CN102539499A - Composite nano material modified screen print electrode and method for detecting Fumonisin B1 - Google Patents
Composite nano material modified screen print electrode and method for detecting Fumonisin B1 Download PDFInfo
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- CN102539499A CN102539499A CN201210002405XA CN201210002405A CN102539499A CN 102539499 A CN102539499 A CN 102539499A CN 201210002405X A CN201210002405X A CN 201210002405XA CN 201210002405 A CN201210002405 A CN 201210002405A CN 102539499 A CN102539499 A CN 102539499A
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Abstract
The invention discloses a composite nano material modified screen print electrode and a method for detecting Fumonisin B1. A working electrode of the screen print electrode is coated with a multi-walled carbon nano tube-colloid gold-chitosan mixture film. The preparation process includes the following steps; activating the multi-walled carbon nano tube; preparing the colloid gold; uniformly mixing the multi-walled carbon nano tube and the colloid gold through the chitosan; coating the mixture on the surface of the working electrode; and completing the preparation after the working electrode is dried at 37 DEG C and the film is formed. The method for detecting Fumonisin B1 provided by the invention is realized in the way that if FB1 exists in a to-be-detected sample, the FB1 competes with FB1 antibodies in the combination with FB1-OVA, so that the antibody amount combined with the FB1-OVA on the electrode is reduced, the amount of HRP-goat-anti-mouse second antibodies on the electrode is reduced, and the current variation value delta I is influenced; and then the content of FB1 in the to-be-detected sample of grains can be judged as per the standard curve drawn through an FB1 standard product. According to the invention, the detection object is single, the pertinence is strong, the accuracy is high, and the sensitivity is strong.
Description
Technical field
The invention belongs to the detection technique field, be specifically related to a kind of screen printing electrode of modifying with composite nano materials (CNT and collaurum) and the method that detects fumonisin B1.
Background technology
(Fumonisin B1 is the fusarium moniliforme secondary metabolite that breeding is produced under certain humidity and temperature conditions FB1) to fumonisin B1, in worldwide, distributes extensively.1988, Gelderblom etc. isolated fumonisin first from the fusarium moniliforme nutrient solution.Fumonisin can pollute corn and goods thereof, and in some products that with cereal are raw material like noodles, beer, flavouring, even in asparagus, also detected fumonisin.It is reported, fumonisin and horse white matter of brain malacosis, the pulmonary edema syndrome of pig, diseases such as rat liver cancer are relevant.Except that above-mentioned disease, find that in the area, Transkei and the area studies of Chinese Lin County in South Africa the corn product that edible fumonisin pollutes is relevant with human cancer of the esophagus.The toxin that fusarium moniliforme produces by international cancer research association (International Agency for Research on Cancer IARC) is divided into the 2B class--possible human carcinogenic substance.After the accession to WTO; The quantum of international trade of agricultural product and relevant food increases day by day; Also increasingly high to the requirement of the biological safety of importing and exporting product thereupon; For the smooth listing that guarantees this series products and eater's health, departments such as entry and exit quarantine, customs, manufacturing enterprise, superintendent office press for a kind of special, quick, easy fumonisin detection method.
Electrochemical sensor is the device that the physical quantity of impression, information such as chemistry amount etc. is converted according to certain rules to the electric signal of being convenient to measure and transmitting.It as recognition component, produces signal that be interrupted or continuous through the physics and chemistry transducer by immobilized biological sensitive materials, is amplified or directly collection and treatment by receiving device again.Because signal intensity and analyte concentration that transducer produces are proportional, so electrochemical sensor to a certain extent can the accurate quantification measured object.
Along with the development of electrochemical detection method, thereby utilize nano material to improve electron transfer speed, amplification detection signal, improve detection sensitivity, become the key areas of electrochemical sensor as the electrode modification thing.Wherein, multi-walled carbon nano-tubes is owing to having large surface area, more can strengthening electron transfer, widespread use on Study of Electrochemical Sensors for electron transfer provides better channels, opening structure.And the screen printing electrode common glass-carbon electrode of comparing, except having good galvanochemistry performance, also have following advantage: 1. three-electrode system is incorporated on the less substrate of volume; 2. jettisonable need not grind once more; 3. can use in a large number, be beneficial to extensive on-the-spot the detection; 4. can modify arbitrarily, and preserve the long period.
Several different methods has been set up in detection about fumonisin both at home and abroad.The method that detects fumonisin at present is mainly high performance liquid chromatography (HPLC) and enzyme linked immunosorbent assay (ELISA).Yet; Because fumonisin itself had not both had special ultraviolet absorption group; There is not simultaneously fluorescent characteristic yet; But fumonisin can form the derivant with fluorescence with some substance reaction under certain condition, so the accuracy and the sensitivity of the selection of fluorescence derivating agent and deriving method and HPLC detection fumonisin have substantial connection.This method need be carried out strict pre-service to test sample in addition, also needs expensive instruments such as high performance liquid chromatograph, requires to have the operating personnel of specialty simultaneously, is unfavorable for on-the-spot conventional sense use.Though and the ELISA method is simple than HPLC, can mass detection, sensitivity is limited, and should not carry out the scene and detect.This method uses the instrument volume little, in light weight, is easy to carry, and is suitable for on-the-spot the detection, has the detection sensitive characteristics simultaneously.
Summary of the invention
The objective of the invention is to overcome the deficiency that above-mentioned prior art exists, provide a kind of composite nano materials to modify the method for screen printing electrode and detection fumonisin B1.This method is used the modification system of multi-walled carbon nano-tubes/collaurum/chitosan compound as electrode; Utilized multi-walled carbon nano-tubes owing to have large surface area; Thereby can provide characteristics that better channels, opening structure can strengthen electron transfer and collaurum owing to have sphere structure for electron transfer; The characteristics that strengthen electron transfer are arranged after on attached to multi-walled carbon nano-tubes, realized effective amplification detection signal, thereby improved the sensitivity that detects fumonisin B1.
The present invention realizes through following technical scheme:
The present invention relates to a kind of composite Nano and modify screen printing electrode, the perform region of the working electrode of said screen printing electrode is coated with multi-walled carbon nano-tubes-collaurum-chitosan compound rete.
Preferably, said collaurum is 16～18nm.
The invention still further relates to a kind of method that above-mentioned composite Nano is modified screen printing electrode for preparing, comprise the steps:
Preferably, the activation method of multi-walled carbon nano-tubes described in the step 1 is specially: it is 1: 3 HNO that the 5mg multi-walled carbon nano-tubes is dissolved in the 15ml volume ratio
3And H
2SO
4In the activated solution that mixes and get, behind the ultrasonic 30min, the centrifugal 5min of 9000rpm is behind the removal supernatant; Add the ultrapure water washing, the centrifugal 5min of 9000rpm removes supernatant, merges residue; 60 ℃ of oven for drying add 5ml water, are mixed with 1mg/ml solution, and 4 ℃ of storages are subsequent use.
Preferably, the preparation method of collaurum described in the step 1 is specially: with the HAuCl of 100ml
4Solution is heated to boiling, adds the citric acid three sodium solution of 1.2ml afterwards, boils 7～10min, adds tri-distilled water at last to 100ml, promptly gets colloidal gold solution; To account for the number percent of overall solution volume be 1% to the trisodium citrate quality in the said citric acid three sodium solution, said HAuCl
4HAuCl in the solution
4The number percent that quality accounts for overall solution volume is 0.01%.
Preferably, shitosan described in the step 1 was specially with mixing of multi-walled carbon nano-tubes and collaurum: with volume ratio is that 1: 5: 5 multi-walled carbon nano-tubes solution, colloidal gold solution and chitosan solution mixes ultrasonic mixing; To account for the number percent of overall solution volume be 2% to chitosan mass in the said chitosan solution.
The invention still further relates to the above-mentioned composite Nano of a kind of usefulness and modify the method that screen printing electrode detects fumonisin B1, comprise the steps:
Step 5 is used 0.01M PBST washing working electrode, dries, and the sheep anti mouse two that drips 6 μ lHRP marks is anti-, places 30min for 37 ℃;
Step 6 is used 0.01M PBST washing working electrode, and is dried, and screen printing electrode is put into the damping fluid that 10ml pH is 7.4 0.01M PBS, measures background current, is recorded as I
0
Step 7 is taken out screen printing electrode, uses 0.01M PBST washing working electrode; And dry, put into 10mlpH and be the damping fluid of 7.4 0.01M PBS, add hydrogen peroxide and quinhydrones again; Make its concentration be respectively 2mM and 0.1mM, behind the agitating solution 20min, stop to stir; Measure electric current, be recorded as I, the electric current changing value
ΔI=I-I
0
Step 8; Use Excel software, with FB1 standard items variable concentrations and corresponding
ΔI drawing standard curve, testing sample is corresponding
ΔI brings in the typical curve, obtains FB1 concentration, and multiply by dilution gfactor, is FB1 content in the testing sample.
Preferably, in the step 2, said centrifugal be 3000 to leave the heart 15 minutes.
Preferably, in the step 2, the volume ratio of methyl alcohol and water is 80: 20 in the said methanol solution.
Compared with prior art, the present invention has following beneficial effect: detected object of the present invention is single and with strong points, and accuracy rate is high, and sensitivity is strong, and sensitivity is greater than enzyme linked immunosorbent assay commonly used; Can satisfy grain store inspection departments such as marketing organization, entry and exit, customs fast, the requirement of the FB1 content of toxins that judges rightly, and be convenient to that basic unit promotes and utilization.
Description of drawings
Fig. 1 is an embodiment of the invention synoptic diagram;
Wherein, 1 is contrast electrode, and 2 is working electrode, and 3 is to electrode, and 4 is collaurum, and 5 is multi-walled carbon nano-tubes, and 6 is OVA-FB1, and 7 is anti-FB1 monoclonal antibody, and 8 is that sheep anti mouse HRP mark two is anti-.
Embodiment
Below in conjunction with accompanying drawing embodiments of the invention are elaborated: present embodiment provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment being to implement under the prerequisite with technical scheme of the present invention.Among the following embodiment, the experimental technique of unreceipted actual conditions, usually according to normal condition, or the condition of advising according to manufacturer.
Embodiment
The synoptic diagram of the embodiment of the invention is as shown in Figure 1, and said screen printing electrode comprises contrast electrode 1, working electrode 2, to electrode 3; The perform region of said working electrode 2 is coated with multi-walled carbon nano-tubes 5-collaurum 4-chitosan compound rete; During with the fumonisin B1 (FB1) in the said screen printing electrode test sample; At first OVA-FB16 is dripped on the working electrode that is coated with mixture film; The mixed solution that adds anti-FB1 monoclonal antibody 7 and testing sample extract after the drying again; The sheep anti mouse two that adds the HRP mark after the drying again resists 8, adds substrate solution H at last
20
2And HQ.
Prepare composite nano materials of the present invention and modify screen printing electrode and following with its method concrete steps that detect fumonisin B1:
The 5mg CNT is dissolved in 15ml mixed acid solution (HNO
3With H
2SO
4Volume ratio is 1: 3) in, ultrasonic mixing 30 minutes.With the solution packing, place hydro-extractor, 9000 rev/mins centrifugal 5 minutes, remove supernatant after, more once with the ultrapure water washing, be specially 9000 rev/mins centrifugal 5 minutes.After removing supernatant, merge residue, place 60 ℃ of oven for drying.Add 5ml water at last, 4 ℃ store for future use.
0.001% (W/V) HAuCl4 solution with 100ml is heated to boiling earlier, adds 1% (W/V) trisodium citrate aqueous solution of 1.3ml rapidly, begins some blueness; Light blue then, blue; Redness appears in heating again, boils 7～10min and transparent claret occurs, adds tri-distilled water at last to 100ml.Use the Electronic Speculum microscopy, guarantee that the gold grain for preparing makes its size consistent as far as possible, evenly, particle diameter is at 16～18nm.
10 μ l carbon nano-tube solution and 50 μ l colloidal gold solutions are added in 50 μ l 2% (W/V) chitosan solutions.Ultrasonic mixing was got 6 μ l and is dripped on the working electrode of screen printing electrode after 5 minutes, and drying at room temperature got final product in 30 minutes.
Get the 0.75g testing sample, add 3ml methanol solution (volume ratio of methyl alcohol and water is 80: 20), behind the concussion 15min, leave standstill 10min, 3000 left the heart after 15 minutes, used 5 times of 0.01M PBS dilutions, and it is subsequent use to get 5 μ l.
Step 5, testing process
6 μ l FB1-OVA are dripped in the working electrode zone, place 30min for 37 ℃.Use 0.01M PBST washing working electrode, and dry, drip 8 μ l 1% (W/V) BSA solution,, place 30min for 37 ℃ with the sealing working electrode.
To resist the FB1 monoclonal antibody to mix with FB1 standard items (0,0.1,0.5,1,5,10,50,100ng/ml) and testing sample dilution respectively, hatch 60min for 37 ℃.Use 0.01M PBST washing working electrode, and dry, drip the mixed solution of the anti-FB1 monoclonal antibody of 6 μ l and FB1 standard items or testing sample dilution, place 30min for 37 ℃.Use 0.01M PBST washing working electrode, and dry, the sheep anti mouse two that drips 6 μ l HRP marks is anti-, places 30min for 37 ℃.Use 0.01M PBST washing working electrode, and dry, screen printing electrode is put into the damping fluid of 10ml 0.01M PBS (pH 7.4), measure background current, be recorded as I
0Screen printing electrode is taken out, use 0.01M PBST washing working electrode, and dry, put into the damping fluid of 10ml 0.01M PBS (pH 7.4), add hydrogen peroxide (H again
2O
2) and quinhydrones (HQ), make its concentration be respectively 2mM and 0.1mM, behind the agitating solution 20min, stop to stir, measure electric current, be recorded as I, the electric current changing value
ΔI=I-I
0Use Excel software, with FB1 standard items variable concentrations and corresponding
ΔI drawing standard curve, testing sample is corresponding
ΔI brings in the typical curve, obtains FB1 concentration, and * dilution gfactor (4 * 5), be FB1 content in the testing sample (μ g/kg).
In sum; In the test sample of the present invention the method for FB1 be in view of testing sample if the FB1 toxin is arranged, then combines with anti-FB1 antibody competition, make with electrode on the antibody amount minimizing that combines of FB1-OVA; And the amounts that HRP-sheep anti mouse two resists on the electrode are also reduced, and influence the electric current changing value
ΔI; The typical curve that this method is drawn according to the FB1 standard items is judged the content of FB1 in the cereal test sample.Can find out, but the FB1 in the method direct quantitative test sample of present embodiment does not need professional training, easy to operate, quick.
Claims (9)
1. a composite Nano is modified screen printing electrode, it is characterized in that the perform region of the working electrode of said screen printing electrode is coated with multi-walled carbon nano-tubes-collaurum-chitosan compound rete.
2. composite Nano as claimed in claim 1 is modified screen printing electrode, it is characterized in that said collaurum is 16～18nm.
3. one kind prepares the method that composite Nano as claimed in claim 1 is modified screen printing electrode, it is characterized in that, comprises the steps:
Step 1, with the multi-walled carbon nano-tubes activation, and the preparation collaurum, utilize shitosan that multi-walled carbon nano-tubes and collaurum are mixed;
Step 2, the potpourri that step 1 is made are coated in the working electrode surface of screen printing electrode, and 37 ℃ of drying and forming-films promptly get.
4. composite Nano as claimed in claim 3 is modified the preparation method of screen printing electrode, and it is characterized in that the activation method of multi-walled carbon nano-tubes described in the step 1 is specially: the 5mg multi-walled carbon nano-tubes is dissolved in the 15ml activated solution, behind the ultrasonic 30min; The centrifugal 5min of 9000rpm behind the removal supernatant, adds the ultrapure water washing, the centrifugal 5min of 9000rpm; Remove supernatant, merge residue, 60 ℃ of oven for drying; Add 5ml water, be mixed with 1mg/ml solution, 4 ℃ of storages are subsequent use; Said activated solution is that volume ratio is 1: 3 HNO
3And H
2SO
4
5. composite Nano as claimed in claim 4 is modified the preparation method of screen printing electrode, it is characterized in that the preparation method of collaurum described in the step 1 is specially: with the HAuCl of 100ml
4Solution is heated to boiling, adds the citric acid three sodium solution of 1.2ml afterwards, boils 7～10min, adds tri-distilled water at last to 100ml, promptly gets colloidal gold solution; To account for the number percent of overall solution volume be 1% to the trisodium citrate quality in the said citric acid three sodium solution, said HAuCl
4HAuCl in the solution
4The number percent that quality accounts for overall solution volume is 0.01%.
6. composite Nano as claimed in claim 5 is modified the preparation method of screen printing electrode; It is characterized in that; Shitosan described in the step 1 was specially with mixing of multi-walled carbon nano-tubes and collaurum: 10 μ l multi-walled carbon nano-tubes solution and 50 μ l colloidal gold solutions are added in the 50 μ l chitosan solutions ultrasonic mixing; To account for the number percent of overall solution volume be 2% to chitosan mass in the said chitosan solution.
7. a method of modifying screen printing electrode detection fumonisin B1 with composite Nano as claimed in claim 1 is characterized in that, comprises the steps:
Step 1 drips 6 μ l FB1-OVA in said working electrode zone, places 30min for 37 ℃; Use 0.01MPBST washing working electrode, dry, drip 8 μ l BSA solution,, place 30min for 37 ℃ with the sealing working electrode; To account for the number percent of overall solution volume be 1% to the BSA quality in the said BSA solution;
Step 2 is got the 0.75g testing sample, adds the 3ml methanol solution, shakes, leaves standstill, centrifugal, uses 5 times of 0.01MPBS dilutions, and it is subsequent use to get 5 μ l;
Step 3 is mixed anti-FB1 monoclonal antibody respectively with the FB1 standard items and the testing sample dilution of variable concentrations, hatch 60min for 37 ℃;
Step 4 is used 0.01M PBST washing working electrode, dries, and drips the anti-FB1 monoclonal antibody and the mixed solution of FB1 standard items or testing sample dilution that 6 μ l step 3 make, 37 ℃ of placement 30min;
Step 5 is used 0.01M PBST washing working electrode, dries, and the sheep anti mouse two that drips 6 μ 1HRP marks is anti-, places 30min for 37 ℃;
Step 6 is used 0.01M PBST washing working electrode, and is dried, and screen printing electrode is put into the damping fluid that 10ml pH is 7.4 0.01M PBS, measures background current, is recorded as I
0
Step 7 is taken out screen printing electrode, uses 0.01M PBST washing working electrode; And dry, put into 10mlpH and be the damping fluid of 7.4 0.01M PBS, add hydrogen peroxide and quinhydrones again; Make its concentration be respectively 2mM and 0.1mM, behind the agitating solution 20min, stop to stir; Measure electric current, be recorded as I, the electric current changing value
ΔI=I-I
0
Step 8; Use Excel software, with FB1 standard items variable concentrations and corresponding
ΔI drawing standard curve, testing sample is corresponding
ΔI brings in the typical curve, obtains FB1 concentration, and multiply by dilution gfactor, is FB1 content in the testing sample.
8. composite Nano as claimed in claim 7 is modified the method that screen printing electrode detects fumonisin B1, it is characterized in that, centrifugal described in the step 2 is 3000 to leave the heart 15 minutes.
9. composite Nano as claimed in claim 7 is modified the method that screen printing electrode detects fumonisin B1, it is characterized in that, described in the step 2 in the methanol solution methyl alcohol and water volume ratio be 80: 20.
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| CN107764887A (en) * | 2017-10-26 | 2018-03-06 | 天津科技大学 | A kind of 24 site microarray screen-printing electrochemical sensing devices and its application |
| CN111426849A (en) * | 2020-04-13 | 2020-07-17 | 云南万魁生物科技有限公司 | Method for determining 14-3-3 protein expression level in soluble total protein |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106706733A (en) * | 2016-11-14 | 2017-05-24 | 江苏大学 | Preparation method of electrochemical adapter sensor for detecting fumonisin B1 ( FB1) |
| CN106896147A (en) * | 2017-04-26 | 2017-06-27 | 重庆医科大学 | A kind of electrochemical sensor of Rapid Determination of Plasma indoles |
| CN106896147B (en) * | 2017-04-26 | 2019-07-05 | 重庆医科大学 | A kind of electrochemical sensor of Rapid Determination of Plasma indoles |
| CN107764887A (en) * | 2017-10-26 | 2018-03-06 | 天津科技大学 | A kind of 24 site microarray screen-printing electrochemical sensing devices and its application |
| CN107764887B (en) * | 2017-10-26 | 2024-05-10 | 天津科技大学 | 24-Site microarray screen printing electrochemical sensing device and application thereof |
| CN111426849A (en) * | 2020-04-13 | 2020-07-17 | 云南万魁生物科技有限公司 | Method for determining 14-3-3 protein expression level in soluble total protein |
| CN111426849B (en) * | 2020-04-13 | 2024-02-09 | 云南万魁生物科技有限公司 | Method for measuring 14-3-3 protein expression level in soluble total protein |
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