CN101196486A - Bionic molecular recognition nano sensing film of electrochemical transducer and method for producing the same - Google Patents
Bionic molecular recognition nano sensing film of electrochemical transducer and method for producing the same Download PDFInfo
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Abstract
The invention relates to an electrochemical sensor molecular bionics recognizing nanometer transduction membrane and the manufacturing method, which is a transduction membrane with special molecular bionics recognizing capability to template molecule and is characterized in that the transduction membrane is produced on the surface of the metal electrode of the electrochemical sensor molecular bionics, glass or quartz material and the transduction membrane is a membrane with controllable thickness in 5 to 50nm.The thickness of the transduction membrane is controlled by reversible addition-chain transfer free radical polymerization reaction time, and forms linear relation with time. The metal electrode is made of gold or platinum. The manufacturing method comprises conducting chlorsilane deriving, transferring the chlorsilane of the end surface of the optical fiber into chain transfer agent, then conducting molecular engram polyreaction to the optical fiber with the end surface of chain transfer agent, and finally eluting the template molecule in molecular bionics recognizing nanometer transduction membrane. The sensitivity of the sensor manufactured by the invention is high and the stability is good, which will be widely used in metallurgy, chemical industry, environment protection and other related fields.
Description
Technical field
The invention belongs to bionical molecular recognition and electrochemical sensor field, relate to a kind of bionic molecular recognition nano sensing film of electrochemical transducer and preparation method thereof or rather.
Background technology
Molecular recognition is meant in the mixture system of complexity, and molecule or molecule fragment are discerned another molecule or molecule fragment specifically, and mutually combines and form a compound or supramolecular phenomenon.The notion of molecular recognition be to the research of biomolecule such as enzyme, acceptor, antibody progressively development come out, it is prevalent among the various vital movements, as antigen and antibody, protein hormone, phytolectin, lectin or medicine and acceptor, proteinase and protein substrate, all have the molecular recognition phenomenon between protein and the nucleic acid, so molecular recognition plays crucial effects to vital movement.Biomolecule identification at present and correlation technique have been widely used in fields such as clinical examination, Pharmaceutical Analysis, new drug development, gene diagnosis, treatment of cancer and environmental evaluation.Molecular imprinting (the MolecularlyImprinted Technology that grows up as numerous branch superiorities such as collecting Polymer Synthesizing, MOLECULE DESIGN, molecular recognition, bionical bioengineering in recent years, MIT), exactly from the bionics angle, adopt the method for synthetic to obtain on space and binding site, to mate fully with a certain specific molecular (microsphere, template molecule), have the macromolecular material of specificity in conjunction with this specific molecular, thereby realize bionical molecular recognition, promptly realize bionic function.Resulting molecularly imprinted polymer (Molecularly Imprinted Polymers, MIPs) promptly have recognition performance and the selectivity same with natural antibody, have again and the same high stability of macromolecule and the ability of anti-adverse environment, thereby be widely used in various fields such as biomimetic sensor, bioengineering, clinical medicine, environmental monitoring, food industry, become new century one of the most potential new material.
At present, with sensitive material/recognition unit---the sensing membrane of molecularly imprinted polymer as sensor, the preparation biomimetic sensor has become the research direction of molecular engram and sensor field common concern, and has become common research focus.Concrete research contents is that molecularly imprinted polymer is fixed on the transducer face of sensor with the form of film by suitable mode, converts to by means such as various electricity, heat, light, matter then and can survey signal, is used for the quantitative test all cpds.In numerous sensors, electrochemical sensor is low in energy consumption because of having, highly sensitive, reliable and stable, volume is little, detection speed is fast, accurate, portable, can on-the-spot directly detect and many distinct advantages such as continuous detecting, more and more be subjected to people's generally attention, be widely used in fields such as metallurgy, chemical industry, environmental protection, in various measurement, brought into play own original effect.Existing sensing membrane film build method commonly used such as pressed film method, spin-coating method etc., the thickness of made sensing membrane have a strong impact on mass transfer velocity and the equilibration time of target molecule (template molecule) in sensing membrane inside mostly in μ m level; Made sensing membrane comes off from the transducer of sensor easily, influences the stability of sensor, has limited the application and the development of molecularly imprinted polymer biomimetic sensor.Therefore, directly the surperficial synthetic molecules trace nanometer film of the metal electrode of biomimetic sensor (＜50nm), improve the mass transfer velocity of target molecule (template molecule), reduce equilibration time and become one of focus of biomimetic sensor research field forefront in molecular imprinted polymer membrane inside.Though existing bibliographical information adopts grafting copolymerization process at carrier surface synthesis of nano film (Sulitzly C., Ruckert B., hour all A.J.et al..Macromolecules, 2002,35:79-91.), but, make it can't be widely used because grafting copolymerization process can't be broken away from the even shortcoming of non-uniform film thickness of solution copolymerization, formation always.Key is the bionical sensing membrane that classic method makes, and thickness is big, homogeneity is relatively poor, thickness is uncontrollable (generally reaching micron order), and comes off from surface of metal electrode easily, has a strong impact on sensitivity, stability and the range of application of biomimetic sensor; Recently surface modification method is not still broken away from shortcomings such as the non-uniform film thickness of solution copolymerization, formation is even, has seriously limited the application of molecularly imprinted polymer biomimetic sensor.
Summary of the invention
The present invention aims to provide a kind of bionic molecular recognition nano sensing film of electrochemical transducer and preparation method thereof, improves sensitivity, stability and the range of application of electrochemical sensor, remedies the deficiencies in the prior art.
Technical conceive of the present invention is by molecular imprinting is combined with reversible addition-fragmentation chain transfer free radical polymerization reacting phase, make on surface of metal electrode " growth " go out to microsphere have bionical molecule distinguishability, thickness can be by the nano sensing film of reversible addition-fragmentation chain transfer free radical polymerization reaction time control, as the recognition means of electrochemical sensor, and be widely used in fields such as metallurgy, chemical industry, environmental protection.
Therefore, bionic molecular recognition nano sensing film of the present invention, it is the sensing membrane that has the single-minded bionical molecule distinguishability of template molecule, it is characterized in that this sensing membrane is that to be created on the surface of metal electrode of electrochemical sensor, controllable thickness, thickness range be the film of 5～50nm, thickness only is Nano grade.
The thickness of sensing membrane of the present invention is by reversible addition-chain transfer Raolical polymerizable time control, and linear with the time.Described metal electrode is to be the metal electrode of material with gold or platinum.
Preparation method of the present invention is: at first the surface of metal electrode at electrochemical sensor connects amino, alcohol radical isoreactivity group, reactive group is converted into the chain-transferring agent precursor of alkyl halide as reversible addition-fragmentation chain transfer free radical polymerization, under the effect of Grignard reagent, alkyl halide is converted into chain-transferring agent, afterwards under action of evocating, cause reversible addition-fragmentation chain transfer free radical polymerization reaction (the molecular engram polyreaction takes place simultaneously), generate the bionic molecular recognition nano sensing film that formation comprises template molecule at surface of metal electrode, obtain the bionic molecular recognition nano sensing film of controllable thickness through the template molecule wash-out.
The concrete grammar of the present invention's preparation is as follows:
A. surface of metal electrode being carried out alkyl halide derives:
(1) metal electrode is placed airtight glassware, with Piranha solution (98%H
2SO
4: 30%H
2O
2=7: 3, volume ratio) under 90 ℃ of conditions, soaked 5 minutes, use ultrapure water, absolute ethyl alcohol drip washing then successively, nitrogen dries up; Metal electrode is placed another airtight glassware again, soaked 2～48 hours in concentration is the ethanol solution of sulfhydryl compound of 0.01～10mmol/L, take out with absolute ethyl alcohol drip washing totally, nitrogen dries up, and obtains the metal electrode of sulfhydrylation;
(2) metal electrode with above-mentioned sulfhydrylation places airtight glassware, adds dry methylene chloride 10～100mL successively, triethylamine 10～500 μ L, and dimethyl aminopyridine 5～50mg, ice-water bath is cooled to 0～5 ℃ with reaction vessel, obtains system A; Dibromo dimethyl propyl acylbromide 10～500 μ L are dissolved in the methylene chloride of other 10～100mL drying, dropwise add among the system A; Keep and continue under the condition of ice bath to stir 1～24 hour, transferred stirring at room afterwards to 1～48 hour; It is clean with absolute ethyl alcohol drip washing to take out metal electrode, and nitrogen dries up, and obtains the metal electrode of alkyl halide derivatization;
B. the alkyl halide with above-mentioned surface of metal electrode is converted into chain-transferring agent
5～50mL Grignard reagent is dissolved in the super dry tetrahydrofuran of 10～100mL, and oil bath is preheated to 30～60 ℃, dropwise adds dry carbon disulphide 100～1000 μ L, keeps 50 ℃ of reactions of oil bath 1～24 hour; The metal electrode that adds above-mentioned alkyl halide derivatization afterwards, logical nitrogen 10 minutes, the following 50 ℃ of oil baths reaction of nitrogen protection afterwards 1～48 hour; Reaction finishes the back metal electrode and uses tetrahydrofuran successively, and methyl alcohol, acetone respectively wash 3 times, and 60 ℃ of vacuum drying obtain the metal electrode of chain-transferring agent derivatization;
C. be that the metal electrode of chain-transferring agent carries out the molecular engram polyreaction to above-mentioned surface again
(1) add pore-foaming agent in the airtight glassware, add function monomer, template molecule, crosslinking chemical successively, stirring makes it to dissolve fully, obtains the transparence solution B; The metal electrode that adds the above-mentioned derivatization of chain-transferring agent;
(2) in the airtight glassware of another one, initiating agent added to stir in the pore-foaming agent and make it to dissolve fully, add above-mentioned solution B then and mix, obtain solution C;
(3) above-mentioned solution C is led to nitrogen 1～60 minute, remove deoxidation and moisture; Then airtight glassware is placed 50～120 ℃ oil bath, stirring reaction 1～72 hour; Can on surface of metal electrode, obtain the bionic molecular recognition nano sensing film that contains template molecule that thickness range is 5～50nm by control time length;
D. the template molecule in the last wash-out bionic molecular recognition nano sensing film
With the metal electrode of the bionic molecular recognition nano sensing film that contains template molecule of above-mentioned preparation, clean with absolute ethyl alcohol drip washing; Be that 10/1～1/10 mixed solution soaks with acetate/methyl alcohol volume ratio then, vibrated 1～72 hour; Use the methyl alcohol cyclic washing until neutrality after the elimination soak solution, then in 50～120 ℃, dry to constant weight with vacuum drying chamber, obtain the metal electrode of surface biomimetic molecular recognition nano sensing film derivatization at last, promptly obtain bionic molecular recognition nano sensing film at surface of metal electrode.Wherein above-mentioned
Metal electrode is: with gold, platinum etc. is the electrode of material;
Sulfhydryl compound: 2 mercapto ethanol, 2-mercaptoethylmaine, 2-mercaprol, 2-sulfydryl propylamine, the positive undecyl alcohol of sulfydryl, the positive undecylamine of sulfydryl;
Grignard reagent is: phenyl-magnesium-bromide (PMB), phenyl-magnesium-chloride (PMC);
Chain-transferring agent is: dithioesters compounds such as dithiobenzoic acid ester;
Pore-foaming agent is: benzene and derivant thereof, methine halide, tetrahydrofuran, N, dinethylformamide (DMF), dimethyl sulfoxide (DMSO), acetonitrile;
Template molecule is: compounds such as amino acid and derivant thereof, chipal compounds, medicine, agricultural chemicals;
Function monomer is: acrylic acid with and ester class, vinylpyridine class;
Crosslinking chemical is: ethylene glycol dimethacrylate (EGDMA), divinylbenzene (DVB);
Initiating agent is: azoisobutyronitrile azo-initiators such as (AIBN).
Bionic molecular recognition nano sensing film of electrochemical transducer of the present invention combines the bionical specific recognition ability of molecularly imprinted polymer, the controllability dual nature of reversible addition-fragmentation chain transfer free radical polymerization technology, direct surperficial synthetic molecules trace nano sensing film at metal electrode, make the thickness of sensing membrane reach the nm rank, transducer sensitivity height, good stability that the present invention makes will be applied in association areas such as metallurgy, chemical industry, environmental protection widely.
Embodiment
Bionic molecular recognition nano sensing film of the present invention, it is the sensing membrane that has the single-minded bionical molecule distinguishability of template molecule, it is characterized in that this sensing membrane is that to be created on the surface of metal electrode of electrochemical sensor, controllable thickness, thickness range be the film of 5～50nm, thickness only is Nano grade.
The thickness of described sensing membrane is by reversible addition-chain transfer Raolical polymerizable time control, and linear with the time.Described metal electrode is to be the metal electrode of material with gold or platinum.
The concrete grammar of the present invention's preparation further describes with four following embodiment:
Embodiment 1.
A. at first surface of metal electrode being carried out alkyl halide derives
(1) metal electrode is placed airtight glassware, with Piranha solution (98%H
2SO
4: 30%H
2O
2=7: 3, volume ratio) under 90 ℃ of conditions, soaked 5 minutes, use ultrapure water, absolute ethyl alcohol drip washing successively, nitrogen dries up; Metal electrode is placed another airtight glassware again, soaked 48 hours in concentration is the ethanol solution of 2 mercapto ethanol of 0.1mmol/L, take out with absolute ethyl alcohol drip washing totally, nitrogen dries up, and obtains the metal electrode of sulfhydrylation;
(2) metal electrode with above-mentioned sulfhydrylation places airtight glassware, adds dry methylene chloride 10mL successively, triethylamine 10 μ L, and dimethyl aminopyridine 5mg, ice-water bath is cooled to 0～5 ℃ with reaction vessel, obtains system A; Dibromo dimethyl propyl acylbromide 10 μ L are dissolved in the methylene chloride of other 10mL drying, dropwise add among the system A; Keep and continue under the condition of ice bath to stir 2 hours, transferred stirring at room afterwards to 6 hours; It is clean with absolute ethyl alcohol drip washing to take out metal electrode, and nitrogen dries up, and obtains the metal electrode of alkyl halide derivatization;
B. the alkyl halide with above-mentioned surface of metal electrode is converted into chain-transferring agent
The 5mL Grignard reagent is dissolved in the super dry tetrahydrofuran of 10mL, and oil bath is preheated to 40 ℃, dropwise adds dry carbon disulphide 100 μ L, keeps 50 ℃ of reactions of oil bath 2 hours; The metal electrode that adds precursor-derivedization of chain-transferring agent afterwards, logical nitrogen 10 minutes, the following 50 ℃ of oil baths reaction of nitrogen protection afterwards 6 hours; Reaction finishes the back metal electrode and uses tetrahydrofuran successively, and methyl alcohol, acetone respectively wash 3 times, and 60 ℃ of vacuum drying obtain the metal electrode of chain-transferring agent derivatization;
C. be that the metal electrode of chain-transferring agent carries out the molecular engram polyreaction to above-mentioned surface
(1) add pore-foaming agent tetrahydrofuran 20mL in the airtight glassware, add function monomer methacrylic acid 1mmol, template molecule theophylline 20mg, crosslinking chemical EGDMA 5mmol successively, stirring makes it to dissolve fully, obtains the transparence solution A; Add the metal electrode of chain-transferring agent derivatization;
(2) in the airtight glassware of another one with initiator A IBN, add to stir in the pore-foaming agent tetrahydrofuran and make it to dissolve fully, add solution A then and mix, obtain solution B;
(3) the logical nitrogen of solution B is 10 minutes, removes deoxidation and moisture; Airtight then glassware places 60 ℃ oil bath, stirring reaction 6 hours; Obtain containing the bionic molecular recognition nano sensing film of template molecule at surface of metal electrode;
D. the template molecule in the wash-out bionic molecular recognition nano sensing film
With the metal electrode of the bionic molecular recognition nano sensing film derivatization that contains template molecule of above-mentioned preparation, clean with absolute ethyl alcohol drip washing; Be that 10/1 mixed solution soaks with acetate/methyl alcohol volume ratio then, vibrated 1 hour; Use the methyl alcohol cyclic washing until neutrality after the elimination soak solution, then in 50 ℃, dry to constant weight with vacuum drying chamber, obtain the metal electrode of bionic molecular recognition nano sensing film derivatization, promptly the surface of metal electrode at electrochemical sensor obtains bionic molecular recognition nano sensing film.
The electrochemical sensor of prepared surface of metal electrode bionic molecular recognition nano sensing film derivatization, sensing membrane is for having the nano sensing film of bionical molecule distinguishability to theophylline, and its dimensional thickness is controlled at 1.8nm; Identification to theophylline is rolled off the production line (detection line) for 3nmol/L, and the range of linearity is 10～500nmol/L; Because contrast molecule, its separation factor to theophylline is 6.92 with coffee.
Embodiment 2.
A. electrode surface being carried out alkyl halide derives
(1) metal electrode is placed airtight glassware, with Piranha solution (98%H
2SO
4: 30%H
2O
2=7: 3, volume ratio) under 90 ℃ of conditions, soaked 5 minutes, use ultrapure water, absolute ethyl alcohol drip washing successively, nitrogen dries up; Then metal electrode is placed another airtight glassware, soaked 24 hours in concentration is the ethanol solution of 2-mercaptoethylmaine of 1mmol/L, take out with absolute ethyl alcohol drip washing totally, nitrogen dries up, and obtains the metal electrode of sulfhydrylation; (2) metal electrode with above-mentioned sulfhydrylation places airtight glassware, adds dry methylene chloride 20mL successively, triethylamine 20 μ L, and dimethyl aminopyridine 10mg, ice-water bath is cooled to 0～5 ℃ with reaction vessel, obtains system A; Dibromo dimethyl propyl acylbromide 20 μ L are dissolved in the methylene chloride of other 20mL drying, dropwise add among the system A; Keep and continue under the condition of ice bath to stir 6 hours, transferred stirring at room afterwards to 12 hours; It is clean with absolute ethyl alcohol drip washing to take out metal electrode, and nitrogen dries up, and obtains the metal electrode of alkyl halide derivatization;
B. the alkyl halide with above-mentioned surface of metal electrode is converted into chain-transferring agent
The 10mL Grignard reagent is dissolved in the super dry tetrahydrofuran of 20mL, and oil bath is preheated to 50 ℃, dropwise adds dry carbon disulphide 200 μ L, keeps 50 ℃ of reactions of oil bath 6 hours; The metal electrode that adds precursor-derivedization of chain-transferring agent afterwards, logical nitrogen 10 minutes, the following 50 ℃ of oil baths reaction of nitrogen protection afterwards 12 hours; Reaction finishes the back metal electrode and uses tetrahydrofuran successively, and methyl alcohol, acetone respectively wash 3 times, and 60 ℃ of vacuum drying obtain the metal electrode of chain-transferring agent derivatization;
C. be that the metal electrode of chain-transferring agent carries out the molecular engram polyreaction to above-mentioned surface
(1) add pore-foaming agent methylene chloride 20mL in the airtight glassware, add function monomer 4-vinylpridine 1mmol, template molecule caffeine 20mg, crosslinking chemical crosslinking chemical DVB 5mmol successively, stirring makes it to dissolve fully, obtains the transparence solution A; Add the metal electrode of chain-transferring agent derivatization;
(2) in the airtight glassware of another one with initiator A IBN, add to stir in the pore-foaming agent methylene chloride and make it to dissolve fully, add solution A then and mix, obtain solution B;
(3) the logical nitrogen of solution B is 20 minutes, removes deoxidation and moisture; Airtight then glassware places 60 ℃ oil bath, and stirring reaction 12 hours obtains containing the bionic molecular recognition nano sensing film of template molecule at surface of metal electrode;
D. the template molecule in the wash-out bionic molecular recognition nano sensing film
With the metal electrode of the bionic molecular recognition nano sensing film that contains template molecule of preparation, clean with absolute ethyl alcohol drip washing; Be that 1/1 mixed solution soaks with acetate/methyl alcohol volume ratio then, vibrated 5 hours; Use the methyl alcohol cyclic washing until neutrality after the elimination soak solution, then in 60 ℃, dry to constant weight with vacuum drying chamber, obtain the metal electrode of bionic molecular recognition nano sensing film derivatization, promptly the surface of metal electrode at electrochemical sensor obtains bionic molecular recognition nano sensing film.
The electrochemical sensor of prepared surface of metal electrode bionic molecular recognition nano sensing film derivatization, sensing membrane is for having the nano sensing film of bionical molecule distinguishability to caffeine, and its dimensional thickness is controlled at 6nm; Identification to theophylline is rolled off the production line (detection line) for 1.5nmol/L, and the range of linearity is 5～500nmol/L; With the theophylline is the contrast molecule, and its separation factor to caffeine is 7.38.
Embodiment 3.
A. surface of metal electrode being carried out alkyl halide derives
(1) metal electrode is placed airtight glassware, with Piranha solution (98%H
2SO
4: 30%H
2O
2=7: 3, volume ratio) under 90 ℃ of conditions, soaked 5 minutes, use ultrapure water, absolute ethyl alcohol drip washing successively, nitrogen dries up; Then metal electrode is placed another airtight glassware, soaked 12 hours in concentration is the ethanol solution of the positive undecyl alcohol of sulfydryl of 5mmol/L, take out with absolute ethyl alcohol drip washing totally, nitrogen dries up, and obtains the metal electrode of sulfhydrylation; (2) metal electrode with above-mentioned sulfhydrylation places airtight glassware, adds dry methylene chloride 50mL successively, triethylamine 200 μ L, and dimethyl aminopyridine 20mg, ice-water bath is cooled to 0～5 ℃ with reaction vessel, obtains system A; Dibromo dimethyl propyl acylbromide 200 μ L are dissolved in the methylene chloride of other 50mL drying, dropwise add among the system A; Keep and continue under the condition of ice bath to stir 12 hours, transferred stirring at room afterwards to 24 hours; It is clean with absolute ethyl alcohol drip washing to take out metal electrode, and nitrogen dries up, and obtains the metal electrode of alkyl halide derivatization;
B. the alkyl halide with above-mentioned surface of metal electrode is converted into chain-transferring agent
The 20mL Grignard reagent is dissolved in the super dry tetrahydrofuran of 50mL, and oil bath is preheated to 50 ℃, dropwise adds dry carbon disulphide 500 μ L, keeps 50 ℃ of reactions of oil bath 12 hours; The metal electrode that adds precursor-derivedization of chain-transferring agent afterwards, logical nitrogen 10 minutes, the following 50 ℃ of oil baths reaction of nitrogen protection afterwards 24 hours; Reaction finishes the back metal electrode and uses tetrahydrofuran successively, and methyl alcohol, acetone respectively wash 3 times, and 60 ℃ of vacuum drying obtain the metal electrode of chain-transferring agent derivatization;
C. be that the metal electrode of chain-transferring agent carries out the molecular engram polyreaction to above-mentioned surface
(1) add pore-foaming agent acetonitrile 40mL in the airtight glassware, add successively function monomer 4-vinylpridine 1mmol, template molecule 4,4 '-DDT 50mg, crosslinking chemical crosslinking chemical EGDMA 5mmol, stir and make it to dissolve fully, obtain the transparence solution A; Add the metal electrode of chain-transferring agent derivatization;
(2) in the airtight glassware of another one with initiator A IBN, add to stir in the pore-foaming agent acetonitrile and make it to dissolve fully, add solution A then and mix, obtain solution B;
(3) the logical nitrogen of solution B is 30 minutes, removes deoxidation and moisture; Airtight then glassware places 50 ℃ oil bath, and stirring reaction 48 hours obtains containing the bionic molecular recognition nano sensing film of template molecule at surface of metal electrode;
D. the template molecule in the wash-out bionic molecular recognition nano sensing film
With the metal electrode of the bionic molecular recognition nano sensing film derivatization that contains template molecule of preparation, clean with absolute ethyl alcohol drip washing; Be that 1/5 mixed solution soaks with acetate/methyl alcohol volume ratio then, vibrated 10 hours; Use the methyl alcohol cyclic washing until neutrality after the elimination soak solution, then in 60 ℃, dry to constant weight with vacuum drying chamber, obtain the metal electrode of bionic molecular recognition nano sensing film derivatization, promptly the surface of metal electrode at electrochemical sensor obtains bionic molecular recognition nano sensing film.
The electrochemical sensor of prepared surface of metal electrode bionic molecular recognition nano sensing film derivatization, sensing membrane be to 4,4 '-DDT has the nano sensing film of bionical molecule distinguishability, its dimensional thickness is controlled at 15nm; To 4,4 '-identification of DDT roll off the production line (detection line) be 1nmol/L, the range of linearity is 5～500nmol/L; With 2,4 '-DDT is the contrast molecule, its to 4,4 '-separation factor of DDT is 4.16.
Embodiment 4.
A. surface of metal electrode being carried out alkyl halide derives
(1) metal electrode is placed airtight glassware, with Piranha solution (98%H
2SO
4: 30%H
2O
2=7: 3, volume ratio) under 90 ℃ of conditions, soaked 5 minutes, use ultrapure water, absolute ethyl alcohol drip washing successively, nitrogen dries up; Then metal electrode is placed another airtight glassware, soaked 6 hours in concentration is the ethanol solution of the positive undecylamine of sulfydryl of 10mmol/L, take out with absolute ethyl alcohol drip washing totally, nitrogen dries up, and obtains the metal electrode of sulfhydrylation; (2) metal electrode with above-mentioned sulfhydrylation places airtight glassware, adds dry methylene chloride 100mL successively, triethylamine 500 μ L, and dimethyl aminopyridine 50mg, ice-water bath is cooled to 0～5 ℃ with reaction vessel, obtains system A; Dibromo dimethyl propyl acylbromide 500 μ L are dissolved in the methylene chloride of other 100mL drying, dropwise add among the system A; Keep and continue under the condition of ice bath to stir 24 hours, transferred stirring at room afterwards to 48 hours; It is clean with absolute ethyl alcohol drip washing to take out metal electrode, and nitrogen dries up, and obtains the metal electrode of alkyl halide derivatization;
B. the alkyl halide with above-mentioned surface of metal electrode is converted into chain-transferring agent
The 50mL Grignard reagent is dissolved in the super dry tetrahydrofuran of 100mL, and oil bath is preheated to 60 ℃, dropwise adds dry carbon disulphide 1000 μ L, keeps 50 ℃ of reactions of oil bath 24 hours; The metal electrode that adds precursor-derivedization of chain-transferring agent afterwards, logical nitrogen 10 minutes, the following 50 ℃ of oil baths reaction of nitrogen protection afterwards 48 hours; Reaction finishes the back metal electrode and uses tetrahydrofuran successively, and methyl alcohol, acetone respectively wash 3 times, and 60 ℃ of vacuum drying obtain the metal electrode of chain-transferring agent derivatization;
C. be that the metal electrode of chain-transferring agent carries out the molecular engram polyreaction to above-mentioned surface
(1) add pore-foaming agent toluene 40mL in the airtight glassware, add function monomer 4-vinylpridine 1mmol, template molecule 3-chlorophenol 20mg, crosslinking chemical crosslinking chemical EGDMA 5mmol successively, stirring makes it to dissolve fully, obtains the transparence solution A; Add the metal electrode of chain-transferring agent derivatization;
(2) in the airtight glassware of another one with initiator A IBN, add to stir in the pore-foaming agent toluene and make it to dissolve fully, add solution A then and mix, obtain solution B;
(3) the logical nitrogen of solution B is 60 minutes, removes deoxidation and moisture; Airtight then glassware places 100 ℃ oil bath, and stirring reaction 72 hours obtains containing the bionic molecular recognition nano sensing film of template molecule at surface of metal electrode;
D. the template molecule in the wash-out bionic molecular recognition nano sensing film
With the metal electrode of the bionic molecular recognition nano sensing film derivatization that contains template molecule of preparation, clean with absolute ethyl alcohol drip washing; Be that 1/10 mixed solution soaks with acetate/methyl alcohol volume ratio then, vibrated 12 hours; Use the methyl alcohol cyclic washing until neutrality after the elimination soak solution, then in 60 ℃, dry to constant weight with vacuum drying chamber, obtain the metal electrode of bionic molecular recognition nano sensing film derivatization, promptly the surface of metal electrode at electrochemical sensor obtains bionic molecular recognition nano sensing film.
The electrochemical sensor of prepared surface of metal electrode bionic molecular recognition nano sensing film derivatization, sensing membrane is for having the nano sensing film of bionical molecule distinguishability to the 3-chlorophenol, and its dimensional thickness is controlled at 26nm; Identification to the 3-chlorophenol is rolled off the production line (detection line) for 0.6nmol/L, and the range of linearity is 3～500nmol/L; With the 5-chlorophenol is the contrast molecule, and its separation factor to the 3-chlorophenol is 8.23.
Claims (7)
1. bionic molecular recognition nano sensing film of electrochemical transducer, be the sensing membrane that has the single-minded bionical molecule distinguishability of template molecule, it is characterized in that this sensing membrane is that to be created on the surface of metal electrode of electrochemical sensor, controllable thickness, thickness range be the film of 5～50nm.
2. bionic molecular recognition nano sensing film of electrochemical transducer as claimed in claim 1 is characterized in that described metal electrode is is the metal electrode of material with gold or platinum.
3. bionic molecular recognition nano sensing film of electrochemical transducer as claimed in claim 1, the thickness that it is characterized in that sensing membrane are by reversible addition-chain transfer Raolical polymerizable time control, and linear with the time.
4. the preparation method of a bionic molecular recognition nano sensing film of electrochemical transducer comprises following a-d step:
A. surface of metal electrode being carried out alkyl halide derives:
(1) metal electrode is placed airtight glassware, with Piranha solution (98%H
2SO
4: 30%H
2O
2=7: 3, volume ratio) under 90 ℃ of conditions, soaked 5 minutes, use ultrapure water, absolute ethyl alcohol drip washing then successively, nitrogen dries up; Metal electrode is placed another airtight glassware again, soaked 2～48 hours in concentration is the ethanol solution of sulfhydryl compound of 0.01～10mmol/L, take out with absolute ethyl alcohol drip washing totally, nitrogen dries up, and obtains the metal electrode of sulfhydrylation;
(2) metal electrode with above-mentioned sulfhydrylation places airtight glassware, adds dry methylene chloride 10～100mL successively, triethylamine 10～500 μ L, and dimethyl aminopyridine 5～50mg, ice-water bath is cooled to 0～5 ℃ with reaction vessel, obtains system A; Dibromo dimethyl propyl acylbromide 10～500 μ L are dissolved in the methylene chloride of other 10～100mL drying, dropwise add among the system A; Keep and continue under the condition of ice bath to stir 1～24 hour, transferred stirring at room afterwards to 1～48 hour; It is clean with absolute ethyl alcohol drip washing to take out metal electrode, and nitrogen dries up, and obtains the metal electrode of alkyl halide derivatization;
B. the alkyl halide with above-mentioned surface of metal electrode is converted into chain-transferring agent
5～50mL Grignard reagent is dissolved in the super dry tetrahydrofuran of 10～100mL, and oil bath is preheated to 30～60 ℃, dropwise adds dry carbon disulphide 100～1000 μ L, keeps 50 ℃ of reactions of oil bath 1～24 hour; The metal electrode that adds above-mentioned alkyl halide derivatization afterwards, logical nitrogen 10 minutes, the following 50 ℃ of oil baths reaction of nitrogen protection afterwards 1～48 hour; Reaction finishes the back metal electrode and uses tetrahydrofuran successively, and methyl alcohol, acetone respectively wash 3 times, and 60 ℃ of vacuum drying obtain the metal electrode of chain-transferring agent derivatization;
C. be that the metal electrode of chain-transferring agent carries out the molecular engram polyreaction to above-mentioned surface:
(1) add pore-foaming agent in the airtight glassware, add function monomer, template molecule, crosslinking chemical successively, stirring makes it to dissolve fully, obtains the transparence solution B; The metal electrode that adds the above-mentioned derivatization of chain-transferring agent;
(2) in the airtight glassware of another one, initiating agent added to stir in the pore-foaming agent and make it to dissolve fully, add above-mentioned solution B then and mix, obtain solution C;
(3) above-mentioned solution C is led to nitrogen 1～60 minute, remove deoxidation and moisture; Then airtight glassware is placed 50～120 ℃ oil bath, stirring reaction 1～72 hour; Can on surface of metal electrode, obtain the bionic molecular recognition nano sensing film that contains template molecule that thickness range is 5～50nm by control time length;
D. the template molecule in the last wash-out bionic molecular recognition nano sensing film:
With the metal electrode of the bionic molecular recognition nano sensing film that contains template molecule of above-mentioned preparation, clean with absolute ethyl alcohol drip washing; Be that 10/1～1/10 mixed solution soaks with acetate/methyl alcohol volume ratio then, vibrated 1～72 hour; Use the methyl alcohol cyclic washing until neutrality after the elimination soak solution, then in 50～120 ℃, dry to constant weight with vacuum drying chamber, obtain the metal electrode of surface biomimetic molecular recognition nano sensing film derivatization at last, promptly obtain bionic molecular recognition nano sensing film at surface of metal electrode.
5. as claim 1 or 4 described bionic molecular recognition nano sensing film of electrochemical transducer, it is characterized in that described template molecule is one of them of theophylline, caffeine, chlorophenol or DDT molecule.
6. bionic molecular recognition nano sensing film of electrochemical transducer as claimed in claim 4 is characterized in that described chain-transferring agent is the dithiobenzoic acid ester.
7. bionic molecular recognition nano sensing film of electrochemical transducer is applied on the surface of metal electrode of electrochemical sensor.
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