CN102520168A - Immunosensor for detecting aspergillus parasiticus used for producing aflatoxin and preparing method thereof - Google Patents
Immunosensor for detecting aspergillus parasiticus used for producing aflatoxin and preparing method thereof Download PDFInfo
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- CN102520168A CN102520168A CN2011103817260A CN201110381726A CN102520168A CN 102520168 A CN102520168 A CN 102520168A CN 2011103817260 A CN2011103817260 A CN 2011103817260A CN 201110381726 A CN201110381726 A CN 201110381726A CN 102520168 A CN102520168 A CN 102520168A
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- aspergillus parasiticus
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- halfcystine
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Abstract
The invention discloses an immunosensor for detecting aspergillus parasiticus used for producing aflatoxin and a preparing method thereof. The immunosensor is obtained by the following step that: nano-Au, L- cysteine and aspergillus parasiticus polyoxin are self-assembled and modified on a gold electrode. According to the invention, the chemical amplification of the nano-Au and the specificity of the immunosensor are combined, and the advantages of the nano-Au and the immunosensor are also merged, so that the immunosensor simultaneously has the specificity of immune reaction and the quickness and sensitivity of electrochemistry analysis, and can accurately detect the low-content aspergillus parasiticus used for producing the aflatoxin.
Description
Technical field
The invention belongs to the pathomycete rapid detection technical field, specifically be based on antigen and antibody response, be used to unmarked type immunity biosensor that detects the Aspergillus of producing aflatoxin and preparation method thereof.
Background technology
Aflatoxin is a kind of severe toxicity and strong carcinogen, for the most stable a kind of in the various mycotoxins of finding so far, promptly is decided to be I level carcinogenic substance in 1988, and its toxicity is 10 times of potassium cyanide, 68 times of arsenic.Aflatoxin widely dispersed, nearly all cereal, feed and various food (comprising livestock products) all can be used as the growth matrix that produces the aflatoxin bacterium, serious harm human beings'health.
And the harm of prevention aflatoxin, the most important condition is to detect the pollution whether cereal, food etc. are produced the aflatoxin Aspergillus rapidly and accurately.At present, the detection method of product aflatoxin Aspergillus mainly comprises traditional differential medium method, EUSA (ELISA), PCR and RT-PCR technology.The differential medium method is drawn materials conveniently, but sample pretreatment is complicated, operating process is more loaded down with trivial details, sense cycle is long, and susceptibility is low, time-consuming effort again.ELISA needs special instrument and equipment, and the detection step is comparatively loaded down with trivial details, expense is high, detection time is long, and is prone to false positive.Though the development of the detection method of the molecular level of multiplex PCR, real-time fluorescence quantitative PCR has superiority than classic method; But required instrument and equipment is expensive, testing process is complicated, cost is higher, to testing environment and operating personnel's professional technique have relatively high expectations, employed reagent all has bigger harm to human body and environment, and makes the method specificity not good enough owing to produce the complicacy of virus gene.
Therefore to set up a kind of sensitivity, detection method quick, easy, that specificity is high and economic be production and operation enterprise, Quality Control personnel, import and export the powerful guarantee with food, Environmental security that presses for of inspection merchant, government administration section.
Summary of the invention
The present invention is directed to the above-mentioned deficiency that prior art exists; A kind of immunosensor that is used to detect the aspergillus parasiticus that produces aflatoxin is provided; Be many anti-the modifications successively on the gold electrode of L-halfcystine, nm of gold and aspergillus parasiticus bacterium that will contain sulfydryl, obtain immune electrochemical sensor.
Preferably, described L-halfcystine dissolves with the 0.1-0.3mol/L acetate buffer.
Preferably, described nm of gold is the nanogold particle of 10nm-20nm.
Further; How anti-described aspergillus parasiticus bacterium can buy commercial product or self-control obtains; Concrete grammar document (the Yong and Cousin that sees reference; 2003.Detection of moulds producing aflatoxins in maize and peanuts by an immunoassay.International Journal of Food Microbiology 65 (27)); Be to utilize shaking table to cultivate to produce aspergillus parasiticus mycelium protein immunogene, the male new zealand white rabbit of immune health and the polyclonal antibody for preparing.
Further, described gold electrode is meant: diameter be 2mm and place Piranha solution to soak 10min after, use the Al of 0.3 μ m, 0.05 μ m respectively
2O
3After the burnishing powder polishing, the gold electrode of each ultrasonic 5min in absolute ethyl alcohol, ultrapure water.
The present invention also provides a kind of preparation method of above-mentioned immune electrochemical sensor, is gold electrode is immersed in the L-halfcystine solution, and insulation is soaked.Then, the depositing nano gold, 2-6 ℃ of hold over night after the taking-up, washed, dried up with ultrapure water.Electrode surface is coated with how anti-drip the aspergillus parasiticus bacterium and hatch.Drip skimmed milk-PBS solution at last, seal, use the ultrapure water washes clean as covering reagent.
Said L-halfcystine solution concentration is 0.3%-0.7% (m/v), and preferred concentration is 0.5% (m/v).
The temperature that said insulation is soaked is 35-40 ℃, soaks 3-7h.
Preferred 4 ℃ of said hold over night temperature.
Said being coated with at electrode surface dripped the many anti-3-15 μ L that are preferably of aspergillus parasiticus bacterium.
The said preferred Dropwise 5 % skimmed milk of the reagent closed process-PBS solution 10 μ L that cover use the ultrapure water washes clean after 37 ℃ of reactions.
Aspergillus parasiticus bacterium according to the invention preferably secretes the aspergillus parasiticus GIM3.395 (microorganism fungus kind preservation center, Guangdong http://www.gimcc.net/) of aflatoxin.
The present invention also provides a kind of above-mentioned immunosensor to detect the method for the aspergillus parasiticus that produces aflatoxin, may further comprise the steps:
1) sample to be tested drips the working electrode surface in immunosensor, hatches 30min under 37 ℃;
2) immunosensor places electrolytic cell, and liquid is the potassium ferricyanide damping fluid that contains the 2.5mmol of 0.1mol/LKCl at the bottom of the detection of electrolytic cell;
3) set suitable sweep parameter, carry out cyclic voltammetric (CV) and AC impedance (EIS) and measure.
Experimental result shows that its resistance value increases along with aspergillus parasiticus bacteria concentration ground increases.The resistance value that is defined in sealing immunization electrode afterwards is R
0, the electrode impedance value after the antigen-antibody reaction is R
x, and calculate Δ R
Et=R
x-R
0, with Δ R
EtDilutability mapping to GIM3.395 can obtain linearity curve.The aspergillus parasiticus bacteria concentration has favorable linearity in the 75-1500cfu/mL scope relevant, linearly dependent coefficient R
2=0.99114.LDL can reach 18cfu/mL.
The present invention has following beneficial effect: because nanogold particle has the electron transport that promotes the bio-electrical activity molecule well; And be easy to fixing biological molecules and can keep its activity; And because the effect of L-halfcystine; Antibody is fixed on the modified gold electrode surface effectively, and has kept higher activity.Immunosensor of the present invention is easy and simple to handle, cheap, high specificity, highly sensitive, response time are short, can realize producing the direct detection of malicious Aspergillus aspergillus parasiticus GIM3.395, is fit to basic unit or on-the-spot the detection.
The present invention is applicable to that field fast detecting such as food industry, feedstuff industry and environmental protection go out early stage product aflatoxins Aspergillus, to effectively prevent the generation of aflatoxin.
Description of drawings
Fig. 1 is an immunosensor principle of work synoptic diagram;
Among the figure: (a) absorption (d) skimmed milk of self assembly L-halfcystine unimolecular layer (b) nanogold particle absorption (c) product aflatoxin Aspergillus antibody carries out site sealing (e) antigen and antibody specific association reaction
Fig. 2 is the cyclic voltammogram under the modified electrode different condition;
Among the figure: the modified electrode after (a) gold electrode (d) skimmed milk seals after gold electrode (c) antibody/nm of gold L-cysteine modified after gold electrode (b) the nm of gold L-cysteine modified after the L-cysteine modified
Fig. 3 is the electron scanning micrograph (80000 times) of nano particle.
Fig. 4 is the EIS response of immunosensor to the aspergillus parasiticus GIM3.395 of variable concentrations
Among the figure: (a) 75cfu/mL (b) 88cfu/mL (c) 167cfu/mL (d) 300cfu/mL (e) 1500cfu/mL (f) 3000cfu/mL (g) 6000cfu/mL (h) 7500cfu/mL (i) 10000cfu/mL (j) 15000cfu/mL (k) 30000cfu/mL
Embodiment
Elaborate in the face of embodiments of the invention down, present embodiment has provided detailed embodiment and concrete operating process being to implement under the prerequisite with technical scheme of the present invention.
Embodiment 1
Step 1, the structure of immunosensor: after naked gold electrode polishing, cleaning, be immersed in earlier in the L-halfcystine of 0.3%-0.7% (m/v), 3-7h is soaked in 35-40 ℃ of insulation.Then, 4 ℃ of hold over night depositing nano gold after the taking-up, wash, dry up with ultrapure water.It is how anti-that electrode surface is coated with a 3-15 μ L self-control, hatches 1h for 37 ℃.Last Dropwise 5 % skimmed milk-PBS solution 10 μ L seals the non-specific adsorption site as covering reagent, after 37 ℃ of reactions, uses the ultrapure water washes clean.After the electrode that uses immediately needed not dry up with nitrogen, placing pH was that 7.4 PBS is for use in 4 ℃ of preservations.
Step 2, immunosensor are to the detection of aspergillus parasiticus bacterium spore liquid: the aspergillus parasiticus bacterium was cultivated 5-7 days on the PDA inclined-plane solid medium in 28 ℃, with the mould distilled water wash-out spore that contains 0.05% tween of 10mL, prepared spore suspension.The adjustment spore concentration is 1.5 * 10
6Cfu/mL, aseptic gradient dilution is carried out to spore liquid in the back.Immerse in the dilution of different gradients modifying good immunoelectrode, the antibody on spore liquid antigen and the electrode carries out 37 ℃ of following incubation reaction 30min, in the potassium ferricyanide damping fluid of the 2.5mmol of 0.1mol/LKCl, carries out EIS mensuration.The test condition of EIS: initial potential is 0.2V, amplitude 0.05V, and frequency range is 1～100kHz, and time of repose is 2s, and all tests are all at room temperature carried out.The resistance value that is defined in sealing immunization electrode afterwards is R
0, the electrode impedance value after the antigen-antibody reaction is R
x, and calculate Δ R
Et=R
x-R
0, with Δ R
EtDilutability mapping to the reference culture aspergillus parasiticus can obtain linearity curve, realizes producing the detection of aflatoxin aspergillus aspergillus parasiticus bacterium spore liquid.
Embodiment 2
The mensuration of mark-on aspergillus parasiticus bacterium in the spontaneous fermentation beans sauce actual sample
Step 1, the processing of spontaneous fermentation beans sauce sample: back sterilization that beans sauce sample is levigate, under sterile working, get 2g beans sauce, dilute with 1: 1000 sterilization ultrapure water, and add aspergillus parasiticus bacterium spore standard items, making its ultimate density is 150-1500cfu/mL.And dispose a blank that does not contain the beans sauce of aspergillus parasiticus bacterium spore.
Step 2, the structure of immunosensor: after naked gold electrode polishing, cleaning, be immersed in earlier in the L-halfcystine of 0.5% (m/v), 5h is soaked in 37 ℃ of insulations.Then, 4 ℃ of hold over night depositing nano gold after the taking-up, wash, dry up with ultrapure water.It is how anti-that electrode surface is coated with 10 a μ L self-control, hatches 1h for 37 ℃.Last Dropwise 5 % skimmed milk-PBS solution 10 μ L seals the non-specific adsorption site as covering reagent, after 37 ℃ of reactions, uses the ultrapure water washes clean.After the electrode that uses immediately needed not dry up with nitrogen, placing pH was that 7.4 PBS is for use in 4 ℃ of preservations.
Step 3; The mensuration of mark-on aspergillus parasiticus bacterium GIM3.395 in the spontaneous fermentation beans sauce actual sample: will modify good immunoelectrode and immerse in the different mark-on beans sauce sample liquid; Antibody on mark-on antigen and the electrode carries out 37 ℃ of following incubation reaction 30min, in the potassium ferricyanide damping fluid of the 2.5mmol of 0.1mol/L KCl, carries out EIS and measures.Look into working curve and obtain Aspergillus concentration, calculate recovery rate, recovery result such as table 1.
Table 1 is aspergillus parasiticus bacterium recovery of standard addition and an error result in the immunosensor spontaneous fermentation beans sauce actual sample.
| Mark-on concentration (cfu/mL) | 150 | 300 | 1000 | 1500 |
| The recovery (%) (n=3) | 113.5±4.5 | 81.5±3.8 | 87.9±1.2 | 85.2±1.6 |
Embodiment 3
The mensuration of aspergillus parasiticus bacterium in the corn flour sample
Step 1, the processing of corn flour sample: under sterile working, get the 2g corn flour, and suitably mill.Dilute with certain density sterilization ultrapure water after milling.
Step 2, the structure of immunosensor: after naked gold electrode polishing, cleaning, be immersed in earlier in the L-halfcystine of 0.5% (m/v), 5h is soaked in 37 ℃ of insulations.Then, 4 ℃ of hold over night depositing nano gold after the taking-up, wash, dry up with ultrapure water.Electrode surface is coated with and drips 10 μ L product aflatoxin Aspergillus polyclonal antibody, hatches 1h for 37 ℃.Last Dropwise 5 % skimmed milk-PBS solution 10 μ L seals the non-specific adsorption site as covering reagent, after 37 ℃ of reactions, uses the ultrapure water washes clean.After the electrode that uses immediately needed not dry up with nitrogen, placing pH was that 7.4 PBS is for use in 4 ℃ of preservations.
Step 3, the detection of corn flour sample: will modify good immunoelectrode and immerse in the corn flour sample liquid, 37 ℃ of following incubation reaction 30min carry out EIS and measure in the potassium ferricyanide damping fluid of the 2.5mmol of 0.1mol/LKCl.
Claims (7)
1. an immunity biosensor that detects the aspergillus parasiticus bacterium that produces aflatoxin is characterized in that, is with the L-halfcystine that contains sulfydryl, nm of gold, the many anti-biology sensors that obtain on the gold electrode of modifying successively of aspergillus parasiticus bacterium.
2. according to the said sensor of claim 1, it is characterized in that described L-halfcystine dissolves with the 0.1-0.3mol/L acetate buffer, L-halfcystine solution concentration is 0.3%-0.7%.
3. according to the said sensor of claim 1, it is characterized in that described nm of gold is the nanogold particle of 10 nm-20 nm.
4. according to the said sensor of claim 1, it is characterized in that described gold electrode is meant: diameter be 2mm and place Piranha solution to soak 10 min after, use the Al of 0.3 μ m, 0 .05 μ m respectively
2O
3After the burnishing powder polishing, the gold electrode of each ultrasonic 5min in absolute ethyl alcohol, ultrapure water.
5. a preparation method who detects aspergillus parasiticus bacterial immunity biology sensor is characterized in that, gold electrode is immersed in the L-halfcystine solution, and insulation is soaked; The depositing nano gold, 2-6 ℃ of hold over night after the taking-up, washed, dried up with ultrapure water; Electrode surface is coated with how anti-drip the aspergillus parasiticus bacterium and hatch; Drip skimmed milk-PBS solution, seal, use the ultrapure water washes clean as covering reagent.
6. according to the said method of claim 5, it is characterized in that concrete grammar is following: gold electrode is immersed in 0.5% the L-halfcystine solution, 5 h are soaked in 37 ℃ of insulations; Then, the depositing nano gold, 4 ℃ of hold over night after the taking-up, are washed, are dried up with ultrapure water; It is how anti-that electrode surface is coated with 10 a μ L self-control, hatches 1h for 37 ℃; Last Dropwise 5 % skimmed milk-PBS solution 10 μ L seals as covering reagent, after 37 ℃ of reactions, uses the ultrapure water washes clean.
7. the said application of sensor of claim 1 is characterized in that, may further comprise the steps:
1) sample to be tested drips in the working electrode surface of immunosensor according to claim 1, hatches 30min under 37 ℃;
2) immunosensor places electrolytic cell, and the test fluid of electrolytic cell is the potassium ferricyanide damping fluid that contains the 2.5mmol of 0.1mol/LKCl;
3) set suitable sweep parameter, carry out cyclic voltammetric and AC impedance and measure.
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| CN104076025A (en) * | 2014-06-18 | 2014-10-01 | 深圳职业技术学院 | Antimicrobial peptide electrochemiluminescence sensor and preparation method and detection method thereof |
| RU2534732C1 (en) * | 2013-07-26 | 2014-12-10 | Федеральное государственное бюджетное образовательное учреждение высшего профессионального образования "Национальный исследовательский Томский политехнический университет" | Method for quantitative determination of aflatoxin b1 by differential voltammetry |
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| CN108181360A (en) * | 2017-12-22 | 2018-06-19 | 齐鲁医药学院 | The construction method of acid phosphoric acid ester enzyme biologic sensor based on mercapto-functionalized graphene self assembly |
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| CN103018298A (en) * | 2012-11-29 | 2013-04-03 | 江南大学 | Preparation method and application of L-cysteine/gold nanoparticle composite film cell sensor for allergen detection |
| RU2534732C1 (en) * | 2013-07-26 | 2014-12-10 | Федеральное государственное бюджетное образовательное учреждение высшего профессионального образования "Национальный исследовательский Томский политехнический университет" | Method for quantitative determination of aflatoxin b1 by differential voltammetry |
| CN104076025A (en) * | 2014-06-18 | 2014-10-01 | 深圳职业技术学院 | Antimicrobial peptide electrochemiluminescence sensor and preparation method and detection method thereof |
| CN104076025B (en) * | 2014-06-18 | 2017-04-05 | 深圳职业技术学院 | A kind of antibacterial peptide electrochemical luminous sensor and preparation method thereof and detection method |
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| CN108181360A (en) * | 2017-12-22 | 2018-06-19 | 齐鲁医药学院 | The construction method of acid phosphoric acid ester enzyme biologic sensor based on mercapto-functionalized graphene self assembly |
| CN112179962A (en) * | 2020-09-29 | 2021-01-05 | 陕西科技大学 | Detection method of aflatoxin based on iron ion probe-nanogold/glassy carbon electrode modified electrode |
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Effective date of registration: 20200909 Address after: No. 1800 road 214000 Jiangsu Lihu Binhu District City of Wuxi Province Co-patentee after: NANJING MICRODETECTION BIOLOGICAL TECHNOLOGY Co.,Ltd. Patentee after: Jiangnan University Address before: New Kumho 210061 Nanjing Road, Jiangsu province No. 3-1 in garden building B room 1307 Patentee before: NANJING MICRODETECTION BIOLOGICAL TECHNOLOGY Co.,Ltd. |