Electrochemical immunosensor and preparation and application thereof

Technical Field

The present invention relates to electrochemical field, in particular to a kind of electrochemical immune sensor and its preparation and application.

Background Art

The toxin is reddish brown koji mold and penicillius which belongs to the genus Aspergillus several kind of fungi produce the secondary metabolite, with teratogenic, carcinogenic, mutagenic, amynologic toxicity and other, the International agency for research on cancer (IARC) as toxin reddish brown koji mold 2B types of carcinogen, is divided into reddish brown aspergillin A, B, compounds of similar structure such as C, wherein the toxicity of the toxin reddish brown koji mold A (OTA) to the maximum. OTA producing strain is distributed widely in nature, grain type, grape and wine, Chinese herbal medicine, bean product, beer, tea or other food and of various crops can be OTA pollution. In recent years with respect to the danger of toxin reddish brown koji mold has caused great concern to all countries of the world, many countries to its established maximum allowable amount.

High performance liquid chromatography in the recent OTA International standard system has been in an absolutely dominant position, as a quantitative detection method, laboratory and both at home and abroad by widespread use of the detection mechanism. Adopts the high performance liquid chromatography, or mass spectrometry for the detection of OTA, although with the result is accurate, high recovery rate, good precision, reproducibility, and the like, however the cost is high, the operation process is complex, a long time, is not able to meet the needs of the on-site detection.

Immunologically based principle with simple OTA enzyme-linked immunoradioassay detection, rapid, sensitive, and the like, in order to rapidly large-scale screening provides great help. However, due to the detection of this kind of method is complex substrate sample, a plurality of false positive detection result. Therefore should be positive sample instrument for the method.

Electrochemical immune sensor is the immunological techniques and electrochemical detection technique to combine a new analysis method, using signal converter (electrochemical workstation) molecules recognizer (probe) with the object to be measured of the physical or chemical change can be changed into an electrical signal. The technology with rapid, sensitive, high selectivity, simple operation, and the like, so the applied electrochemical immunosensor the detection in a sample of the OTA has a very important meaning.

Since the electrochemical immune sensor self-establishment, is mainly applied to the detection of the major disease markers. In recent years, the technology is gradually applied to the detection of the fungal toxin in the food, but less relative to the literature, especially in the food an electrochemical biosensor of the OTA the study and application of the detection technology is not yet mature.

The prior art in order to compensate for the deficiency of the OTA detection, aimed at the establishment of a novel electrochemical immunosensor for OTA rapid, sensitive detection.

Content of the invention

In view of the above defects in prior art, 1st aspect of the present invention to provide a detecting reddish brown koji mold toxin A (OTA) electrochemical immune sensor, comprises a working electrode, reference electrode and the counter electrode, the working electrode on the surface of the substrate electrode first modified carboxyl single wall carbon nanotube/chitosan (SWNTs-COOH/CS) composite material, and after-fixing reddish brown koji mold toxin A-bovine serum albumin conjugate the (OTA-BSA).

Preferably, the substrate electrode is selected from the group consisting of glassy carbon electrode.

Preferably, the carboxyl single wall carbon nanotube/chitosan composite material for (SWNTs-COOH/CS) (SWNTs-COOH) the carboxyl single wall carbon nanotube is dispersed in the prepared in chitosan (CS).

Preferably, the carboxyl single wall carbon nanotube/chitosan composite material in (SWNTs-COOH/CS), carboxyl single wall carbon nanotube and chitosan the weight ratio of 1:1-12:1; more preferably 1:1.

Preferably, on each of the working electrode, carboxyl single wall carbon nanotube/chitosan composite material (SWNTs-COOH/CS) carboxyl single wall carbon nanotube in the quality of 1-12ug, more preferably 2ug.

Preferably, on the working electrode, carboxyl single wall carbon nanotube/chitosan composite material in (SWNTs-COOH/CS) carboxyl single wall carbon nanotube and reddish brown koji mold toxin A-ox blood Qingqing protein conjugate (OTA-BSA) mass ratio is 6.67: 1-2400:1, more preferably 6.67: 1-80:1, most preferably 40:1. Furthermore, the electrochemical immune in the sensor reference electrode of the working electrode and the counter electrode a three-electrode system.

Preferably, the reference electrode is selected from saturated calomel electrode or electrode silver silver chloride a randomly (Ag/AgCl); more preferably, the reference electrode is a silver/silver chloride (Ag/AgCl) electrode.

Preferably, the electrode is a platinum wire electrode.

2nd aspect of the invention provides the aforementioned electrochemical immune sensor in the method for preparing the working electrode, the method is modified on the surface of the substrate electrode first carboxyl single wall carbon nanotube/chitosan composite material (SWNTs-COOH/CS), and then fixed reddish brown koji mold toxin A-ox blood Qingqing protein conjugate (OTA-BSA).

Preferably, the working electrode prepared in accordance with the following steps:

1) preparing carboxyl single wall carbon nanotube/chitosan composite material (SWNTs-COOH/CS):

The chitosan dissolved in the acetic acid solution, to prepare chitosan solution ; (SWNTs-COOH) carboxyl single wall carbon nanotube will be dissolved in the obtained chitosan solution, mixed suspension shall SWNTs-COOH/CS composite material;

2) functional modification and of working electrode:

(1) the substrate electrode surface treatment: the substrate is subjected to polishing process the surface of the electrode, so that the smooth surface;

(2) modified single-walled carbon nanotube/chitosan nano-composite material (SWNTs-COOH/CS): step 1) preparation of in SWNTs-COOH/CS the suspension drop spreads to step (1) in the surface of the substrate electrode of the, dry film, to obtain carboxyl single wall carbon nanotube/chitosan composite material modified electrode (SWNTs-COOH/CS/GCE);

(3) in the activation of the activation solution of the resulting SWNTs-COOH/CS/GCE SWNTs-COOH carboxyl groups;

(4) fixing reddish brown koji mold toxin A-ox blood Qingqing protein conjugate (OTA-BSA): of the after activation and SWNTs-COOH/CS/GCE OTA-BSA, incubation, forming OTA-BSA-SWNTs-COOH/CS/GCE, nonspecific adsorption site BSA closed, the working electrode is obtained.

Preferably, step 1) in, the molecular formula of the chitosan (C₆ H₁₁ NO₄)_N, unit of molecular weight 161.2.

Preferably, step 1) in, the concentration of the acetate solution 1%.

Preferably, step 1) in, the concentration of the chitosan solution is 0.5-6 mg/mL, more preferably 1 mg/mL.

Preferably, step 1) in, SWNTs-COOH/CS composite material in suspension, carboxyl single wall carbon nanotube and chitosan the weight ratio of 1:1-12:1; more preferably 1:1.

Preferably, step 1) in, SWNTs-COOH/CS composite material in suspension, SWNTs-COOH final concentration is 0.5-6.0 mg/ml; more preferably 1.0 mg/ml.

Preferably, step (1) in, the base electrode is selected from the group consisting of glassy carbon electrode.

Preferably, step (1) in, can be made of aluminum oxide powder to the substrate electrode is subjected to polishing process.

Preferably, step (2) is modified in the single-walled carbon nanotube/chitosan nano-composite material in (SWNTs-COOH/CS), carboxyl single wall carbon nanotube and chitosan the weight ratio of 1:1-12:1; more preferably 1:1.

Preferably, step (2) in, on each working electrode the modified carboxyl single wall carbon nanotube/chitosan composite material (SWNTs-COOH/CS) carboxyl single wall carbon nanotube in the quality of 1-12ug, more preferably 2ug.

Preferably, step (3) in, the activating liquid is mixed solution of EDC and NHS EDC/NHS. More preferably, the concentration of the EDC EDC/NHS in 10 mg/ml, NHS to a concentration of 4 mg/ml, EDC and NHS mass ratio is 5 mm:2 mm.

Preferably, step (4) in, toxin reddish brown koji mold A-ox blood Qingqing protein conjugate (OTA-BSA) the concentration is 0.5-15.0ug/ml; more preferably, the concentration of the OTA-BSA 5.0ug/ml.

Preferably, step (2) is modified in the single-walled carbon nanotube/chitosan nano-composite material in (SWNTs-COOH/CS) carboxyl single wall carbon nanotube and step (4) fixed in the toxin reddish brown koji mold A-ox blood Qingqing protein conjugate (OTA-BSA) mass ratio is 6.67: 1-2400:1, more preferably 6.67: 1-80:1, most preferably 40:1.

3rd aspect of the invention provides a detection system for the detection of toxin reddish brown koji mold A, the 1st, including the present invention the electrochemical immunosensor, OTA monoclonal antibody, two anti-and immune reaction buffer system.

Preferably, the secondary antibody is alkaline phosphatase labeled secondary antibody.

Furthermore, alkaline phosphatase-labeled selectable ma Kangshu , accuracy, arbitrary source of goat anti-mouse secondary antibody of anti-plague, more preferably the alkaline phosphatase-labeled mouse ma Kang IgG (H+L).

Preferably, the immune reaction buffer system containing α-NP of diethanolamine (DEA) buffer solution.

4th aspect of the invention provides a method of detecting reddish brown koji mold toxin A, as by the electrochemical immune sensor or detection system to sample in the detection of toxin reddish brown koji mold A, in particular the method comprises the following steps:

(A) in the aforesaid construction of the working electrode of the electrochemical immune sensor at the same time a certain amount of sample solution and OTA to monoclonal antibody solution;

(B) adding proper amount of alkaline phosphatase-labeled secondary antibodies, incubation, the OTA monoclonal antibody by selectively binding to the electrode surface;

(C) the working electrode is placed in the immune reaction buffer system, and the working electrode, reference electrode and the electrode is properly connected to the electrochemical workstation, using differential pulse voltammetry (DPV) is measured.

Preferably, step (a) in, OTA the concentration of the monoclonal antibody solution 1.25-20.0ug/ml; more preferably 5.0ug/ml.

Preferably, step (b) in, the alkaline phosphatase-labeled alkaline phosphatase-labeled secondary antibody to mouse ma Kang IgG (H+L).

More preferably, the alkaline phosphatase-labeled mouse ma Kang IgG (H+L) for the dilution ratio of the: 1:50-1:400 ; the best is 1:200.

Preferably, step (c) in, the reference electrode is selected from saturated calomel electrode or electrode silver silver chloride a randomly (Ag/AgCl); more preferably, the reference electrode is a silver/silver chloride (Ag/AgCl) electrode.

Preferably, step (c) in, wherein the electrode is a platinum wire electrode.

Preferably, step (c) in, the immune reaction buffer system containing α-NP of diethanolamine (DEA) buffer solution.

More preferably, the immune reaction buffer system α-NP is in a concentration of 0.25-1.5 mg/ml; preferably 0.75 mg/mL.

5th aspect of the invention provides the aforementioned electrochemical immune sensor or a detection system in the detection of the use of toxin reddish brown koji mold A.

The beneficial results of this invention are:

(1) this invention has developed a single walled carbon nanotube-based material/chitosan composite fixed antigen (OTA-BSA) (SWNTs-COOH/CS) indirect competitive electrochemical immunosensor, OTA can be used for sensitive detection. First of all produced good stability of the single wall carbon nanotube/chitosan composite material to the surface of the modified (SWNTs-COOH/CS) glassy carbon electrode, then will be through the adsorption action reddish brown koji mold toxin-bovine serum albumin crosslinked product (OTA-BSA) fixed to the modified electrode surface. When detecting, with a certain amount of a sample is applied to the OTA (anti-OTA) monoclonal antibody, the OTA in a sample free of a certain amount of working electrode surface immobilization of monoclonal antibody competition binding coupling OTA-BSA, after the competition reaction, adding alkaline phosphatase-labeled secondary antibody, a secondary antibody specific to a surface of the electrode an anti-reaction of the captured, and then alkaline phosphatase catalytic substrate α-naphthyl phosphate hydrolysis, generating an electrical signal on the surface of the electrode. The electrochemical immune sensor for the linear range of the 0.01 [...] 100ng/ml, linear equation for Y=6.3155E^-6 -2.89458E^-6 X, detection limit 4pg/ml, the linear correlation coefficient 0.99984.

(2) as is well known, only the biocompatible OTA-BSA in the environment of its activity can be maintained. The surface of the electrode is directly fixed to the concept of are usually the cause of antibody to change, thus leading to loss of biological activity. Therefore, how to effectively handle OTA-BSA is fixed on the electrode of the present invention the electrochemical immune preparation of sensor a key step. Because the modification of the surface of the working electrode in SWNTs-COOH/CS composite material has a very large specific surface area and good biocompatibility, so can firmly adsorbed OTA-BSA, when detecting, can effectively increase the loading capacity of the antibody, and maintain the biological activity of the antibody.

Furthermore, SWNTs-COOH/CS composite material also has good electronic conduction, and sample solution can be increased between the GCE electron transfer, increase the conductivity, significantly increase the measuring sensitivity of the OTA, very good analysis to reduce the detection lower limit of the OTA.

(3) at the same time, signal amplification is that the impact of the detection sensitivity another important factor. The invention adopted on the basis of the carbon nanotube and alkaline phosphatase double amplification strategy, realize greatly the signal amplification. In the present invention, alkaline phosphatase is the introduction of the surface of the electrode (AP) catalytic α-naphthyl phosphate hydrolysis to generate an electrochemical signal (α-NP), enhances the electrochemical response signal, can be in the sample concentration of the OTA accurate quantitative.

(4) to sum up, the invention has succeeded in constructing the reddish brown koji mold toxin can be used for detecting immune sensor and the electrochemical A (OTA) a detecting system, application of the sensor of the present invention, the determination and display of the OTA high sensitivity, stability, repeatability is good capacity. Compared with the prior art, low cost of the sensor of the present invention, it is simple and convenient to operate, the detection cycle is short, the specificity is good, and the false positive rate of false negative rate. The measurement of the actual sample is, the value is expected to become a practical application of the sensor.

Description of drawings

Figure 1 is the embodiment of the invention 1 for suspension of the composite material SWNTs-COOH/CS S-300N the results of scanning electron microscope observation.

Figure 2 is the embodiment of the invention 1 one step each of the different modified electrode 5 mm potassium ferricyanide solution of the square wave of the volt-ampere response curve, wherein

For a bare GCE;

For b SWNTs-COOH/CS/GCE;

For c OTA-BSA-SWNTs-COOH/CS/GCE;

For d anti-OTA/OTA-BSA-SWNTs-COOH/CS/GCE;

For e AP-anti-antibody/anti-OTA/OTA-BSA-SWNTs-COOH/CS/GCE.

Figure 3 is the embodiment of the invention 1 the different modified electrode of each step corresponding to the cyclic voltammetry response curve of, wherein

For a bare GCE;

For b SWNTs-COOH/CS/GCE;

For c OTA-BSA-SWNTs-COOH/CS/GCE;

For d anti-OTA/OTA-BSA-SWNTs-COOH/CS/GCE;

For e AP-anti-antibody/anti-OTA/OTA-BSA-SWNTs-COOH/CS/GCE.

Figure 4 is the current response curve of each sensor in the comparative test, wherein the curve a, b, c for the carbon electrode bare Bolivia respectively, modified SWNTs-COOH/CS/GCE glassy carbon electrode, the glassy carbon electrode modified SWNTs-COOH/GCE after constructing the sensor on the basis of the response curve of the current signal.

Figure 5 is a different concentration of the construction of the OTA-BSA electrochemical immune sensor differential pulse voltammetry scan results.

Figure 6 is a different concentration of the construction of the anti-OTA electrochemical immune sensor differential pulse voltammetry scan results.

Figure 7 for different dilution ratio of the construction of the AP-anti-antibody electrochemical immune sensor differential pulse voltammetry scan results.

Figure 8 for different concentration of the construction of the α-NP electrochemical immune sensor differential pulse voltammetry scan results.

Figure 9 is OTA incubating in standard substance solution, DPV ip method for the recording current value graph of electrochemical immune sensor of the present invention in five different concentrations (100ng/ml, 10ng/ml, 1ng/ml, 0 . 1ng/ml, 0 . 01ng/ml).

Figure 10 of the present invention the electrochemical immune sensor to detect when the standard curve of the OTA.

Figure 11 is the invention preparation of immune specificity of the analysis of the experiment results.

Mode of execution

Through the particular examples illustrate embodiments of the present invention, the technical personnel in this field revealed by the description of the content of the present invention easily understand other advantages and effects. The invention can also be through the specific implementation in addition different manner or application, the various in this specification details also can be based on different views and applications, without departing from the spirit of this invention various modification or change.

Need-to-know, the following embodiment does not specifically the process equipment or device is the conventional in the field of equipment or device; and the scope of all pressure values are mean absolute pressure.

In addition should be understood, that in this invention the one or more method steps does not exclude the front and back steps in said combination there can also be other method steps or in these explicit reference to the steps of other methods can also be inserted in between the steps, unless otherwise indicated; also should be understood, in the present invention refer to one or more of the combination of equipment/device connection relationship between on said combination does not exclude and equipment/device there can also be other equipment/device or in those explicitly mentioned in the between two equipment/device can also be inserted in other equipment/device, unless otherwise indicated. And, unless otherwise indicated, the number of the steps of the method only the authentication steps of the various methods to facilitate tool, not to limit the arrangement order of steps of the method or to define the scope of this invention can be implemented, the change or adjustment of the relative relations between the, no substantial change in the case of technical content, when the also regarded as the scope of this invention can be implemented.

Example 1 preparing electrochemical immune sensor

1. Materials and methods

1.1 material

Toxin reddish brown koji mold A-ox blood Qingqing protein conjugate (OTA-BSA), OTA (anti-OTA) monoclonal antibody from the Beijing huaan macco biotechnology Company limited, alkaline phosphatase-labeled mouse ma Kang laboratory Vector IgG (H+L) bought from the United States, OTA standard, 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide (EDC), N-hydroxy succinimide (NHS), α-naphthyl phosphate (α-NP), chitosan (CS) are purchased from the United States Company Sigma-Aldrich, carboxyl single wall carbon nanotube from shenzhen nanometer port limited, methanol, acetone and other reagents are purchased from chongqing cyclopentadiene industry chemical reagent limited.

1.2 detection instrument

CHI660D-type electrochemical work station for Shanghai [...] instrument Company's products.

1.3 detection principle

In the glassy carbon electrode is modified by directly coating the surface with a layer of carboxyl single wall carbon nanotube/chitosan (SWNTs-COOH/CS) composite material, and the use of the carboxyl groups of the activating EDC/NHS SWNTs-COOH. The amino terminal through a protein OTA-BSA single wall carbon nanotubes with carboxyl SWNTs-COOH the activation of the carboxyl reaction connection, and at the same time through the antigen-antibody reaction with OTA monomer according to the OTA (anti-OTA) monoclonal antibody competitive adsorption, then adding mouse ma Kang IgG (H+L) alkaline phosphatase-labeled secondary antibody, alkaline phosphatase with the substrate through α-naphthyl phosphate (α-NP) electrochemical signal produced in the reaction to draw the standard curve and measured sample level OTA.

2. Preparation of working electrode

2.1 Carboxy single wall carbon nanotube/chitosan (SWNTs-COOH/CS) preparation of composite material

The chitosan powder is dissolved in 1% acetic acid solution, preparation concentration is 1 mg/ml of the chitosan solution;

The chitosan solution is added into 5.0ml1mg/ml 5.0 mg Carboxy single wall carbon nano-tube ultrasonic dispersion (SWNTs-COOH) 2h shall be uniform and stable dispersion of the composite material mixed suspension SWNTs-COOH/CS, wherein SWNTs-COOH the final concentration of 1 mg/ml. Before in use, for the 4 [...] low-temperature preservation.

2.2 functional modification and of working electrode

(1) glassy carbon electrode surface processing: glassy carbon electrode (GCE) before using to the mirror polishing aluminum oxide powder, successively respectively for UPW and volume ratio of 1:1 in the acetone and nitric acid ultrasonic a few minutes. The electrode again subsequently ultrasonic ultra-pure water for a few minutes, and then flushed with deionized water, dried at room temperature.

(2) the 2 the pre-prepared   L of suspension SWNTs-COOH/CS (1 mg   ml^-1) carefully clean the processing the drop spreads surface of the glassy carbon electrode. Furthermore, the modified at room temperature of the glassy carbon electrode sleepovers drying to obtain carboxyl single wall carbon nanotube/chitosan composite material modified electrode (SWNTs-COOH/CS/GCE).

(3) the prepared SWNTs-COOH/CS/GCE phosphate buffer solution (PBS, 0.01mol/L, pH7 . 4) after the cleaning, immersed in the fresh configuration of the activation solution EDC/NHS (EDC and NHS concentration ratio is, 5 mm:2 mm) in, for the 37 incubated [...] 1h, activation of the carboxyl group on SWNTs-COOH.

(4) activation of the PBS buffer solution for SWNTs-COOH/CS/GCE thorough cleaning. The 10 the the   L OTA-BSA (5     g   ml^-1) surface of the electrode melted immediately, 37 the incubated [...] 1.5h. Completely after the incubation, the preparation of PBS for electrode cleaning. At room temperature in 10 the L1% bovine serum albumin (BSA) is not the surface of the electrode closed in the active site of the reaction. Finally, electrode thorough cleaning to PBS, and shall OTA-BSA-SWNTs/CS/GCE 4 the the [...] for use in the refrigerator.

3. The use of electrochemical immune sensor

(1) will be 5 the PBS   L (pH7.4) for dilution to a certain concentration of OTA (0-500ng   ml^-1) and 5     L10     g   ml^-1 (anti-OTA) of uniformly mixed monoclonal antibody OTA, dropping in modifying the surface of the electrode OTA-BSA OTA-BSA-SWNTs/CS/GCE, the 37 [...] incubate 90 min, shall be anti-OTA-OTA-BSA-SWNTs/CS/GCE. In the reaction process, fixed on the electrode with free OTA-BSA in the mixed solution of the OTA anti-OTA common competition derived.

(2) then using PBS buffer solution (pH7.4) cleaning the electrode, the 10 the L1: 200 dilution of mouse ma Kang IgG alkaline phosphatase-labeled secondary antibody in (H+L), the 37 [...] incubate 90 min, PBS buffer solution (pH7.4) after washing, be AP-anti-antibody/anti-OTA/OTA-BSA-SWNTs-COOH/CS/GCE.

(3) in order to present the prepared containing 0.75 mg   ml^-1 α-NP of diethanolamine (DEA) buffer solution as immune reaction buffer system, the working electrode is placed wherein Ag/AgCl electrode as a reference electrode, the platinum wire electrode as the counter electrode, under the room temperature, differential pulse voltammetry for measuring (DPV).

4. Comparison test

Under the same experimental conditions, to the carbon electrode and the glassy carbon electrode bare Bolivia the surface only carboxyl single wall carbon nanotube GCE (SWNTs-COOH) the resulting working electrode, the construction of the SWNTs-COOH/GCE electrochemical immunosensor as control.

Embodiment 2 electrochemical immunosensor for characterization and testing

1. The characterization of SWNTs-COOH/CS composite material film

For the implementation of an example 1 of the composite material in suspension S-300N SWNTs-COOH/CS for scanning scanning electron microscope observation, as shown in Figure 1a and 1b the display, the nano-composite material is a carbon tube in the tubular, is uniformly dispersed in the solution.

Dispensing surface of the glassy carbon electrode SWNTs-COOH/CS, after drying a film at room temperature, observed by naked eyes in the electrode: SWNTs-COOH/CS/GCE can see the surface of a layer of even film-like material.

2. Different modified electrode electrochemical characterization

As shown in Figure 2, is different in every step of the modified electrode 5 mm potassium ferricyanide solution of the square wave of the volt-ampere response curve:

For a bare GCE;

For b SWNTs-COOH/CS/GCE;

For c OTA-BSA-SWNTs-COOH/CS/GCE;

For d anti-OTA/OTA-BSA-SWNTs-COOH/CS/GCE;

For e AP-anti-antibody/anti-OTA/OTA-BSA-SWNTs-COOH/CS/GCE.

After surface modification GCE SWNTs-COOH/CS when bare , peak current obviously increases (Figure 2-curve b), the result can be promoted by thin film SWNTs-COOH/CS electronic in the solution transfer on the surface of the electrode. When dropping OTA-BSA after activation of the surface of the electrode, the phenomenon of the steric hindrance of the protein and insulating effect, an obstacle to the electronic transfer, the reducing of the peak current presents the obvious (Figure 2-curve c). When the electrode through the further modified after incubation anti-OTA, increased resistance to further reduce the peak current (Figure 2-curve d), shows that the prepared electrochemical immune sensor working electrode to OTA-BSA anti-OTA in a successful identification and binding action. When a certain concentration of AP-anti-antibody is dripped into the incubation after modifying the surface of an electrode, a further reduced peak current (Figure 2-curve e), indicating successful AP-anti-antibody electrochemical immune sensor the introduction of the working electrode.

Figure 3 for each step corresponding to the different modified electrode cyclic voltammetry response curve,

For a bare GCE;

For b SWNTs-COOH/CS/GCE;

For c OTA-BSA-SWNTs-COOH/CS/GCE;

For d anti-OTA/OTA-BSA-SWNTs-COOH/CS/GCE;

For e AP-anti-antibody/anti-OTA/OTA-BSA-SWNTs-COOH/CS/GCE.

Result and Figure 2 presents a good consistency, we have succeeded in effectively demonstrate the electrode to the modification of each step.

3. Contrast experimental:

As shown in Figure 4, curve a, b, c for the carbon electrode bare Bolivia respectively, modified SWNTs-COOH/CS/GCE glassy carbon electrode, the glassy carbon electrode modified SWNTs-COOH/GCE after constructing the sensor on the basis of the response curve of the current signal. Curve a shows that protein can not be directly and effectively fixed on the surface of the carbon electrode bare Bolivia , so responding to the electric current is very low; curve c shows that only carbon nanotube after into a film on the surface of the electrode, through its carboxyl terminal and can form stable binding protein amino, the antigen is fixed on the electrode surface, so that the current response the value compared to uses bare glassy carbon electrode construction of the sensor high, but as shown, is constructed using single SWNTs-COOH modified electrode sensor has a shortcoming, that is, its response to the baseline of the electric current is very high, and unstable; curve b use the modified electrode after SWNTs-COOH/CS composite material constructing sensor, not only can effectively fix detecting antigen, but also can effectively reduce and stabilize the baseline of the current response of the sensor, and the composite material in response to the modified electrode does not affect the magnitude of electric current. Therefore, the modified SWNTs-COOH/CS construct electrode as a working electrode used for measuring electrochemical OTA with incomparable advantages immune sensor.

Example 3 electrochemical immune sensor and the optimization of the conditions of use thereof

In the course of experiment we would also like to several important conditions SWNTs-COOH SWNTs-COOH/CS composite suspension in the final concentration, the concentration of OTA-BSA, the concentration of anti-OTA, the dilution ratio of the AP-anti-antibody, α-NP the several measuring the concentration of the further optimization of conditions. To each of the conditions are from low concentration to high concentration respectively selected to carry out a series of experiments the five points.

1. To study SWNTs-COOH SWNTs-COOH/CS composite material of the suspension in the final concentration of the influence of electrochemical immune sensor, the experiment adopts a different concentrations of (0.5, 1.0, 2.0, 4.0, 6.0 mg/ml) compound suspension SWNTs-COOH/CS constructing electrochemical immune sensor. The result shows that, SWNTs-COOH final concentration is too low, its membrane difference , not very good adsorption OTA-BSA and fixed antibody to, and if the film of dispersed in less SWNTs-COOH, the role of promoting electron transfer is relatively weak; if the final concentration of SWNTs-COOH too large, the film thickness is increased, will also impede electron transfer. The experiment found that, for the final concentration of SWNTs-COOH 0.5-6.0 mg/ml of suspension can realize composite SWNTs-COOH/CS construction meets the requirements of the sensor, wherein the final concentration to SWNTs-COOH 1.0 mg/ml suspension of compound SWNTs-COOH/CS is the best.

2. OTA-BSA to study the concentration of the influence of electrochemical immune sensor, the experiment adopts a different concentrations of (0.5, 1.0, 5.0, 10.0, 15 . 0ug/ml) constructing OTA-BSA immune electrochemical immune sensor, then differential pulse voltammetry scan. As shown in Figure 5 can be seen, in response to the increase of the density of the current with the increased OTA-BSA, the concentration reaches OTA-BSA 5.0ug/ml the subsequent, further increases the concentration of OTA-BSA, the response current is increased is not obvious, note 5.0ug/ml has reached optimum concentration of OTA-BSA.

3. By the same token, the size of the inspection anti-OTA to the use concentration of the influence of electrochemical immune sensor, the experiment adopts a different concentrations of (1.25, 2.5, 5.0, 10.0, 20 . 0ug/ml) anti-OTA, then differential pulse voltammetry scan. As shown in Figure 6 can be seen, the optimum concentration anti-OTA 5.0ug/ml.

4. By the same token, the size of the inspection AP-anti-antibody to the use concentration of the influence of electrochemical immune sensor, the experiment adopting different of the diluting ratio (1:50, 1:100, 1:200, 1:300, 1 : 400V/V) AP-anti-antibody, then differential pulse voltammetry scan. As shown in Figure 7 can be seen, the best AP-anti-antibody the diluting ratio 1 : 200(V/V).

5. By the same token, the size of the inspection α-NP concentration of the impact of the use of immune sensor, the experiment adopts the containing different concentration α-NP (0.25, 0.5, 0.75, 1, 1.5 mg   ml^-1) immune reaction buffer system, then differential pulse voltammetry scan. As shown in Figure 8 can be seen, the optimum concentration of α-NP 0.75 mg   ml^-1.

Example 4 preparation of electrochemical immune chemical sensor performance analysis

In order to assess the performance of the electrochemical immune sensor, PBS (pH7.4) to different concentrations of OTA with a standard for analysis. Specific, under the optimal experimental conditions, (1) will be 5 the PBS   L (pH7.4) for dilution to five different concentrations of OTA (200ng/ml, 20ng/ml, 2ng/ml, 0 . 2ng/ml, 0 . 02ng/ml) and 5     L10     g   ml^-1 (anti-OTA) of uniformly mixed monoclonal antibody OTA, dropping in modifying the surface of the electrode OTA-BSA OTA-BSA-SWNTs/CS/GCE, the 37 [...] incubate 90 min. In the reaction process, fixed on the electrode with free OTA-BSA in the mixed solution of the OTA anti-OTA common competition derived. (2) then using PBS buffer solution (pH7.4) cleaning the electrode, the 10 the L1: 200 dilution of mouse ma Kang IgG alkaline phosphatase-labeled secondary antibody in (H+L), the 37 [...] incubate 90 min, PBS buffer solution (pH7.4) cleaning. DPV ip method for the recording current value (as shown in Figure 9), draw the standard curve, as shown in Figure 10. The experimental results show, when the concentration of OTA 10pg   ml^-1 to 100ng   ml^-1 the between, OTA of the peak current is linearly related to the logarithm of the concentration, the regression equation: Y=6.3155E^-6 -2.89458E^-6 X. The correlation coefficient of 0.999. When only the sensor containing 5ug   ml^-1 of a standard substance that does not contain OTA anti-OTA compete continuous scanning in the blank solution 10 times, according to the blank signal plus 3 times the signal value corresponding to the standard deviation of estimated detection limit, calculating 4pg   ml^-1.

Example 5 preparation of electrochemical immune sensor specificity analysis

Electrochemical immunological specificity of the sensor, without separating the analysis of biological markers in biological sample with an important role when, mainly depends on the specificity of the antibody. Is our use of the OTA monoclonal antibody. In order to evaluate the specificity of the immune sensor, in detecting OTA other may occur when the three fungal toxin aflatoxin B₁ (AFB₁), zearalenon (ZEA), fumonisins B₁ (FB₁) carrying out the detection. Specific, the aflatoxin B₁ (AFB₁), zearalenon (ZEA), fumonisins B₁ (FB₁) all 1     g   ml^-1 and 100ng   ml^-1 OTA are respectively of the same embodiment 4 the construction of the measured electrochemical immune sensor. As a result as shown in Figure 10, the other three of the mycotoxin response current DPV close to the blank sample (containing no OTA), the OTA DPV, however, a marked drop in the response current. These results illustrate the preparation of electrochemical immune sensor can effectively distinguish different types of fungal toxin, with good specificity.

Example 6 preparation of electrochemical immune the stability of the sensor and the reproducibility analysis

Under the optimal experimental conditions, preparing immune sensor. For immune sensor to the different batches 100ng   ml^-1 and 100pg   ml^-1 of the OTA 3 parallel determination, coefficient of variation are 3.56% and 5.20%. Shows that, of the present invention electrochemical immune sensor has good stability and repeatability.

The above-mentioned embodiment example characteristic shows that the principle of this invention and its efficacy, but is not used for limiting of the present invention. Any person familiar with this technology can be subject to the the spirit and scope of the invention, the above-mentioned embodiment to modify or change. Therefore, in all the belonging to the technical field in the holders usually not deviated from the spirit of the invention and the technical thought all accomplished by the equivalent modification or change, the present invention should still be covered by the claim.