CN102520168B - 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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- CN102520168B CN102520168B CN201110381726.0A CN201110381726A CN102520168B CN 102520168 B CN102520168 B CN 102520168B CN 201110381726 A CN201110381726 A CN 201110381726A CN 102520168 B CN102520168 B CN 102520168B
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- aspergillus parasiticus
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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 pathomycete rapid detection technical field, specifically based on antigen and antibody response, for detection of the unmarked type immunity biosensor and preparation method thereof of Aspergillus that produces aflatoxin.
Background technology
Aflatoxin is a kind of severe toxicity and strong carcinogen, for the most stable one in the various mycotoxins of finding so far, within 1988, is decided to be I level carcinogenic substance, its toxicity is potassium cyanide 10 times, 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 aflatoxin bacterium, the serious harm mankind's health.
And the harm of prevention aflatoxin, the most important condition is the pollution that can detect rapidly and accurately cereal, food etc. and whether produced aflatoxin Aspergillus.At present, the detection method of product aflatoxin Aspergillus mainly comprises traditional differential medium method, enzyme linked immunosorbent assay (ELISA), PCR and RT-PCR technology.Differential medium method is drawn materials conveniently, but sample pretreatment complexity, operating process is more loaded down with trivial details, sense cycle is long, and susceptibility is low, time-consumingly requires great effort again.ELISA needs special instrument and equipment, and detecting step is comparatively loaded down with trivial details, expense is high, detection time is long, and is prone to false positive.Although the development of the detection method of the molecular level of multiplex PCR, real-time fluorescence quantitative PCR has superiority than classic method, but the reagent that required instrument and equipment costliness, testing process complexity, cost are higher, testing environment and operating personnel's professional technique are had relatively high expectations, used all has larger harm to human body and environment, and because the complicacy of producing virus gene makes the method specificity not good enough.
Therefore set up a kind of detection method sensitive, quick, easy, that specificity is high and economic and be production and operation enterprise, Quality Control personnel, import and export inspection business, government administration section in the urgent need to the powerful guarantee of food, Environmental security.
Summary of the invention
The present invention is directed to prior art above shortcomings, a kind of immunosensor of the aspergillus parasiticus for detection of product aflatoxin is provided, be to modify successively on gold electrode how anti-to Cys, nm of gold and aspergillus parasiticus bacterium containing sulfydryl, obtain immune-electrochemistry sensor.
Preferably, described Cys dissolves with 0.1-0.3mol/L acetate buffer.
Preferably, the nanogold particle that described nm of gold is 10nm-20nm.
Further, how anti-described aspergillus parasiticus bacterium is 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)), 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 prepared.
Further, described gold electrode refers to: diameter is 2mm and is placed in Piranha solution and soaks after 10min, uses respectively the Al of 0.3 μ m, 0.05 μ m
2o
3after 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-electrochemistry sensor, is gold electrode is immersed in Cys solution heat-insulation soaking.Then, depositing nano gold, 2-6 ℃ of hold over night, after taking-up, with ultrapure water washing, dry up.Electrode surface is coated with an aspergillus parasiticus bacterium and resists and hatch more.Finally drip skimmed milk-PBS solution, seal as covering reagent, use ultrapure water washes clean.
Described Cys solution concentration is 0.3%-0.7% (m/v), and preferred concentration is 0.5% (m/v).
The temperature of described heat-insulation soaking is 35-40 ℃, soaks 3-7h.
Preferably 4 ℃ of described hold over night temperature.
Described at the how anti-3-15 μ L that are preferably of an electrode surface painting aspergillus parasiticus bacterium.
The described reagent closed process of covering preferably drips 5% skimmed milk-PBS solution, 10 μ L, uses ultrapure water washes clean after 37 ℃ of reactions.
Aspergillus parasiticus bacterium of the present 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, comprises the following steps:
1) sample to be tested drips the working electrode surface in immunosensor, hatches 30min at 37 ℃;
2) immunosensor is placed in electrolytic cell, and at the bottom of the detection of electrolytic cell, liquid is the potassium ferricyanide damping fluid containing the 2.5mmol of 0.1mol/LKCl;
3) set suitable sweep parameter, carry out cyclic voltammetric (CV) and AC impedance (EIS) and measure.
Experimental result show along with aspergillus parasiticus bacteria concentration increase, its resistance value increases.The resistance value that is defined in sealing immunization electrode is afterwards R
0, the electrode impedance value after 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.Aspergillus parasiticus bacteria concentration has good linear dependence within the scope of 75-1500cfu/mL, linearly dependent coefficient R
2=0.99114.Lowest detectable limit can reach 18cfu/mL.
The present invention has following beneficial effect: because nanogold particle has the electronics transmission that promotes well bio-electrical activity molecule, and be easy to fixing biological molecules and can keep its activity, and due to the effect of Cys, antibody is fixed on 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, the response time is short, can realize the direct-detection to producing malicious Aspergillus aspergillus parasiticus GIM3.395, is applicable to basic unit or Site Detection.
The present invention is applicable to the field fast detecting such as food industry, feedstuff industry and environmental protection and goes out early stage product aflatoxins Aspergillus, effectively to prevent the generation of aflatoxin.
Accompanying drawing explanation
Fig. 1 is immunosensor principle of work schematic diagram;
In figure: (a) absorption (d) skimmed milk of self assembly Cys 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 modified electrode different condition;
In figure: (a) after Cys modification, gold electrode (b) nm of gold Cys is modified rear gold electrode (c) antibody/nm of gold Cys and modified the modified electrode after rear gold electrode (d) skimmed milk sealing
Fig. 3 is the electron scanning micrograph (80000 times) of nano particle.
Fig. 4 is the EIS response of the aspergillus parasiticus GIM3.395 of immunosensor to variable concentrations
In 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
Below embodiments of the invention are elaborated, the present embodiment is implemented under take technical solution of the present invention as prerequisite, has provided detailed embodiment and concrete operating process.
Embodiment 1
Step 1, the structure of immunosensor: after naked gold electrode polishing, cleaning, be first immersed in the Cys of 0.3%-0.7% (m/v) 35-40 ℃ of heat-insulation soaking 3-7h.Then, 4 ℃ of hold over night depositing nano gold, after taking-up, with ultrapure water washing, dry up.It is how anti-that electrode surface is coated with a 3-15 μ L self-control, hatches 1h for 37 ℃.Finally drip 5% skimmed milk-PBS solution, 10 μ L, as covering reagent, non-specific adsorption site is sealed, after 37 ℃ of reactions, use ultrapure water washes clean.After the electrode not using immediately need not dry up with nitrogen, be placed in pH and be 7.4 PBS stand-by in 4 ℃ of preservations.
Step 2, the detection of immunosensor to aspergillus parasiticus bacterium spore liquid: aspergillus parasiticus bacterium, in 28 ℃, is cultivated 5-7 days on PDA inclined-plane solid medium, with the mould distilled water wash-out spore containing 0.05% tween of 10mL, prepares spore suspension.Adjusting spore concentration is 1.5 × 10
6cfu/mL, carries out aseptic gradient dilution afterwards to spore liquid.The immunoelectrode of having modified is immersed in the dilution of different gradients, the antibody on spore liquid antigen and electrode carries out incubation reaction 30min at 37 ℃, 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, 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 is afterwards R
0, the electrode impedance value after antigen-antibody reaction is R
x, and calculate Δ R
et=R
x-R
0, with Δ R
etdilutability mapping to reference culture aspergillus parasiticus can obtain linearity curve, realizes the detection to producing aflatoxin aspergillus aspergillus parasiticus bacterium spore liquid.
Embodiment 2
The mensuration of mark-on aspergillus parasiticus bacterium in spontaneous fermentation beans sauce actual sample
Step 1, the processing of spontaneous fermentation beans sauce sample: rear sterilizing that beans sauce sample is levigate, under sterile working, get 2g beans sauce, dilute with the sterilizing ultrapure water of 1: 1000, and add aspergillus parasiticus bacterium spore standard items, making its ultimate density is 150-1500cfu/mL.And configure one not containing the blank of the beans sauce of aspergillus parasiticus bacterium spore.
Step 2, the structure of immunosensor: after naked gold electrode polishing, cleaning, be first immersed in the Cys of 0.5% (m/v) 37 ℃ of heat-insulation soaking 5h.Then, 4 ℃ of hold over night depositing nano gold, after taking-up, with ultrapure water washing, dry up.It is how anti-that electrode surface is coated with a 10 μ L self-control, hatches 1h for 37 ℃.Finally drip 5% skimmed milk-PBS solution, 10 μ L, as covering reagent, non-specific adsorption site is sealed, after 37 ℃ of reactions, use ultrapure water washes clean.After the electrode not using immediately need not dry up with nitrogen, be placed in pH and be 7.4 PBS stand-by in 4 ℃ of preservations.
Step 3, the mensuration of mark-on aspergillus parasiticus bacterium GIM3.395 in spontaneous fermentation beans sauce actual sample: the immunoelectrode of having modified is immersed in different mark-on beans sauce sample liquid, antibody on mark-on antigen and electrode carries out incubation reaction 30min at 37 ℃, in the potassium ferricyanide damping fluid of the 2.5mmol of 0.1mol/L KCl, carries out EIS mensuration.Look into working curve and obtain Aspergillus concentration, calculate recovery rate, recovery result is as table 1.
Table 1 is aspergillus parasiticus bacterium recovery of standard addition and error result in 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 corn flour sample
Step 1, the processing of corn flour sample: get 2g corn flour under sterile working, and suitably mill.After milling, dilute with certain density sterilizing ultrapure water.
Step 2, the structure of immunosensor: after naked gold electrode polishing, cleaning, be first immersed in the Cys of 0.5% (m/v) 37 ℃ of heat-insulation soaking 5h.Then, 4 ℃ of hold over night depositing nano gold, after taking-up, with ultrapure water washing, dry up.Electrode surface is coated with and drips 10 μ L product aflatoxin Aspergillus polyclonal antibodies, hatches 1h for 37 ℃.Finally drip 5% skimmed milk-PBS solution, 10 μ L, as covering reagent, non-specific adsorption site is sealed, after 37 ℃ of reactions, use ultrapure water washes clean.After the electrode not using immediately need not dry up with nitrogen, be placed in pH and be 7.4 PBS stand-by in 4 ℃ of preservations.
Step 3, the detection of corn flour sample: the immunoelectrode modified is immersed in corn flour sample liquid, and incubation reaction 30min at 37 ℃ carries out EIS mensuration in the potassium ferricyanide damping fluid of the 2.5mmol of 0.1mol/LKCl.
Claims (3)
1. an immunity biosensor that detects the aspergillus parasiticus bacterium that produces aflatoxin, is characterized in that, is by the Cys containing sulfydryl, nm of gold, the how anti-biology sensor obtaining on gold electrode of modifying successively of aspergillus parasiticus bacterium; Described Cys dissolves with 0.1-0.3mol/L acetate buffer, and Cys solution concentration is 0.3%-0.7%; Described nm of gold is the nanogold particle of 10nm-20nm.
2. sensor according to claim 1, is characterized in that, described gold electrode refers to: diameter is 2mm and is placed in Piranha solution and soaks after 10min, uses respectively the Al of 0.3 μ m, 0.05 μ m
2o
3after burnishing powder polishing, the gold electrode of each ultrasonic 5min in absolute ethyl alcohol, ultrapure water.
3. an application for sensor described in claim 1, is characterized in that, comprises the following steps:
1) sample to be tested drips in the working electrode surface of immunosensor as claimed in claim 1, hatches 30min at 37 ℃;
2) immunosensor is placed in electrolytic cell, and the test fluid of electrolytic cell is the potassium ferricyanide damping fluid containing the 2.5mmol of 0.1mol/LKCl;
3) set suitable sweep parameter, carry out cyclic voltammetric and Based on AC Impedance Measurements.
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