CN106680346A - Electrochemical detection method for AFB1 (Aflatoxin B1) based on strand displacement amplification and surface proximity hybridization reaction and application thereof - Google Patents
Electrochemical detection method for AFB1 (Aflatoxin B1) based on strand displacement amplification and surface proximity hybridization reaction and application thereof Download PDFInfo
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- 238000009396 hybridization Methods 0.000 title claims abstract description 37
- 238000006073 displacement reaction Methods 0.000 title claims abstract description 34
- 238000000034 method Methods 0.000 title claims abstract description 34
- 238000006243 chemical reaction Methods 0.000 title claims abstract description 14
- 230000003321 amplification Effects 0.000 title claims abstract description 12
- 238000003199 nucleic acid amplification method Methods 0.000 title claims abstract description 12
- OQIQSTLJSLGHID-WNWIJWBNSA-N aflatoxin B1 Chemical compound C=1([C@@H]2C=CO[C@@H]2OC=1C=C(C1=2)OC)C=2OC(=O)C2=C1CCC2=O OQIQSTLJSLGHID-WNWIJWBNSA-N 0.000 title abstract description 22
- 239000002115 aflatoxin B1 Substances 0.000 title abstract description 12
- 229930020125 aflatoxin-B1 Natural products 0.000 title abstract description 12
- 239000000523 sample Substances 0.000 claims abstract description 52
- 239000011324 bead Substances 0.000 claims abstract description 25
- 238000001514 detection method Methods 0.000 claims abstract description 21
- 108091023037 Aptamer Proteins 0.000 claims abstract description 15
- 238000007650 screen-printing Methods 0.000 claims description 33
- 239000000243 solution Substances 0.000 claims description 24
- 238000012986 modification Methods 0.000 claims description 21
- 230000004048 modification Effects 0.000 claims description 21
- 239000007853 buffer solution Substances 0.000 claims description 17
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 claims description 15
- 239000000872 buffer Substances 0.000 claims description 13
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 12
- 239000007788 liquid Substances 0.000 claims description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- KTWOOEGAPBSYNW-UHFFFAOYSA-N ferrocene Chemical compound [Fe+2].C=1C=C[CH-]C=1.C=1C=C[CH-]C=1 KTWOOEGAPBSYNW-UHFFFAOYSA-N 0.000 claims description 9
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- 230000008859 change Effects 0.000 claims description 5
- 229910001487 potassium perchlorate Inorganic materials 0.000 claims description 5
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- XWIYFDMXXLINPU-UHFFFAOYSA-N Aflatoxin G Chemical compound O=C1OCCC2=C1C(=O)OC1=C2C(OC)=CC2=C1C1C=COC1O2 XWIYFDMXXLINPU-UHFFFAOYSA-N 0.000 description 3
- 101100449517 Arabidopsis thaliana GRH1 gene Proteins 0.000 description 3
- 101100434479 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) AFB1 gene Proteins 0.000 description 3
- 239000005409 aflatoxin Substances 0.000 description 3
- WWSYXEZEXMQWHT-WNWIJWBNSA-N aflatoxin B2 Chemical compound C=1([C@@H]2CCO[C@@H]2OC=1C=C(C1=2)OC)C=2OC(=O)C2=C1CCC2=O WWSYXEZEXMQWHT-WNWIJWBNSA-N 0.000 description 3
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- BXFOFFBJRFZBQZ-QYWOHJEZSA-N T-2 toxin Chemical compound C([C@@]12[C@]3(C)[C@H](OC(C)=O)[C@@H](O)[C@H]1O[C@H]1[C@]3(COC(C)=O)C[C@@H](C(=C1)C)OC(=O)CC(C)C)O2 BXFOFFBJRFZBQZ-QYWOHJEZSA-N 0.000 description 2
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- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 125000001340 2-chloroethyl group Chemical group [H]C([H])(Cl)C([H])([H])* 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
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- 229910019142 PO4 Inorganic materials 0.000 description 1
- RQNWIZPPADIBDY-UHFFFAOYSA-N arsenic atom Chemical compound [As] RQNWIZPPADIBDY-UHFFFAOYSA-N 0.000 description 1
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- LINOMUASTDIRTM-QGRHZQQGSA-N deoxynivalenol Chemical compound C([C@@]12[C@@]3(C[C@@H](O)[C@H]1O[C@@H]1C=C(C([C@@H](O)[C@@]13CO)=O)C)C)O2 LINOMUASTDIRTM-QGRHZQQGSA-N 0.000 description 1
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- 229910052697 platinum Inorganic materials 0.000 description 1
- NNFCIKHAZHQZJG-UHFFFAOYSA-N potassium cyanide Chemical compound [K+].N#[C-] NNFCIKHAZHQZJG-UHFFFAOYSA-N 0.000 description 1
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- 231100000765 toxin Toxicity 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- LINOMUASTDIRTM-UHFFFAOYSA-N vomitoxin hydrate Natural products OCC12C(O)C(=O)C(C)=CC1OC1C(O)CC2(C)C11CO1 LINOMUASTDIRTM-UHFFFAOYSA-N 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- MBMQEIFVQACCCH-QBODLPLBSA-N zearalenone Chemical compound O=C1O[C@@H](C)CCCC(=O)CCC\C=C\C2=CC(O)=CC(O)=C21 MBMQEIFVQACCCH-QBODLPLBSA-N 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
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- Electrochemistry (AREA)
- Spectroscopy & Molecular Physics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
The invention belongs to the technical field of the quality safety detection of agricultural products, and relates to the detection of AFB1 (Aflatoxin B1) in the agricultural products, in particular to an electrochemical detection method for the AFB1 based on a strand displacement amplification and surface proximity hybridization reaction and application thereof. A hybrid complex formed by a biotin-labelled AFB1 aptamer and a Block probe is prefixed on a surface of a magnetic bead; in the presence of a target, the AFB1 and the Block probe are combined with the aptamer in a competitive manner; the replaced Block probe can trigger a strand displacement amplification reaction so as to produce a lot of amplicons; and the amplicons can cooperatively participate in the surface proximity hybridization reaction on a surface of a screen-printed electrode, so as to introduce an electroactive probe to generate a corresponding electrical signal. Compared with specific experiments and conventional ELISA (Enzyme-linked Immuno Sorbent Assay) methods, the electrochemical detection method for the AFB1 disclosed by the invention has the advantages of good specificity, high sensitivity, reliability and accuracy, and is expected to provide a technical selection for large-scale screening of the AFB1 in agricultural products including corn.
Description
Technical field
The invention belongs to agricultural product quality and safety detection technique field, is related to aflatoxin B in agricultural product1（AFB1）'s
Detection, and in particular to a kind of to amplify the AFB that hybridization is closed on surface based on strand displacement1Electrochemical detection method and application.
Background technology
Secondary metabolism thing of the aflatoxin (aflatoxin, AF) as many mycetes, in field and storing process
Most of agricultural product can be polluted, the such as plurality of cereals such as corn, Semen arachidis hypogaeae, Semen Maydiss, oilseeds and feedstuff have become impact food
The huge killer of safety, harm human and animal's health.There is the aflatoxin having now been found that induced mutation, suppression to exempt from
Epidemic disease and carcinogenesis, delimited as I class carcinogen by the Agency for Research on Cancer of World Health Organization (WHO), it has now been found that have
AFB1、AFB2、AFG1、AFG2、AFM1And AFM2Deng 18 kinds, wherein with AFB1The most common, hazardness is also most strong.It is reported that, AFB1
Toxicity be 30 times, 20 times of 6-(10-hydroxy-6-oxo-trans-1-undecenyl)-.beta.-resorcylic acid lactone, 10 times of potassium cyanide, 68 times of arsenicum of vomitoxin respectively.Therefore, very
Many countries have worked out strict AF limit standards, wherein especially with the harshest of European Union, it is stipulated that AFB in Semen arachidis hypogaeae1Content must not surpass
2 ppb are crossed, AFB in baby food1Content must not be higher than 0.1 ppb（European Commission regulation
1881/2006）.Current aflatoxin B1（AFB1）Detection method mainly have high performance liquid chromatography (HPLC), thin layer chromatography
Method (TLC), euzymelinked immunosorbent assay (ELISA) (ELISA) etc..Because in most cases, AFB in agricultural product1Content it is extremely micro, so opening
Send out simple to operate, sensitivity is high, accuracy good, detect danger of the high detection method of flux for reduction AF to human life's safety
Do harm to, ensure the unimpeded with extremely important effect of International import-export trade.
Aptamer is used as single stranded oligonucleotide（20~60 bases）, it is referred to as " chemical antibody ", has many advantages, such as：
Specificity is high, and compatibility is good, good stability and is easily-synthesized, easy labelling.Electrochemical biosensor detection method has quick, spirit
The peculiar advantage such as quick, instrument and equipment is simple.
The content of the invention
The present invention is the current AFB of solution1The sensitivity of detection method is low, and operation difficulty is big, and the high technology of testing cost is difficult
Topic, discloses a kind of AFB for amplifying based on strand displacement and hybridization being closed on surface1Electrochemical detection method and application.
To solve above-mentioned technical barrier, the present invention is employed the following technical solutions：
It is a kind of to amplify the AFB that hybridization is closed on surface based on strand displacement1Electrochemical detection method, the detection method will
AFB1Neucleic acid aptamers as molecular recognition elements, screen printing electrode as transducer, using constant temperature strand displacement amplification side
After method carries out signal amplification, then hybridization is closed on by surface carry out signal output.
The AFB1The sequence of neucleic acid aptamers such as SEQ ID NO：Shown in 1.
Amplify the AFB that hybridization is closed on surface based on strand displacement1Electrochemical detection method, comprises the following steps：
（1）The surface modification of magnetic bead：By the AFB of biotin labeling1Aptamer and Block probe storing solutions, difference Jing 95
Then DEG C 5min, the pretreatment of 0 DEG C of 10min use AFB11 × BB buffer is diluted to 200nM, and hybridizes in room temperature
30min, is obtained hybrid mixed liquid；Meanwhile, 50 L magnetic beads are taken with 500 L AFB11 × BB buffer solutions are twice；Take 200
L hybrid mixed liquid adds washed magnetic bead in centrifuge tube, is incubated 15min at room temperature, finally again with 500 L AFB1 1
× BB buffer, washs the magnetic bead being incubated twice, completes the surface modification of magnetic bead；
（2）The pretreatment of screen printing electrode and the preparation of sensing interface：It is clean in Jing dehydrated alcohol and deionized water wash
On the electrode surface of screen printing electrode, 40 L of Deca, 0.1 M H2SO4Solution, activates 60s under 2V DC voltages, is living
On screen printing electrode after change, 4 L of Deca contains the buffer solution of 1 μM of sulfydryl modification probe, assembles under the conditions of 30 DEG C
3h, uses AFB1Screen printing electrode after the assembling of 1 × BB buffer solution for cleaning, 4 L of last Deca, 1 mM6- sulfydryl hexanol solution
30min is closed in room temperature, AFB is used1After 1 × BB buffer solution for cleaning 3 times, screen printing electrode is dried up standby in 4 DEG C；
（3）AFB1Hybridization is closed on reference to the strand displacement and surface for mediating：In AFB150 L steps are added in target solution（1）
The middle magnetic bead modified, gently concussion is incubated 30 min at room temperature, and subsequent Magneto separate takes 10 L of supernatant, adds 10 L 10
× NEB buffer 3.1,2 L, 0.5 μM of strand displacing probes, 2 L, 10 mM dNTPs, 4 L, 2 U/ L polymerases and 2
10 U/ L nicking restriction endonucleases of L, deionized water supplement reaction system to 100 L, at 55 DEG C react 1h；Then add
10 L ferrocene label probes, after mix homogeneously, take 40 L and are added drop-wise to（2）In, the screen printing electrode room surface modified
Temperature 30 min of reaction, finally with 1 × AFB1Buffer solution is clean and is dried up with nitrogen；
（4）Electrochemical Detection：To Jing steps（3）40 L of screen printing electrode surface Deca for processing contains 10 mM
MgCl20.1 M KClO4Solution, then carries out differential pulse Electrochemical Detection.
The step（1）The sequence of middle Block probes such as SEQ ID NO：Shown in 2, the concentration of Block probe liquid storages is
10 µM；The AFB1The formula of 1 × BB buffer is：10 mM Hepes, 120 mM NaCI, 5 mM KCI, 5 mM
MgCl2, pH 7.0。
The step（2）The sequence of middle sulfydryl modification probe such as SEQ ID NO：Shown in 3, the buffering of sulfydryl modification probe is molten
The formula of liquid is：1 μM of sulfydryl modification probe, 1M NaCl and 1mM TCEP.
The step（3）The sequence of middle strand displacing probes such as SEQ ID NO：Shown in 4, ferrocene label probe such as SEQ ID
NO：Shown in 5.
The step（4）In Electrochemical Detection carry out on electrochemical workstation, design parameter be window sweep length
For 0-0.5 V, pulse width is 50 mV, and 0.2 s of silence period, pulse width are 0.06 s.
Amplify the AFB that hybridization is closed on surface based on strand displacement1Electrochemical detection method, as in detection crop
AFB1Application.
The beneficial effects of the present invention is：
（1）The present invention adopts AFB1Aptamer is further improved as molecular recognition elements, specificity；Meanwhile, because of nucleic acid
Synthesis cost it is very low, and commercialization greatly reduces AFB1Testing cost.
（2）The present invention utilizes the high specific feature of " chemical antibody " aptamer, puts with reference to strand displacement amplified signal
Big strategy, while the mode signal output of hybridization is closed on using surface, constructs a kind of measure aflatoxin B1Electrochemistry
Aptamer sensor, has the advantages that simple and fast, highly sensitive, high specific.
（3）The present invention transducer is made using screen printing electrode, have the advantages that it is simple to operate, be easy to batch quantity analysis.
Description of the drawings
Fig. 1 is the screen printing electrode characteristic peaks figure after activation.
Fig. 2 is Cleaning Principle schematic diagram.
Fig. 3 is detection method specificity analyses figure.
Fig. 4 is standard working curve and range of linearity figure.
Specific embodiment
It is a kind of to amplify the AFB that hybridization is closed on surface based on strand displacement1Electrochemical detection method, the detection method
By AFB1Neucleic acid aptamers as molecular recognition elements, screen printing electrode as transducer, using constant temperature strand displacement amplification
After method carries out signal amplification, then hybridization is closed on by surface carry out signal output.
The AFB1The sequence of neucleic acid aptamers such as SEQ ID NO：Shown in 1.
Amplify the AFB that hybridization is closed on surface based on strand displacement1Electrochemical detection method, comprises the following steps：
（1）The surface modification of magnetic bead：By the AFB of biotin labeling1Aptamer and Block probe storing solutions, difference Jing 95
Then DEG C 5min, the pretreatment of 0 DEG C of 10min use AFB11 × BB buffer is diluted to 200nM, and hybridizes in room temperature
30min, is obtained hybrid mixed liquid；Meanwhile, 50 L magnetic beads are taken with 500 L AFB11 × BB buffer solutions are twice；Take 200
L hybrid mixed liquid adds washed magnetic bead in centrifuge tube, is incubated 15min at room temperature, finally again with 500 L AFB1 1
× BB buffer, washs the magnetic bead being incubated twice, completes the surface modification of magnetic bead；
（2）The pretreatment of screen printing electrode and the preparation of sensing interface：It is clean in Jing dehydrated alcohol and deionized water wash
On the electrode surface of screen printing electrode, 40 L of Deca, 0.1 M H2SO4Solution, activates 60s under 2V DC voltages, is living
On screen printing electrode after change, 4 L of Deca contains the buffer solution of 1 μM of sulfydryl modification probe, assembles under the conditions of 30 DEG C
3h, uses AFB1Screen printing electrode after the assembling of 1 × BB buffer solution for cleaning, 4 L of last Deca, 1 mM6- sulfydryl hexanol solution
30min is closed in room temperature, AFB is used1After 1 × BB buffer solution for cleaning 3 times, screen printing electrode is dried up standby in 4 DEG C；
（3）AFB1Hybridization is closed on reference to the strand displacement and surface for mediating：In AFB150 L steps are added in target solution（1）
The middle magnetic bead modified, gently concussion is incubated 30 min at room temperature, and subsequent Magneto separate takes 10 L of supernatant, adds 10 L 10
× NEB buffer 3.1,2 L, 0.5 μM of strand displacing probes, 2 L, 10 mM dNTPs, 4 L, 2 U/ L polymerases and 2
10 U/ L nicking restriction endonucleases of L, deionized water supplement reaction system to 100 L, at 55 DEG C react 1h；Then add
10 L ferrocene label probes, after mix homogeneously, take 40 L and are added drop-wise to（2）In, the screen printing electrode room surface modified
Temperature 30 min of reaction, finally with 1 × AFB1Buffer solution is clean and is dried up with nitrogen；
（4）Electrochemical Detection：To Jing steps（3）40 L of screen printing electrode surface Deca for processing contains 10 mM
MgCl20.1 M KClO4Solution, then carries out differential pulse Electrochemical Detection.
The step（1）The sequence of middle Block probes such as SEQ ID NO：Shown in 2, the concentration of Block probe liquid storages is
10 µM；The AFB1The formula of 1 × BB buffer is：10 mM Hepes, 120 mM NaCI, 5 mM KCI, 5 mM
MgCl2, pH 7.0。
The step（2）The sequence of middle sulfydryl modification probe such as SEQ ID NO：Shown in 3, the buffering of sulfydryl modification probe is molten
The formula of liquid is：1 μM of sulfydryl modification probe, 1M NaCl and 1mM TCEP.
The step（3）The sequence of middle strand displacing probes such as SEQ ID NO：Shown in 4, ferrocene label probe such as SEQ ID
NO：Shown in 5.
The step（4）In Electrochemical Detection carry out on electrochemical workstation, design parameter be window sweep length
For 0-0.5 V, pulse width is 50 mV, and 0.2 s of silence period, pulse width are 0.06 s.
Amplify the AFB that hybridization is closed on surface based on strand displacement1Electrochemical detection method, as in detection crop
AFB1Application.
Below in conjunction with specific embodiment, the present invention is described in detail.
1. it is a kind of to amplify the aflatoxin B that Hybridization Strategy is closed on surface based on strand displacement1（AFB1）Electrochemical Detection side
Method, comprises the following steps：
（1）The surface modification of magnetic bead
The AFB of biotin labeling1Aptamer such as SEQ IDNO：Shown in 1, which 3 ' terminal modified has Biotin groups and 10 μM
Block probes such as SEQ ID NO：Shown in 2, storing solution first passes through 95 DEG C of 5 min, the pretreatment of 0 DEG C of 10 min, Ran Houyong
AFB11 × BB buffer（10 mM Hepes, 120 mM NaCI, 5 mM KCI, 5 mM MgCl2, pH 7.0）It is diluted to
200 nM, and hybridize 30 min in room temperature.Meanwhile, take 50 L MP-SA-10 magnetic bead AFB11 × BB is washed twice, and 500
L/ time, then take the good mixed solution of the above-mentioned hybridization of 200 L in 1 mL centrifuge tubes with the magnetic bead of washes clean at room temperature
15 min are incubated, AFB is finally used1Twice, 500 L/ time, 4 DEG C of storages are standby for the magnetic bead modified of 1 × BB washings.
（2）The pretreatment of screen printing electrode and the preparation of sensing interface
New screen printing electrode first uses dehydrated alcohol and deionized water wash clean, 40 L of Deca, 0.1 M H2SO4In electricity
Pole surface, and apply DC voltage 60 S of activation of+2 V.Toward 40 L of screen printing electrode surface Deca, 0.1 M after activation
H2SO4, voltammetric scan is circulated, window sweep length is 0 ~ 1.5 V, and sweep speed is 100 mV/s, as a result as shown in Figure 1.
Cyclic voltammetric characteristic peak of the characteristic peak of the screen printing electrode after activation to gold electrodes in sulphuric acid is extremely similar.It is past living
4 L of Deca contains 1 μM of sulfydryl modification probe such as SEQ ID NO to electrode surface after change immediately：Shown in 3,3 ' terminal modified have SH bases
Buffer solution（Containing 1 M NaCl and 1 mM tricresyl phosphates (2- chloroethyls) ester（TCEP））, 3 h are assembled at 30 DEG C, AFB is used1
1×BB （10 mM Hepes, 120 mM NaCI, 5 mM KCI, 5 mM MgCI2, pH 7.0）Clean up.Finally drip
Plus in 4 L, 1 mM6- sulfydryl hexanol solution, room temperature closes 30 min, is cleaned with 1 × BB after 3 times, after electrode clean clean
Dry up 4 DEG C it is standby.
（3）AFB1Hybridization and Electrochemical Detection are closed on reference to the strand displacement and surface for mediating
Principle such as Fig. 2 institutes of the AFB1 electrochemical detection method that Hybridization Strategy is closed on surface are amplified based on strand displacement
Show.The AFB of 3 ' end biotin labelings1The hybridization complex that aptamers are formed with Block probes is fixed on magnetic bead surfaces in advance,
AFB1In the presence of, Block probes and AFB1Competition is combined with aptamers.Because of AFB1It is more than with the adhesion of aptamers
The adhesion of Block probes and aptamers, Block probes are competed.After centrifugation, the Block probes collected in supernatant add
Enter strand displacement amplification system, triggering starts strand displacement amplification reaction so as to produce substantial amounts of amplicon.
Hybridization is closed on the surface that the amplicon can cooperate with participation subsequent：Sulfydryl modification probe, ferrocene labelling are visited
Pin and the concerted reaction of amplicon three, so as to introduce electroactive probe on screen printing electrode surface, produce corresponding telecommunications
Number.If AFB1Do not exist, AFB would not occur1Hybridization is closed on reference to the strand replacement reaction and surface for mediating, naturally not
Current signal can be produced.Therefore, the method is a kind of signal-on detection patterns.The introducing of strand displacement amplification, it is ensured that
The method has enough sensitivity；Surface is closed on the signal output strategy of hybridization and the Cleaning Principle is transformed into signal-on
Pattern, so as to further increase the sensitivity of method；The use of screen printing electrode, it is ensured that the method can be parallel on a large scale
Test, it is ensured that enough detection flux.
Specific implementation step is：Certain density AFB1Target adds 50 L above-mentioned（1）In the middle magnetic bead modified, in
Under room temperature, gently concussion is incubated 30 min, and subsequent Magneto separate takes 10 L of supernatant, 10 10 × NEB of L buffer, 3.1 (1 M
NaCl, 500 mM Tris-HCl, 100 mM MgCl2, 1 mg/mL BSA, pH 7.9), 2 0.5 μM of L strand displacements
Probe (SDA probe, such as SEQ ID NO：Shown in 4), 2 L dNTPs (10 mM), 4 L Vent exo-Polymerase
(2 U/ L), 2 L Nt.Bst NBI (10 U/ L) nicking restriction endonuclease, and reactant is adjusted with the deionized water of sterilizing
Product reacts 1 h to 100 L at 55 DEG C.Then the probe such as SEQ ID NO of 10 L ferrocene labellings are added：Shown in 5, which 5 '
It is terminal modified to have ferrocene group mix homogeneously, take 40 L and be added drop-wise to（2）The middle electrode surface room temperature reaction 30 modified
Min, finally with 1 × AFB1Buffer solution is clean and is dried up with nitrogen.
（4）Electrochemical Detection
And then 40 L of Deca contains 10 mM MgCl20.1 M KClO4Solution is to above-mentioned（3）The electrode table modified
Face, carries out differential pulse（DPV）Electrochemical Detection.Window sweep length is 0 ~ 0.5 V, and pulse width is 50 mV, when mourning in silence
Between 0.2 s, pulse width be 0.06 s.Electrochemical Detection is carried out on UI5020 type electrochemical workstations under room temperature condition：
Electrochemistry experiment uses general screen printing electrode：Working electrode is the gold electrode modified, and platinum electrode is auxiliary electrode, chlorine
Change silver and make reference electrode.
For the specificity of verification method, it is specifically designed for polluting common symbiosis toxin such as FB in Semen Maydiss1, AFB2, OTA,
Disturbed test is carried out with T-2 toxin, as a result as shown in Figure 3.The specificity of the detection method is very high, to FB1, AFB2, OTA
With T-2 toxin almost without response；Even if being directed to and AFB1The extremely similar AFB of structure2Toxin, also only produces about 20%
Interference signal.
（5）Set up standard curve
Contain 10 mM MgCl toward 40 L of electrode surface Deca for having modified20.1 M KClO4Solution, carries out DPV electrochemical
Detection is learned, peak point current is recorded.The AFB of other concentration1Titer（0.1 pM、1 pM、10 pM、100 pM、1 nM、10 nM）
More than repeating（3）、（4）Experimental implementation carries out DPV measurements successively, and records corresponding peak point current.Vertical seat is made with peak point current
Mark, with AFB1Concentration makees abscissa mapping, sets up and determines AFB1Standard curve.
With optimal conditions, to AFB1Quantitative determined, as a result as shown in figure 4, the detection range of the method is 0.1
PM ~ 10 nM, detection are limited to 0.1 pM（3 times of background signal intensities）, linear equation is y=16.89 ln (CAFB1) +
89.63（R² = 0.991）.
（6）Corn sample is analyzed
The corn sample that 5 g have been crushed is weighed, 15 mL acetonitriles are added：Water（4:6, v/v）Extracting solution, shaken at room temperature extract 20
Min, 4 DEG C of centrifugations（4000 g）10 min, supernatant aperture are to dilute 4 with 1 × BB solution after the qualitative filter paper of 0.22 mm is filtered
Times.Then according to above-mentioned（3）、（4）Operation, carries out DPV Electrochemical Detection, records peak point current.Using above-mentioned（5）Set up
Aflatoxin B1Standard curve, calculates the AFB in corn sample solution1Concentration, and enter with business-like ELISA kit
Row contrast, for the Screening analysis of actual corn sample.
<110>Henan Academy of Agricultural Sciences's Agricultural development quality standard and detection technique institute
<120>It is a kind of to amplify AFB1 electrochemical detection methods and the application that hybridization is closed on surface based on strand displacement
<160> 5
<210> 1
<211> 47
<212> DNA
<213>Artificial sequence
<220>
<221> misc_difference
<222>（1）…（47）
<400> 1
gttgggcacg tgttgtctct ctgtgtctcg tgcccttcgc taggccc 47
<210> 2
<211> 21
<212> DNA
<213>Artificial sequence
<220>
<221> misc_difference
<222>（1）…（21）
<400> 2
acacgtgccc aacaatctgg t 21
<210> 3
<211> 14
<212> DNA
<213>Artificial sequence
<220>
<221> misc_difference
<222>（1）…（14）
<400> 3
gcagcaaaga gtcg 14
<210> 4
<211> 50
<212> DNA
<213>Artificial sequence
<220>
<221> misc_difference
<222>（1）…（50）
<400> 4
gcagcaacaa tctggacagt ttttgactca ccagattgtt gggcacgtgt 50
<210> 5
<211> 17
<212> DNA
<213>Artificial sequence
<220>
<221> misc_difference
<222>（1）…（17）
<400> 5
cgactctcaa tctggac 17
Claims (8)
1. it is a kind of to amplify the AFB that hybridization is closed on surface based on strand displacement1Electrochemical detection method, it is characterised in that：It is described
Detection method is by AFB1Neucleic acid aptamers as molecular recognition elements, screen printing electrode as transducer, using constant temperature chain
After displacement amplification method carries out signal amplification, then hybridization is closed on by surface carry out signal output.
2. it is as claimed in claim 1 to amplify the AFB that hybridization is closed on surface based on strand displacement1Electrochemical detection method, its
It is characterised by：The AFB1The sequence of neucleic acid aptamers such as SEQ ID NO：Shown in 1.
3. it is as claimed in claim 1 to amplify the AFB that hybridization is closed on surface based on strand displacement1Electrochemical detection method, its
It is characterised by, comprises the following steps：
（1）The surface modification of magnetic bead：By the AFB of biotin labeling1Aptamer and Block probe storing solutions, 95 DEG C of Jing of difference
Then 5min, the pretreatment of 0 DEG C of 10min use AFB11 × BB buffer is diluted to 200nM, and hybridizes 30min, system in room temperature
Obtain hybrid mixed liquid；Meanwhile, 50 L magnetic beads are taken with 500 L AFB11 × BB buffer solutions are twice；Take 200 L hybridization mixed
Close liquid and washed magnetic bead is added in centrifuge tube, be incubated 15min at room temperature, finally again with 500 L AFB11 × BB delays
Liquid is rushed, the magnetic bead being incubated is washed twice, is completed the surface modification of magnetic bead；
（2）The pretreatment of screen printing electrode and the preparation of sensing interface：It is clean in Jing dehydrated alcohol and deionized water wash
On the electrode surface of screen printing electrode, 40 L of Deca, 0.1 M H2SO4Solution, activates 60s under 2V DC voltages, is living
On screen printing electrode after change, 4 L of Deca contains the buffer solution of 1 μM of sulfydryl modification probe, assembles under the conditions of 30 DEG C
3h, uses AFB1Screen printing electrode after the assembling of 1 × BB buffer solution for cleaning, 4 L of last Deca, 1 mM6- sulfydryl hexanol solution
30min is closed in room temperature, AFB is used1After 1 × BB buffer solution for cleaning 3 times, screen printing electrode is dried up standby in 4 DEG C；
（3）AFB1Hybridization is closed on reference to the strand displacement and surface for mediating：In AFB150 L steps are added in target solution（1）
The middle magnetic bead modified, gently concussion is incubated 30 min at room temperature, and subsequent Magneto separate takes 10 L of supernatant, adds 10 L 10
× NEB buffer 3.1,2 L, 0.5 μM of strand displacing probes, 2 L, 10 mM dNTPs, 4 L, 2 U/ L polymerases and 2
10 U/ L nicking restriction endonucleases of L, deionized water supplement reaction system to 100 L, at 55 DEG C react 1h；Then add
10 L ferrocene label probes, after mix homogeneously, take 40 L and are added drop-wise to（2）In, the screen printing electrode room surface modified
Temperature 30 min of reaction, finally with 1 × AFB1Buffer solution is clean and is dried up with nitrogen；
（4）Electrochemical Detection：To Jing steps（3）40 L of screen printing electrode surface Deca for processing contains 10 mM MgCl2
0.1 M KClO4Solution, then carries out differential pulse Electrochemical Detection.
4. it is as claimed in claim 2 to amplify the AFB that hybridization is closed on surface based on strand displacement1Electrochemical detection method, its
It is characterised by：The step（1）The sequence of middle Block probes such as SEQ ID NO：Shown in 2, the concentration of Block probe liquid storages
For 10 M；The AFB1The formula of 1 × BB buffer is：10 mM Hepes, 120 mM NaCI, 5 mM KCI, 5 mM
MgCl2, pH 7.0。
5. it is as claimed in claim 2 to amplify the AFB that hybridization is closed on surface based on strand displacement1Electrochemical detection method, its
It is characterised by：The step（2）The sequence of middle sulfydryl modification probe such as SEQ ID NO：Shown in 3, the buffering of sulfydryl modification probe
The formula of solution is：1 μM of sulfydryl modification probe, 1M NaCl and 1mM TCEP.
6. it is as claimed in claim 2 to amplify the AFB that hybridization is closed on surface based on strand displacement1Electrochemical detection method, its
It is characterised by：The step（3）The sequence of middle strand displacing probes such as SEQ ID NO：Shown in 4, ferrocene label probe such as SEQ
ID NO：Shown in 5.
7. it is as claimed in claim 2 to amplify the AFB that hybridization is closed on surface based on strand displacement1Electrochemical detection method, its
It is characterised by：The step（4）In Electrochemical Detection carry out on electrochemical workstation, design parameter be window sweep length
For 0-0.5 V, pulse width is 50 mV, and 0.2 s of silence period, pulse width are 0.06 s.
8. it is as claimed in claim 1 or 2 to amplify the AFB that hybridization is closed on surface based on strand displacement1Electrochemical Detection side
Method, as AFB in detection crop1Application.
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