CN105567836A - Signal amplification nucleic acid aptamer sensor for determining aflatoxin B1 and preparation method of sensor - Google Patents

Signal amplification nucleic acid aptamer sensor for determining aflatoxin B1 and preparation method of sensor Download PDF

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CN105567836A
CN105567836A CN201610069597.4A CN201610069597A CN105567836A CN 105567836 A CN105567836 A CN 105567836A CN 201610069597 A CN201610069597 A CN 201610069597A CN 105567836 A CN105567836 A CN 105567836A
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electrode
afb
sensor
nucleic acid
sulfydryl
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王红旗
刘继红
张迪
尹海燕
俎建英
孙江南
祁玉峰
张玲
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Institute Of Agricultural Quality Standards And Testing Technology Henan Academy Of Agricultural Sciences
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Institute Of Agricultural Quality Standards And Testing Technology Henan Academy Of Agricultural Sciences
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/682—Signal amplification

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Abstract

The invention belongs to the technical field of agricultural product quality safety detection, and particularly relates to a signal amplification nucleic acid aptamer sensor for determining aflatoxin B1 and a preparation method of the sensor. A sulfydryl-modified probe in the signal amplification nucleic acid aptamer sensor is bonded to a gold electrode of a screen-printed electrode through a gold-sulfur key, an AFB1 nucleic acid aptamer is hybridized and assembled to the electrode through the sulfydryl-modified probe bonded to the surface of the electrode, and exonuclease participates in a reaction on the surface of the electrode. According to the sensor and the preparation method thereof, the AFB1 nucleic acid aptamer is adopted as a molecule recognition element, and the very good specificity is achieved compared with an antibody-based immunodetection technique; meanwhile, due to the fact that nucleic acid is very low in synthesis cost and is commercialized, the detection cost of the AFB1 is greatly reduced; the screen-printed electrode is adopted as a transducer, an enzyme digestion circulating signal amplification strategy of the exonuclease is adopted, and the advantages of being simple in structure, easy and convenient to process, low in cost, convenient to massively prepare, high in detection sensitivity and the like are achieved.

Description

A kind of mensuration AFB 1signal amplify aptamer sensor and preparation method thereof
Technical field
The invention belongs to agricultural product quality and safety detection technique field, be specifically related to a kind of mensuration AFB 1signal amplify aptamer sensor and preparation method thereof.
Background technology
Aflatoxin (aflatoxin, AF) is mycotoxins pollutent common in the plurality of cereals such as cereal, peanut, corn, oilseeds and feed, has become the huge killer affecting food safety, harm humans and animal health.Aflatoxin is the derivative of the dihydrofuran tonka bean camphor that a class is produced by flavus and Aspergillus parasiticus, and what have been found that at present has AFB 1, AFB 2, AFG 1, AFG 2, AFM 1and AFM 2deng 18 kinds, have induced mutation, Immunosuppression and carcinogenesis, being delimited by the Agency for Research on Cancer of the World Health Organization is I class carcinogenic substance.With AFB in natural food 1the most common, its hazardness is also the strongest.AFB 1toxicity than the strong toxicity 30 times of vomitoxin, than the strong toxicity 20 times of zearalenone; AFB 1acute toxicity be 10 times of potassium cyanide, 68 times of arsenic, chronic toxicity can bring out canceration, and carciongenic potency is 75 times of N-nitrosodimethylamine, higher than dimethyl diimide benzene 900 times, and the primary hepatocarcinoma of people is also probably relevant with aflatoxin.Therefore, a lot of country proposes strict limit standard to the AF content in food and feed in the world at present, wherein especially harsh with the standard of European Union's proposition, regulation AFB 1content must not more than 2ppb, and total AF content must not more than 4ppb.AFB 1quick, sensitive, accurate analysis for reducing the harm of AF to human life's safety, ensureing that the unimpeded of International import-export trade has extremely important effect.Current AFB 1detection method mainly contain high performance liquid chromatography (HPLC), thin layer chromatography (TLC), euzymelinked immunosorbent assay (ELISA) (ELISA) etc.Though these methods achieve greater advance, but still there is the different defects such as operational requirement is high, step is too loaded down with trivial details, sensitivity is poor, false drop rate is high, expensive equipment.
Aptamer is one section of single stranded oligonucleotide (20 ~ 60 base), can form distinctive three-dimensional structure, thus be combined with target molecule high-affinity, high specific by folding.Compared with antibody, the advantage of aptamer is: specificity is high, easily carry out chemosynthesis, transformation with mark, biological chemistry good stability, reversibly carry out Denaturation and Renaturation, the follow-up signals such as toolenzyme carries out increasing, shearing can also be made full use of and amplify process.The various analysis based on aptamer, detection method and technology have presented the advantages such as quick, sensitive, convenient, low cost, have demonstrated wide application prospect in biomedical applications, but relatively less in the application of mycotoxins context of detection.
Electrochemical biosensor detection method has the peculiar advantages such as quick, sensitive, plant and instrument is simple.Recent domestic investigator is to AFB 1the structure of electrochemica biological sensor carried out many-sided effort and trial, have studied the electrochemica biological sensor using antibody, DNA and enzyme as recognition element respectively, and the technology such as nanotechnology, ionic liquid, conductive polymers be applied to AFB 1analysis in, greatly enriched research contents and the thinking of electrochemica biological sensor and pathogenic eukaryotes.
So the present invention utilizes novel molecular recognition component---the high specific feature of aptamer, the signal amplifying function that bind nucleic acid excision enzyme cyclophorase is cut, utilizes electrochemistry simple and fast, high-sensitive advantage simultaneously, constructs a kind of mensuration AFB 1signal amplify aptamer sensor.
Summary of the invention
In order to solve current AFB 1the shortcoming that testing cost is high, false positive rate is high, detection time is long, the invention provides and measure AFB 1signal amplify aptamer sensor, adopt AFB 1neucleic acid aptamers is as molecular recognition elements, and screen printing electrode is as transverter, and exonuclease is as signal magnify tool.In the present invention, AFB 1the appearance enzyme that can cause exonuclease cut circulation amplify reaction, produce stronger electrochemical signals amplification effect.
A kind of mensuration AFB 1signal amplify aptamer sensor, sulfydryl modification probe is attached on the gold electrode of screen printing electrode by gold-sulfide linkage, AFB 1aptamer, by being attached to the sulfydryl modification probe hybridization of electrode surface, is assembled on electrode, and exonuclease participates in reaction at electrode surface.
Described sulfydryl modification probe is 3 ' terminal modifiedly have sulfydryl, the 5 ' terminal modified sulfydryl probe having ferrocene, and its sequence is as shown in SEQIDNO:1; AFB 1nucleic acid aptamer sequence is as shown in SEQIDNO:2.
A kind of mensuration flavus B 1signal amplify the preparation method of aptamer sensor, preparation process is as follows:
(1) drip in the gold electrode surfaces of screen printing electrode 1 × AFB that 3 μ L contain 1 μM of sulfydryl modification probe 1binding buffer solution, self-assembly 12h at 4 DEG C; Then, electrode 1 × BB solution cleans 3 times to remove the probe of weakly stable; Subsequently, electrode room temperature in 1mM mercaptoethanol solution closes 30min; Then the AFB of 1 μM of 1 × BB dilution is dripped toward electrode surface 1aptamers toxin soiutions 50 μ L, hybridizes 2h at 37 DEG C, after then using 1 × BB to clean 3 times, dries up, obtain the electrode modified after electrode clean is clean in 4 DEG C;
(2) drip 50 μ L at the electrode surface modified and contain AFB 1with 1 × BB solution of exonuclease, room temperature reaction 20min, can carry out differential pulse Electrochemical Detection after cleaning 3 times with 1 × BB.
Described 1 × AFB 1binding buffer solution is identical with 1 × BB solution, and component is 10mMHepes, 120mMNaCl, 5mMKCl, 5mMMgCl 2, pH7.0.
A kind of mensuration flavus B 1signal amplify aptamer sensor as AFB in detection corn 1application.Beneficial effect of the present invention is:
(1) the present invention adopts AFB 1aptamer, as molecular recognition elements, compared with the immunoassay technology based on antibody, has extraordinary specificity; Meanwhile, because the synthesis cost of nucleic acid is very cheap, and commercialization, greatly reduce AFB 1testing cost.
(2) the present invention adopts AFB 1adopt screen printing electrode to make transverter, adopt exonuclease enzyme to cut circulation amplify signal and amplify strategy, have structure simple, process easy, with low cost, be convenient to batch preparation, detection sensitivity high.
Accompanying drawing explanation
Fig. 1 is for measuring AFB 1signal amplify aptamer sensor Cleaning Principle figure;
Fig. 2 is for measuring AFB 1signal amplify the phenomenon figure of aptamer sensor;
Fig. 3 is standard working curve and linearity range.
Embodiment
A kind of mensuration AFB 1signal amplify aptamer sensor (as shown in Figure 1), sulfydryl modification probe is attached on the gold electrode of screen printing electrode by gold-sulfide linkage, AFB 1aptamer is assembled on electrode by the sulfydryl modification probe hybridization being attached to electrode surface, and exonuclease participates in reaction at electrode surface, plays signal and amplifies.
Described probe is 3 ' terminal modifiedly have sulfydryl, the 5 ' terminal modified sulfydryl probe having ferrocene, and its sequence is as shown in SEQIDNO:1; AFB 1nucleic acid aptamer sequence is as shown in SEQIDNO:2.
A kind of mensuration flavus B 1signal amplify the preparation method of aptamer sensor, preparation process is as follows:
(1) drip in the gold electrode surfaces of screen printing electrode 1 × AFB that 3 μ L contain 1 μM of sulfydryl modification probe 1binding buffer solution, self-assembly 12h at 4 DEG C; Then, electrode 1 × BB solution cleans 3 times to remove the probe of weakly stable; Subsequently, electrode room temperature in 1mM mercaptoethanol solution closes 30min; Then the AFB of 1 μM of 1 × BB dilution is dripped toward electrode surface 1aptamers toxin soiutions 50 μ L, hybridizes 2h at 37 DEG C, after then using 1 × BB to clean 3 times, dries up, obtain the electrode modified after electrode clean is clean in 4 DEG C;
(2) drip 50 μ L at the electrode surface modified and contain AFB 1with 1 × BB solution of exonuclease, room temperature reaction 20min, can carry out differential pulse Electrochemical Detection after cleaning 3 times with 1 × BB.
Described 1 × AFB 1binding buffer solution is identical with 1 × BB solution, and component is 10mMHepes, 120mMNaCl, 5mMKCl, 5mMMgCl 2, pH7.0.
A kind of mensuration flavus B 1signal amplify aptamer sensor as AFB in detection corn 1application, concrete steps are as follows:
(1) electrochemical signals amplification detection
Electrochemical Detection is carried out on CHI660E type electrochemical workstation under room temperature condition: drip 50 μ L1 × BB solution toward the electrode surface after cleaning; Electrochemistry experiment uses general screen printing electrode: working electrode is the gold electrode modified, and platinum electrode is supporting electrode, and reference electrode made by silver chloride.
The signal amplification effect of this detection method as shown in Figure 2, works as AFB 1in non-existent situation, exonuclease can not cut double-strand hybridization complex.The formation of DNA double chain has certain rigidity effect, because the distance on red probe between ferrocene and electrode is larger, cause the transfer transport between ferrocene electroactive group to electrode more difficult or cannot carry out, therefore on electrode, current signal is less or negligible.Work as AFB 1deposit in case, strand replacement reaction, i.e. AFB occur 1and the bonding force of aptamer is greater than the hybridization power between hybridization complex, thus causes dissociating of heteroduplex mixture, form AFB 1with aptamer in conjunction with mixture, the ferrocene electroactive group discharging strand modifies probe, this probe self is easy to form hairpin structure, thus the distance furthered between ferrocene and electrode, impel the ratio of the transfer transport between ferrocene electroactive group to electrode to be easier to, therefore current signal appears in electrode.Exonuclease can identify AFB 1with aptamer in conjunction with mixture, thus degraded aptamer discharge AFB 1, AFB 1with the reaction of heteroduplex mixture, so can circulate again, realize signal and amplify.
(2) AFB is utilized 1standardized solution Criterion curve
Drip 50 μ L at the electrode surface modified and contain 0.1pMAFB 1with 1 × BB solution of exonuclease, room temperature reaction 20min, then after using 1 × BB to clean 3 times, carry out DPV Electrochemical Detection, record peak point current; The AFB of other concentration 1reference liquid (1pM, 10pM, 100pM, 1nM, 10nM, 20nM, 40nM, 60nM, 80nM, 100nM) carries out DPV measurement successively by above experimental implementation, and it is as shown in the table to record corresponding peak point current;
Table 1 different concns AFB 1reference liquid DPV take off data is analyzed
Ordinate zou is made, with AFB with peak point current 1concentration does X-coordinate mapping, sets up and measures AFB 1signal amplify the typical curve (Fig. 3 A is the range of dynamic measurement of method, and Fig. 3 B is the linearity range of method) of aptamer sensor.
(3) detect containing AFB 1corn actual sample
Take the corn sample that 20g has pulverized, add 100mL phosphate buffered saline buffer, mechanical shaking extraction 1h, filter, it is for subsequent use with 1 × BB solution dilution to get appropriate filtrate; The past electrode surface modified drips the AFB that 50 μ L contain unknown concentration 1with 1 × BB solution of exonuclease, room temperature reaction 20min, then after using 1 × BB to clean 3 times, carry out DPV Electrochemical Detection, record peak point current.
Utilize the AFB that above-mentioned (2) are set up 1typical curve, calculates the AFB of described unknown concentration 1aFB in solution 1concentration is as shown in table 2.
AFB in table 2 corn actual sample 1detection
The present invention also can be applicable to AFB in the plurality of cereals such as cereal, peanut, oilseeds 1detection.

Claims (5)

1. one kind measures flavus B 1signal amplify aptamer sensor, it is characterized in that: sulfydryl modification probe is attached on the gold electrode of screen printing electrode by gold-sulfide linkage, AFB 1aptamer is assembled on electrode by the sulfydryl modification probe hybridization being attached to electrode surface, and exonuclease participates in reaction at electrode surface.
2. measure AFB as claimed in claim 1 1signal amplify aptamer sensor, it is characterized in that: described sulfydryl modification probe is 3 ' terminal modifiedly have sulfydryl, the 5 ' terminal modified sulfydryl probe having ferrocene, and its sequence is as shown in SEQIDNO:1; AFB 1nucleic acid aptamer sequence is as shown in SEQIDNO:2.
3. one kind measures flavus B 1signal amplify the preparation method of aptamer sensor, it is characterized in that: preparation process is as follows:
(1) drip in the gold electrode surfaces of screen printing electrode 1 × AFB that 3 μ L contain 1 μM of sulfydryl modification probe 1binding buffer solution, self-assembly 12h at 4 DEG C; Then, electrode 1 × BB solution cleans 3 times to remove the probe of weakly stable; Subsequently, electrode room temperature in 1mM mercaptoethanol solution closes 30min; Then the AFB of 1 μM of 1 × BB dilution is dripped toward electrode surface 1aptamers toxin soiutions 50 μ L, hybridizes 2h at 37 DEG C, after then using 1 × BB to clean 3 times, dries up, obtain the electrode modified after electrode clean is clean in 4 DEG C;
(2) drip 50 μ L at the electrode surface modified and contain AFB 1with 1 × BB solution of exonuclease, room temperature reaction 20min, can carry out differential pulse Electrochemical Detection after cleaning 3 times with 1 × BB.
4. the mensuration AFB as described in claim 1 or 3 1signal amplify aptamer sensor, it is characterized in that: described 1 × AFB 1binding buffer solution is identical with 1 × BB solution, and component is 10mMHepes, 120mMNaCl, 5mMKCl, 5mMMgCl 2, pH7.0.
5. one kind measures flavus B 1signal amplify aptamer sensor as AFB in detection corn 1application.
CN201610069597.4A 2016-02-01 2016-02-01 Signal amplification nucleic acid aptamer sensor for determining aflatoxin B1 and preparation method of sensor Pending CN105567836A (en)

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CN111060576A (en) * 2019-12-26 2020-04-24 中国科学院生态环境研究中心 Electrochemical sensor and method for detecting aflatoxin B1
CN111307907A (en) * 2020-04-07 2020-06-19 中国科学院生态环境研究中心 Reproducible electrochemical sensor and method for detecting aflatoxin
CN111793622A (en) * 2020-07-24 2020-10-20 江苏省原子医学研究所 Hairpin probe group based on enzyme-assisted cascade cyclic amplification, preparation method and application
CN113234800A (en) * 2021-05-13 2021-08-10 中山大学 Detection method of aflatoxin M1 and application thereof
CN115341015A (en) * 2022-09-06 2022-11-15 天津大学 Fluorescence biosensor for detecting aflatoxin B1, preparation method and application

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106680346A (en) * 2016-12-28 2017-05-17 河南省农业科学院农业质量标准与检测技术研究所 Electrochemical detection method for AFB1 (Aflatoxin B1) based on strand displacement amplification and surface proximity hybridization reaction and application thereof
CN111060576A (en) * 2019-12-26 2020-04-24 中国科学院生态环境研究中心 Electrochemical sensor and method for detecting aflatoxin B1
CN111307907A (en) * 2020-04-07 2020-06-19 中国科学院生态环境研究中心 Reproducible electrochemical sensor and method for detecting aflatoxin
CN111307907B (en) * 2020-04-07 2021-06-29 中国科学院生态环境研究中心 Reproducible electrochemical sensor and method for detecting aflatoxin
CN111793622A (en) * 2020-07-24 2020-10-20 江苏省原子医学研究所 Hairpin probe group based on enzyme-assisted cascade cyclic amplification, preparation method and application
CN113234800A (en) * 2021-05-13 2021-08-10 中山大学 Detection method of aflatoxin M1 and application thereof
CN113234800B (en) * 2021-05-13 2023-01-10 中山大学 Detection method of aflatoxin M1 and application thereof
CN115341015A (en) * 2022-09-06 2022-11-15 天津大学 Fluorescence biosensor for detecting aflatoxin B1, preparation method and application

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Application publication date: 20160511