CN108872340A - A kind of electrochemica biological sensor for super sensitivity detection organophosphorus pesticide - Google Patents
A kind of electrochemica biological sensor for super sensitivity detection organophosphorus pesticide Download PDFInfo
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Abstract
The present invention relates to analytical chemistry fields, specifically include a kind of electrochemica biological sensor for super sensitivity detection organophosphorus pesticide.In the way of LBL self-assembly, by amino acid ion liquid-carbon mano-tube composite, the organophosphor hydrolytic enzyme of mineralising, polymeric membrane modification in electrode surface, to obtain the working electrode of the electrochemica biological sensor of super sensitivity detection organophosphorus pesticide.The electrochemica biological sensor has very strong selectivity to organophosphorus pesticide, and the range of linearity is 10 ~ 800 nmol/L, coefficient R2It is 0.9928, the calculation shows that lowest detection is limited to 0.7 fmol/L.It follows that the electrochemica biological sensor has many advantages, such as that sensitivity superelevation, preparation are simple and convenient to operate, selectivity is high, it can be achieved that detecting the purpose of organophosphorus pesticide under negative potential, for organophosphorus pesticide it is quick detect to provide a kind of have promising technology.
Description
Technical field
The present invention relates to analytical chemistry field, specifically includes a kind of electrochemica biological for super sensitivity detection organophosphorus pesticide
Sensor.
Background technique
In the following period of time that the past is very long, organophosphorus pesticide is widely used in disease and pest control.However, organic phosphorus agriculture
Medicine is extensively in large quantities using having seriously threatened the mankind and living environment.Under normal conditions, organophosphorus pesticide is very low, with
It cannot be detected in crops or environment easily as people, and the residual complicated component of the agriculture in actual sample.Cause
This, hypersensitive, highly selective detection residues of organophosphate pesticides are particularly important now.
Electrochemica biological sensor is by bioactive substance such as biological enzyme, antigen/antibody, DNA etc. as molecular recognition
Substance is fixed on the electrode, is the analysis device for detecting signal with electrochemical signals.Electrochemica biological sensor is with its selectivity
Good, high sensitivity, the advantages that response is fast, easy to operate, it is online to can be achieved, in-vivo analysis, enjoy great popularity, in grinding for analytical chemistry
Increasingly important role is played in studying carefully, and is widely used in the fields such as life science, environmental analysis, Pharmaceutical Analysis, in recent years,
In terms of being widely used in Pesticides Testing.
In various detection methods（Such as spectral detection, enzyme inhibition and chromatography detection）In, due to electrochemical enzymatic
Sensor has the characteristics that rapid detection, high sensitivity, easy to operate and micromation, therefore extensive concern has been obtained.?
Detect most commonly used there are two main classes enzyme in the enzyme biologic sensor research of organophosphorus pesticide：One kind is that activity can be organic phosphorus
Pesticide can not retroactive inhibition cholinesterase, such as acetylcholinesterase（AChE）It is based on organic phosphorus Deng, the recognition principle of this fermentoid
Inhibiting effect of the pesticide to enzyme activity；Another kind of is the organophosphor hydrolytic enzyme of energy directly catalyzing hydrolysis organophosphorus pesticide（OPH）, accordingly
Recognition principle be based on enzymatic hydrolysis organophosphorus pesticide.Up to now, the electrochemical student of most of detection organophosphorus pesticides
Object sensor is all based on the inhibition principle of enzyme, and such as biosensor based on acetylcholinesterase is using substrate to biological enzyme
Inhibiting effect reduce enzymatic activity to achieve the purpose that detect pesticide.Subramanian Viswanathan et al. exists
《Electrochemical biosensor for pesticides based on
acetylcholinesteraseimmobilized on polyaniline deposited on vertically
assembled carbonnanotubes wrapped with ssDNA》（Biosensors & Bioelectronics
2009,24,2772）It is used in one text and polyaniline and carbon nanotube and ssDNA orthogonal sets is fixed on based on acetylcholinesterase
The electrochemica biological sensor of dress detects parathion-methyl and chlopyrifos pesticides, wherein parathion-methyl and the minimum inspection of chlopyrifos
Survey limit（Limit of detection, LOD）It is 10-3nmol/L.Muhammet Guler et al. exists《Electrochemical
detection of malathion pesticideusing acetylcholinesterase biosensor based on
glassycarbon electrode modified with conducting polymerfilm》（Environ Sci Pollut Res2016,23,12343-12351）Glass-carbon electrode based on modified conducting polymeric membrane in one text constructs acetyl
Cholinesterase electrochemica biological sensor detects malathion pesticide, and LOD reaches 4.08 nmol/L.Hui-Fang Cui
Et al.《A highly stable acetylcholinesterase biosensor based on chitosan-TiO2-
graphene nanocomposites for detection of organophosphate pesticides》
（Biosensors & Bioelectronics 2017,99,223-229）Chitosan-TiO is based in one text2Graphene nano
Compound is used for the detection of organophosphorus pesticide DDVP, LOD for the stable acetylcholinesterasebiosensor biosensor of platform construction
Up to 29 nmol/L, detection time is up to 25 min.In addition, there are also it is some be dual-enzyme coupling biosensor building, such as acetyl gallbladder
Alkali esterase and choline oxidase（ChOx）Coupling provides the relatively easy effective multienzyme fixed platform of one kind to realize that detection has
Machine phosphorus insecticide residual.XianweiMeng et al. exists《A simple and sensitive fluorescence biosensor
for detectionof organophosphorus pesticides using H2O2-sensitive quantumdots/
bi-enzyme》（Biosensors and Bioelectronics 2013,47,402-407）Based on to hydrogen peroxide in one text
Quantum dot, the double enzymes of sensitivity（Acetylcholinesterase and choline oxidase）Biological sensor is constructed to detect organic phosphorus agriculture
Medicine, wherein the LOD of organophosphorus pesticide DDVP is 4.49 nmol/L.However, due to acetylcholinesterase autospecific difference and
Suppression mechanism based on enzyme usually makes electrochemica biological sensor selectively low, detects limit for height and the response time is slow, in certain journey
The development and application of electrochemistry organophosphorus pesticide biosensor are limited on degree.Moreover, the oxidation current based on analyte, greatly
Most organophosphorus pesticide electrochemical sensors are applied to positive potential, therefore do not avoid more already present in detection architecture
Oxide（Such as uric acid, ascorbic acid）Interference.
In order to overcome the above problems, OPH is introduced into Electrochemical Detection organophosphorus pesticide biosensor to substitute enzyme suppression
The biosensor of system.Because it is nitrophenols electroactive material, tool that OPH, which can specifically hydrolyze substrate organophosphorus pesticide paraoxon,
Selective good, detecting step is simple, the advantages that repeatedly using, and enzyme can fundamentally be overcome to inhibit in biosensor
Preparation of samples is cumbersome, the low disadvantage of selectivity.Cabarcas et al. exists《Chitosan nanocomposite modified
OPH-Based amperometric sensor fororganophosphorus pesticides determination》
（Current Analytical Chemistry2018,14,75-82）Chitosan nano in one text using modification OPH is compound
Object constructs Ampere currents type sensor and is used to detect pesticide, and wherein the LOD of paraoxon is 20 nmol/L.Xiangjiang
Tang et al. exists《Sensitive electrochemical microbial biosensor forp-
nitrophenylorganophosphates based on electrode modified with cellsurface-
displayed organophosphorus hydrolase and ordered mesoporecarbons》（Biosensors and Bioelectronics 2014,60,137-142）It is constructed in conjunction with order mesoporous silicon nano material in one text using OPH
Sensitive electrochemical microsensor is used to detect organophosphorus pesticide（Paraoxon, parathion and parathion-methyl）, wherein right
The LOD of oxygen phosphorus is 9 nmol/L.In research before, we are prepared for the allosteric effect based on biomineralization OPH-Cell
OPH-Cell- cobalt phosphate biologic inorganic hybrid material is used to enhance the catalytic activity of organophosphor hydrolytic enzyme.However, at the same time,
Biomineralization and entire cell itself can also reduce to a certain extent OPH-Cell- cobalt phosphate mixed catalyst conductivity and
Electro-chemical activity.Therefore, although the analysis method based on OPH usually illustrates good selectivity and simple analytic process,
But the electrochemica biological sensor based on OPH is but seldom reported, and have not been reported at present detected under negative potential it is organic phosphorus
The electrochemica biological sensor of pesticide, and negative potential detection can reduce the interference of antioxidant in actual sample.Thus to detection
The electrochemical sensor of pesticide improves, and establishes hypersensitive and highly selective organophosphorus pesticide electrochemica biological sensor is aobvious
It obtains very necessary.
It is well known that carbon nanotube has the characteristics that biggish surface area, conductivity are high, stability is good, has solid enzyme ability,
Carbon nanotube（Single-walled carbon nanotube and multi-walled carbon nanotube）It is organic to detect to be widely used in establishment electrochemica biological sensor
Phosphorus pesticide improves the operating characteristics of electrochemical sensor.Saroja Mantha et al. exists《Electrochemical
biosensing of organophosphatesusing vertically aligned multiwall carbon
nanotubes》（Seventh International Conference on Sensing Technology 2013,71-74）
It is combined in one text using the multi-walled carbon nanotube of vertical alignment and organophosphor hydrolytic enzyme and constructs the preferably electrochemical student of sensitivity
Object sensor is used to detect paraoxon, LOD 20nmol/L.However, carbon nanotube poorly soluble, during electrode modification
Aggregation can be generated, enzyme surface cannot be uniformly covered on and influence electrode modification effect, greatly reduces the detection of modified electrode
Performance.
Ionic liquid（IL）It is a kind of completely as composed by zwitterion in room temperature or close presentation liquid at room temperature
Salt ion compound, and it is conductive, it is difficult volatilization, do not burn, be electrochemically stable, potential window is than other electrolyte aqueous solutions
Greatly, it easily separated with other materials, have the advantages that good thermal stability and chemical stability.Wherein, amino acid ion liquid
（AAIL）Belong to a kind of novel ionic liquid, it not only has the advantages that general ionic liquid（Such as intrinsic mobility, high electricity
Conductance, hardly possible volatilization, low flammability, high electrochemical activity and good stability etc.）, but also there are some unique properties,
Such as strong hydrogen bond network, environmental-friendly, good biocompatibility and biological degradability etc..It there is no AAIL-CNT compound at present
Synthesis report.
In conclusion reported that the sensor of detection organophosphorus pesticide, LOD are generally the nmol/L of 1 pmol/L ~ 29,
And the pesticide residue in crops or environment is less, needs the efficient detection device of denier.Negative electricity is had not been reported at present
The electrochemica biological sensor of position detection organophosphorus pesticide, and have not been reported based on AAIL-CNT compound or biomineralization
OPH electrochemica biological sensor.
Summary of the invention
An object of the present invention is to provide a kind of Novel electric that can be used for super sensitivity detection organophosphorus pesticide under negative potential
Chemical biosensor, with high sensitivity, stability it is good and selectivity it is high the features such as；The second object of the present invention is to provide
A kind of preparation method of the electrochemica biological sensor for super sensitivity detection organophosphorus pesticide, method preparation is simple, easy to operate,
The electrochemica biological sensor of preparation has preferable selectivity and higher sensitivity.
To achieve the above object, the technical solution adopted by the present invention is：
A kind of electrochemica biological sensor for super sensitivity detection organophosphorus pesticide, using layer by layer on the electrode pre-processed
The mode of self assembly, successively by amino acid ion liquid-carbon mano-tube composite of functionalization, mineralising organophosphor hydrolytic enzyme,
Polymeric membrane is modified in electrode surface, to obtain the work electricity of the electrochemica biological sensor of super sensitivity detection organophosphorus pesticide
Pole, to realize that hypersensitive is high under negative potential, selectively detect the purpose of organophosphorus pesticide.
It is characterized in that, this method comprises the following steps：
A amino acid ion liquid and carbon nanotube are mixed in a certain proportion, and functionalization can be obtained by Non-covalent binding in ultrasound
Amino acid ion liquid-carbon mano-tube composite（AAIL-CNT, AC）；
B. step A amino acid ion liquid-carbon mano-tube composite hanging drop that functionalization is made is added to and has been pre-processed
On electrode surface, room temperature is dried；
C. by the organophosphor hydrolytic enzyme of mineralising（M-Cell）It drips on resulting electrode surface in stepb, it is dry at 4 DEG C；
D drips one layer of polymeric membrane solution on the electrode surface obtained in step C, be made described after dry and examine for hypersensitive
Survey the electrochemica biological sensor of organophosphorus pesticide.
Preferably, the polymeric membrane can be perfluoro sulfonic acid membrane（Nafion, 0.5 wt%）, poly-dopamine, polyphenyl formic acid
Ammonium, cyclodextrin polymer etc.；The amino acid ion liquid can be by native aminos such as glycine, valine, lysine and tryptophans
Acid is synthesized using anion exchange method with bromination -1- butyl -3- methylimidazole and is made；The carbon nanotube can be single wall carbon and receive
Mitron, multi-walled carbon nanotube；The electrode is naked glassy carbon electrode, gold electrode or carbon paper；The mineralising organophosphor hydrolytic enzyme
It is to show that biomineralization prepared by the cell surface mineralising of organophosphor hydrolytic enzyme is entirely thin as phosphoric acid cobalt salt or phosphoric acid mantoquita
Born of the same parents' catalyst.
It is further preferred that, the polymeric membrane used in the present invention is perfluoro sulfonic acid membrane（Nafion, 0.5 wt%）, ion
Liquid is glycine ionic liquid, and electrode is glass-carbon electrode.
Preferably, a kind of electrochemica biological sensor for super sensitivity detection organophosphorus pesticide, which is characterized in that described
It is as follows to detect organic phosphorus step：When detection, using three-electrode system, to phosphate buffer solution（PBS, pH 7.4）In be added dropwise
The organic phosphorus sample of various concentration, carries out cyclic voltammetry under the electrochemical window of -0.8 ~ 0 V, at -0.5 ~ -0.2 V
Reduction peak current response it is organic phosphorus to qualitatively or quantitatively determine.
Preferably, a kind of electrochemica biological sensor for super sensitivity detection organophosphorus pesticide, feature exist
In：The three-electrode system includes the electrode that the organophosphor hydrolytic enzyme of mineralising is modified with described in claim 1-3（Work electricity
Pole）, saturated calomel electrode or silver/silver chloride electrode（Reference electrode）, platinum electrode（To electrode）；It is described organic phosphorus for oxygen
Phosphorus, Acetamiprid, sevin, 2-dichloroethylk dimethyl phosphate, pyrrole worm miaow and Nitenpyram.
The present invention by adopting the above technical scheme, has the following advantages that compared with prior art：Using layer by layer on glass-carbon electrode
The mode of self assembly, successively by amino acid ion liquid-carbon mano-tube composite of functionalization, mineralising organophosphor hydrolytic enzyme,
Nafion（0.05 wt%）Modification is on the glass-carbon electrode pre-processed, to obtain the electrification of super sensitivity detection organophosphorus pesticide
The working electrode of Biosensors, thus the purpose realized hypersensitive under negative potential, detect organophosphorus pesticide with high selectivity,
Minimum detection limit is suitble to up to 0.7 fmol/L, the electrochemical sensor sensitivity with higher of building and good selectivity
Rapid sensitive detects on site.
Effect of the invention is：
1. the present invention utilizes the organophosphor hydrolytic enzyme of the carbon nano tube compound material of amino acid ion liquid coating and mineralising for the first time
LBL self-assembly mode, the electrochemica biological sensor of building hypersensitive, highly selective detection organophosphorus pesticide.
2. it is compound that the present invention carries out amino acid ion liquid and carbon nanotube using Non-covalent binding method for the first time, wherein ammonia
For base acid ion liquid as stabilizer and modifying agent, method is easy, and greatly improve dispersibility of the carbon nanotube in water phase and
Biocompatibility.
3. the present invention carries out mineralising to the organophosphor hydrolytic enzyme obtained by Cell surface display, resulting mineralising has
Machine phosphorus hydrolase has very strong catalytic activity to organophosphorus pesticide, and catalytic activity is much higher than activity when non-mineralising, thus
Greatly improve the sensitivity of the electrochemical sensor based on this.
4. the purpose for detecting organophosphorus pesticide under negative potential may be implemented in the present invention, detection architecture can be excluded under negative potential
In some oxides（Such as uric acid, ascorbic acid etc.）Interference, open up for the overdelicate Pesticides Testing of electrochemica biological sensor
One new approach.
5. the minimum detection limit for the electrochemica biological sensor that the present invention constructs can reach 0.7 fmol/L, than reported
The minimum detection limit of organophosphorus pesticide analysis method（1 pmol/L~29 nmol/L）Low three to six orders of magnitude, and the biology
Sensor shows detection organophosphorus pesticide highly selective and highly sensitive.
Detailed description of the invention
Fig. 1 is the transmission electron microscope photo of M-Cell hybrid material provided in an embodiment of the present invention；
Fig. 2 is the transmission electron microscope photo of AC composite material provided in an embodiment of the present invention；
Fig. 3 is the scanning electron microscope photograph of AC composite material provided in an embodiment of the present invention and M-Cell hybrid material LBL self-assembly
Piece；
Fig. 4 is verifying M-Cell hybrid material activity cyclic voltammetric effect picture provided in an embodiment of the present invention；
Fig. 5 is the cyclic voltammetric effect picture provided in an embodiment of the present invention for proving M-Cell hybrid material advantage；
Fig. 6 is the electrochemica biological sensor quantitative detection of super sensitivity detection organophosphorus pesticide provided in an embodiment of the present invention to oxygen
The standard working curve of phosphorus；
Fig. 7 is the electrochemica biological sensor of super sensitivity detection organophosphorus pesticide provided in an embodiment of the present invention to different organic phosphorus
The selective effect picture of pesticide.
Specific embodiment
In order in depth illustrate the contents of the present invention, some embodiments will be further enumerated below, but the present invention does not limit to
In cited embodiment.Specific experiment condition or method are such as not specified in the following example, press the normal condition of this field
Or method carries out.
Embodiment 1
Biomineralization cell surface display eight hypophosphite monohydrate cobalt of OPH-（M-Cell）The preparation of hybrid material：
(1) firstly, surface display the preparation of the microorganism of OPH the gene of anchorin and OPH are specifically inserted into matter
Competent escherichia coli cell is converted in grain and with this（E. coli BL21）, by the engineering bacteria in resistance containing kanamycin
37 DEG C of shaken cultivations in LB culture medium；When culture solution is in the absorbance of 600 nm（Abbreviation OD600 nm）When about 0.6, it is added
The expression of isopropyl-β-d- thiogalactoside induction anchorin and OPH are simultaneously showed in cell surface；37 DEG C of shaken cultivations
After 10 hours, centrifugation（Revolving speed 6000 rpm, 5 minutes）Receive bacterium；
(2) after, by the E. coli of surface display organophosphor hydrolytic enzyme（Whole nm=6 OD600）It is added to phosphate buffer
（PBS, pH 7.4）In and mix；
(3) cobalt chloride solution is added into above-mentioned mixed solution（0.5 mmol/L）And mix, after placing one day at room temperature,
The phosphoric acid cobalt salt of generation is evenly distributed on antimicrobial surface, to form M-Cell hybrid material；
(4) above-mentioned reaction solution is centrifuged, removes the supernatant containing unreacted component, collect precipitating, 4 DEG C of preservations are standby
Remembered with the quantity of, material with the OD600 nm of corresponding E. coli.
The morphology analysis of M-Cell hybrid catalyst material：
By M-Cell material suspension dilution certain multiple drop on copper mesh, room temperature utilizes tem study after drying
Its pattern.
As shown in Figure 1, prepared M-Cell hybrid material is in spindle-type α-Fe2O3,2.8 μm are about, 1.2 μm of left sides of diameter
It is right.And it can be seen from the figure that the surface of hybrid material is in layer structure, illustrate that the phosphoric acid cobalt salt for having deposition to generate is uniform
It is attached to antimicrobial surface, since maximum energy criterion and mechanics principle make cell that allosteric effect have occurred to form spindle-type knot
Structure.
Embodiment 2
Amino acid ion liquid-carbon nanotube（AAIL-CNT, AC）The preparation of compound：
Firstly, by 10 mg of amino acid ion liquid synthesized and 2 mg carbon nanotubes（MWCNTs）It is mixed in 1 mL secondary water
In, it is vortexed and mixes, 15 min are constantly vibrated under ultrasound, good, the finely dispersed AC compound that finally forms stability.
The morphology analysis of AC compound：
By the hanging drop of AC compound on copper mesh, room temperature utilizes its pattern of tem study after drying.
As shown in Fig. 2, obtained AC compound, wherein AAIL is successfully coated on the outer rim of MWCNTs, and AC diameter is about
20~30nm。
Embodiment 3
Building based on AC compound, the electrochemica biological sensor of M-Cell catalyst：
(1) firstly, being 3 mm glass-carbon electrodes by diameter（GCE）Successively with 1.0,0.3,0.05 μm of Al2O3Polishing powder is in muntjac
Sanding and polishing is carried out on skin and first washes away surface contaminants after polishing every time to being in flat mirror, then moves into ultrasonic water bath and cleans, often
Then secondary 2 ~ 3 min is successively cleaned with dehydrated alcohol, ultrapure water in triplicate, dry stand-by；
(2) the 5 μ L AC complex solution of drop coating on the GCE electrode surface pre-processed, makes it relatively evenly be covered on electrode
Surface layer of glass, room temperature dries；
(3) 5 μ L M-Cell liquid thereafter, is put in the lower 4 DEG C of dryings of refrigerator；
(4) 5 μ L Nafion are dripped in dried electrode surface（0.05 wt%）（To prevent modifier in the detection process from electricity
It falls off on extremely）, to obtain the working electrode Nafion/M-Cell/AC/GCE of the electrochemica biological sensor；
(5) with before, electrode need to be rinsed with secondary water, it is therefore an objective to the modifier for being not fixed to electrode surface is rinsed out,
It is placed on 4 DEG C of refrigerator preservations when not.
The morphology analysis of AC compound and M-Cell layer assembly：
By the hanging drop of AC compound on ITO electro-conductive glass, room temperature is dried, and is then dripping one layer of M-Cell liquid above,
It is dry at 4 DEG C, metal spraying is carried out three times to sample later, utilizes its pattern of scanning electron microscope analysis.
As shown in figure 3, this is the scanning electron microscope diagram for constructing AC and M-Cell layer assembly in sensor process, from
It can be seen that spindle-shaped M-Cell is combined together with AC well by physics Non-covalent binding mode in figure.
Embodiment 4
The active verifying of M-Cell electrochemical catalysis：
The experiment constructs two kinds of biosensors of Nafion/M-Cell/AC/GCE, Nafion/AC/GCE respectively, tests respectively
Modify M-Cell and only electro catalytic activity of the glass-carbon electrode of modification AC to paraoxon.
As shown in figure 4, a is Nafion/M-Cell/AC/GCE biosensor in no PBS that paraoxon substrate is added
（pH 7.4）In, the cyclic voltammetry curve measured under negative potential occurs without reduction peak as seen from the figure, therefore judges that system does not have
There is the generation of reduction reaction；B, c, d are that the working electrode is containing 100 nmol/L, 200 nmol/L, 300 nmol/L respectively
The PBS of paraoxon（pH 7.4）In the cyclic voltammetry curve that measures occur reduction peak at -0.37V as seen from the figure, and with
Paraoxon concentration increase, the reduction reaction electric current occurred gradually increases, and e, f are respectively in only PBS（pH 7.4）
With contain paraoxon（300 nmol/L）PBS（pH 7.4）The cyclic voltammetry curve of middle measurement, from figure, it can be seen that do not appoint
What apparent peak, and electric current is also without significant change.Illustrate M-Cell having compared with forceful electric power catalytic activity to paraoxon in the present invention.
Embodiment 5
The active advantage of M-Cell electrochemical catalysis proves：
The experiment constructs Nafion/M-Cell/GCE, Nafion/M-Cell/CNT/GCE, Nafion/Cell/AC/ respectively
Tetra- kinds of biosensors of GCE, Nafion/M-Cell/AC/GCE, added with paraoxon（300 nmol/L）PBS（pH 7.4）
Middle measurement cyclic voltammetry curve tests M-Cell and Cell to the electro catalytic activity of paraoxon respectively.
As shown in figure 5, a is Nafion/M-Cell/GCE biosensor, find occur from figure without reduction peak, and
Overall current is smaller, and explanation is that M-Cell itself has biggish resistance value, hinders the electron transmission of glassy carbon electrode surface, therefore electricity
It flows smaller；B is Nafion/M-Cell/CNT/GCE biosensor, is found from figure, and curve overall current obviously increases, and
There is reduction peak appearance, illustrates that CNT itself has fine conductivity, the electron transmission efficiency of glassy carbon electrode surface can be increased, mentioned
The sensitivity of high sensor；C and d is Nafion/Cell/AC/GCE and Nafion/M-Cell/AC/GCE bio-sensing respectively
Device, from graph discovery, d curve ratio c curve has better peak current, and sensitivity is very high, therefrom illustrates M-Cell to paraoxon
Catalytic activity will be substantially better than the catalytic activity of Cell.
Embodiment 6
Electrochemical sensing standard measure detects organophosphorus pesticide：
The novel electrochemical Biosensors Nafion/M-Cell/AC/GCE constructed with the present invention carries out quantitative inspection to paraoxon
It surveys.
As shown in fig. 6, corresponding reduction peak current value is also with negatively correlated line with the increase of paraoxon concentration of substrate
Property increase, thus fit linear work curvilinear equation y=- 0.018x -1.7703, R2It is 0.9928, detection range 1
Fmol/L ~ 1 μm ol/L, LOD are 0.7 fmol/L, than it is universal it has been reported that machine phosphorus insecticide biosensor detection（1
pmol/L ~29 nmol/L）Low one arrives an amount order of magnitude（Include electrochemica biological activity and non-electrochemical biochemical method）.
Embodiment 7
Selective determination of the Nafion/M-Cell/AC/GCE electrochemica biological sensor to different organophosphorus pesticides：
This experiment has chosen 6 kinds of organophosphorus pesticides, including paraoxon（Paraoxon）, Acetamiprid（Acetamiprid）, sevin
（Carbaryl）, 2-dichloroethylk dimethyl phosphate（Dibrom）, pyrrole worm miaow（Imidacloprid）And Nitenpyram（Nitenpyran）, use this hair
The novel electrochemical Biosensors Nafion/M-Cell/AC/GCE of bright building carries out quantitative inspection to different organophosphorus pesticides
It surveys.
As shown in fig. 7, the electrochemica biological sensor is only in PBS（pH 7.4）When middle detection, without any peak current
Response, when in PBS（pH 7.4）In be separately added into after the organophosphorus pesticide of same concentrations, which begins with different peaks
Current-responsive.Wherein, which has good selectivity to paraoxon, and response current is obviously high
In the response to other organophosphorus pesticides.Illustrate M-Cell catalyst to the better catalytic activity of paraoxon, and to organic phosphorus agriculture
Medicine paraoxon has good selectivity.
Claims (5)
1. a kind of electrochemica biological sensor for super sensitivity detection organophosphorus pesticide, which is characterized in that using layer by layer from group
The mode of dress successively modifies amino acid ion liquid-carbon mano-tube composite, the organophosphor hydrolytic enzyme of mineralising, polymeric membrane
At the electrode surface, to obtain the working electrode of the electrochemica biological sensor of super sensitivity detection organophosphorus pesticide, thus real
Hypersensitive is high under existing negative potential, selectively detects the purpose of organophosphorus pesticide.
2. a kind of electrochemica biological sensor for super sensitivity detection organophosphorus pesticide according to claim 1, special
Sign is that preparation step is as follows：
A. amino acid ion liquid and carbon nanotube are mixed in a certain proportion, ultrasound, by Non-covalent binding obtain amino acid from
Sub- liquid-carbon mano-tube composite；
B. amino acid ion liquid obtained in step A-carbon mano-tube composite hanging drop is added to the electrode pre-processed
On surface, room temperature is dried；
C. the organophosphor hydrolytic enzyme of mineralising is dripped on resulting electrode surface in stepb, it is dry at 4 DEG C；
D. one layer of polymeric membrane solution of drop coating on the electrode surface obtained in step C is made described and is used for oversoul after dry
The electrochemica biological sensor of quick detection organophosphorus pesticide.
3. a kind of electrochemica biological sensor for super sensitivity detection organophosphorus pesticide according to claim 1 or 2,
It is characterized in that, in the preparation step method：The polymeric membrane can be perfluoro sulfonic acid membrane（Nafion, 1 ~ 0.05 wt%）,
Poly-dopamine, polyphenyl ammonium formate, cyclodextrin polymer etc.；The amino acid ion liquid can by glycine, valine, lysine and
The natural amino acids such as tryptophan are synthesized by anion exchange method with bromination -1- butyl -3- methylimidazole and are made；The carbon nanometer
Pipe can be single-walled carbon nanotube, multi-walled carbon nanotube；The electrode is naked glassy carbon electrode, gold electrode or carbon paper；It is described
Mineralising organophosphor hydrolytic enzyme is to be made by phosphoric acid cobalt salt or phosphoric acid mantoquita in the cell surface mineralising for showing organophosphor hydrolytic enzyme
The biomineralization whole-cell catalyst obtained.
4. a kind of electrochemica biological sensor for super sensitivity detection organophosphorus pesticide according to claim 1 to 3,
It is characterized in that, it is as follows to detect organic phosphorus step using the sensor：When detection, using three-electrode system, to phosphate-buffered
Solution（PBS, pH 7.4）In be added dropwise various concentration organic phosphorus sample, cyclic voltammetry is carried out under the electrochemical window of -0.8 ~ 0 V
Measurement, qualitatively or quantitatively determines organic phosphorus according to the response of the reduction peak current at -0.5 ~ -0.2 V.
5. a kind of electrochemica biological sensor for super sensitivity detection organophosphorus pesticide described in -4 according to claim 1,
It is characterized in that：The three-electrode system includes the electrode that the organophosphor hydrolytic enzyme of mineralising is modified with described in claim 1-3
（Working electrode）, saturated calomel electrode or silver/silver chloride electrode（Reference electrode）, platinum electrode（To electrode）；It is described organic phosphorus
For paraoxon, Acetamiprid, sevin, 2-dichloroethylk dimethyl phosphate, pyrrole worm miaow and Nitenpyram.
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