composite electrode fabricated based on a two- Factorial design [J] .Analytica chimica acta, 2007,594 (2)：184-191.

(5) He Q, Yuan S, Chen C, et al.Electrochemical properties of estradiol at glassy carbon electrode modified with nano-Al₂O₃film[J].Materials Science and Engineering：C, 2003,23 (5)：621-625.

(6)Salc₁B, Biryol I.Voltammetric investigation of β-estradiol [J] .Journal of pharmaceutical and biomedical analysis, 2002,28 (3)：753-759.

(7) Song J C, YangJi, Hu X M.Electrochemical determination of estradiol using a poly(L-serine)film-modified electrode[J].Journal of Applied Electrochemistry, 2008,38 (6)：833-836.

(8) Janegitz B C, Santos F A D, Ronaldo C, et al.Electrochemical determination of estradiol using a thin film containing reduced graphene oxide and dihexadecyl phosphate[J].Materials Science and Engineering：C, 2014, 37：14-19.

11st, the comparison of this method and traditional immunoblot method

This experiment is prepared for function monomer using new poly ion liquid and is divided based on the surface of new PIL-MWCNTs Sub- imprinted polymer electrochemical sensor, in order to prove the superiority of this experiment intermediate ion liquid, we under the same conditions, with E₂For template molecule, respectively with MAA, 4-NP and AM as function monomer, EGDMA as cross-linking agent, AIBN is initiator, by tradition Blotting has been respectively synthesized corresponding imprinted material, they is used soxhlet extraction eluting template molecule therein simultaneously.Then Weighing 6mg respectively adds 2mL0.5% chitosan solution ultrasonic so as to be uniformly dispersed, and takes 5 μ L drop coatings respectively in glass-carbon electrode table Face, under identical conditions, to 5 μM of E₂PBS (0.1M pH 6.0) solution detected, result as shown in figure 11, this sensing The DPV current-responsive of device is 1.5 times of MAA, 4-NP and AM, 1.9 times and 2.4 times respectively.This illustrates this PIL/MWCNTs-MIP Sensor is to template molecule E₂There is more preferable adsorptivity and electric conductivity, be attributable to the satisfactory electrical conductivity of MWCNTs and big ratio Surface area, and the good electric conductivity of novel ion liquid and biocompatibility.

12nd, interference experiment

In order to verify that this method is applied to the detection of 17 beta estradiols in environmental water sample, investigate in environmental water sample and there may be Some interfering ions, phenolic compound to measure E₂Impact.Maximum limitation is allowed to be E caused by this amount₂DPV peak current Size variation is within the scope of ± 5%.As shown in table 3, K⁺、Ca²⁺、Na⁺、Al³⁺、SO₄ ^2-、Cl^-Plasma is right within 500 times Detection does not produce interference, to Mg²⁺、Fe³⁺And Cu²⁺、Mn²⁺Do not produce interference within 300 times and 100 times respectively, p-nitrophenyl exists Interference is not produced, nitrophenols do not produce interference within 20 times within 40 times.Result proves, this sensor selectivity preferably, is fitted For E in environmental water sample₂Detection.

Table 3 PIL-MWCNTs-MIP sensor DPV measures E₂Permission interference volume；

Testing conditions, E₂Concentration：1×10^-5M；Supporting electrolyte：0.1M PBS(pH 6.0)；Enrichment time：5min.

13rd, E in actual water sample₂Detection

Environmental water sample takes from the river of Institutes Of Jiaxing school district, and water sample, after centrifugal treating, takes supernatant, and passes through 0.45 μm membrane filtration, gained clear liquor is standby.Tap water is derived from laboratory, is left intact.Then by PIL-MWCNTs- In the detection of environmental water sample, parallel assay 5 times, it is found that no E in water sample for MIP sensor application₂Electrochemical response.Thus The response rate being obtained using standard addition method, to confirm the feasibility of this method.

E in table 4 river and tap water₂Content (each sample parallel assay 5 times)；

Condition determination：The PBS buffer solution of 0.1M, pH 6.0；Adsorption time again：5min.

As shown in Table 4,97.3%～105.7%, result shows PIL-MWCNTs-MIP sensor to the response rate of gained It is suitable for E in actual water sample₂Mensure, and obtain gratifying result.

Claims (8)

1. the surface molecule print poly ion liquid of multi-walled carbon nano-tubes, it be by functionalized multi-wall carbonnanotubes, function monomer, The polymerization under initiator effect of cross-linking agent and template molecule forms and removes the composite of template molecule it is characterised in that institute Stating function monomer is 1- (1 '-hydroxybutane) -3- vinyl imidazole hexafluorophosphoric acid；Described cross-linking agent is ethylene glycol dimethyl third Olefin(e) acid ester and 3,3 '-(1 ', 4 '-butane)-bis- -1- vinyl imidazole hexafluorophosphoric acids；Described template molecule is 17 beta estradiols；

Described 1- (1 '-hydroxybutane) -3- vinyl imidazole hexafluorophosphoric acid, ethylene glycol dimethacrylate, 3,3 '-(1 ', 4 '-butane)-bis- -1- vinyl imidazole hexafluorophosphoric acids mol ratio be 0.3～0.6:1:1.

2. the surface molecule print poly ion liquid of multi-walled carbon nano-tubes as claimed in claim 1 is it is characterised in that described draw Sending out agent is azodiisobutyronitrile.

3. the surface molecule print poly ion liquid of multi-walled carbon nano-tubes as claimed in claim 1 is it is characterised in that described carboxylic The molal of base multi-walled carbon nano-tube and template molecule is than for 0.08～0.1g:0.1mmol.

4. the surface molecule print poly ion liquid of multi-walled carbon nano-tubes as claimed in claim 1 is it is characterised in that described work( Can monomer and initiator molal weight ratio for 0.1mmol:3～6mg.

5. the preparation method of the surface molecule print poly ion liquid of described multi-walled carbon nano-tubes as arbitrary in claim 1-4, bag Include following steps：Template molecule is added in solvent, adds function monomer, cross-linking agent, initiator and carboxylated many walls carbon and receive Mitron, mix homogeneously carries out polyreaction, collects product, the template molecule in removing product, system is finally dried after the completion of reaction Obtain the surface molecule print poly ion liquid of described multi-walled carbon nano-tubes.

6. preparation method as claimed in claim 5 it is characterised in that described polyreaction temperature be 60～80 DEG C, the time For 12～36 hours.

7. the electrification made using the surface molecule print poly ion liquid of the arbitrary described multi-walled carbon nano-tubes of such as claim 1-4 Learn sensor.

8. application in detection 17 beta estradiols for the electrochemical sensor as claimed in claim 7.