CN109030442A - A kind of detection method of detection probe and aflatoxin for detecting aflatoxin AFB1 - Google Patents
A kind of detection method of detection probe and aflatoxin for detecting aflatoxin AFB1 Download PDFInfo
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- CN109030442A CN109030442A CN201810864542.1A CN201810864542A CN109030442A CN 109030442 A CN109030442 A CN 109030442A CN 201810864542 A CN201810864542 A CN 201810864542A CN 109030442 A CN109030442 A CN 109030442A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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Abstract
The detection method of the detection probe and aflatoxin that the present invention relates to a kind of for detecting aflatoxin AFB1.The probe includes the aptamer with the sequence as shown in SEQ ID NO:1 and the aggregation-induced emission molecule AIE as shown in formula (I)；.Detection probe provided by the invention is label-free probe, and probe design is simple, application cost is low, easy to operate, and for detection limit down to 0.08 μ g/L, high specificity can be widely applied to ideal buffer solution system and actual sample (grape wine, coffee, milk).
Description
Technical field
The invention belongs to field of biological detection, more particularly, to a kind of for detecting the detection of aflatoxin AFB1
The detection method of probe and aflatoxin.
Background technique
Mycotoxin be by mycetogenetic toxicity secondary metabolite, to humans and animals have carcinogenic, teratogenesis mutagenesis,
The effects of nephrotoxicity, hepatotoxicity, immunosupress and genital disorders, brings huge to the life and health of humans and animals
Hidden danger.Wherein aflatoxin AFB1 is that the tool that is infected grain or food by aspergillus flavus and aspergillus parasiticus and generated is virose
The general name of secondary metabolite, easily pollution peanut, corn, oilseed, edible vegetable oil and feed, and can remain again dirty
In the tissue of dye, chemical property is extremely stable, toxicity is most strong, is classified as known most strong carcinogenic chemical by the World Health Organization
Substance.Since it has the characteristics that trace, severe toxicity, many kinds of, contamination is complicated, the degree-of-difficulty factor artificially controlled is caused to increase
Add, while also bringing great difficulty to actually detected, so establishing pollution of efficient, the quick detection method for research AFB1
Situation is effectively reduced its harm and is of great significance.
Traditional AFB1 detection method has thin-layered chromatography (TLC), instrumental method (high performance liquid chromatography-HPLC, liquid
Matter combination method-GC/MS etc.), exempt from enzyme linked immunological chemical analysis (ELISA, IAC etc.), electrochemical detection method and surface enhanced
Hand-pulled noodles method etc..However there are various shortcomings in practical applications in these methods, such as: TLC method needs to make in the detection process
With toxic reagent, operating procedure complexity, the not high, poor reproducibility of sensitivity etc., it is far from satisfying inspection of the current social to AFB1
Survey demand；Although HPLC method simplifies analytic process, overcome thermal instability, but sample pre-treatments are complicated, detection time is long,
Limit its practical application；GC/MS method can qualitatively and quantitatively analyze AFB1, but the device is complicated, cumbersome, to behaviour
The professional operation of author is horizontal high；Exempt from enzyme linked immunological chemical analysis in actual operation, since being coated on for solid phase carrier is each
It is extremely difficult between individual unanimously, therefore in being quantitative determined, every batch of sample detection must use same batch various concentration
Standard sample make standard curve under same operating condition, be unable to the ELISA reagent of cross-reference different batches
Box causes testing cost high, simultaneously as enzyme has unstability, so easily occurring false positive and negative result, detection with the method
Sensitivity is not high and needs to prepare specific antibody, and complex steps (coating, incubation and elution etc.), detection time is long.In recent years
Come, fluorescence analysis because its detection sensitivity is high, the selectivity characteristics such as strong, easy to use due to by extensive concern.But traditional is glimmering
Optical analysis detection AFB1 needs to carry out double labelling to probe, not only needs fluorophor, but also need quencher or auxiliary group,
The reliability for increasing testing cost, reducing detection.
Therefore, studying the short detection method of a kind of low testing cost, high reliablity, detection time, there is important research to anticipate
Justice and application value.
Summary of the invention
It is an object of the invention to overcome in the prior art AFB1 detection method testing cost is high, reliability is low, detection when
Between long defect and deficiency, provide a kind of for detecting the detection probe of aflatoxin.Detection probe provided by the invention is
Label-free probe, probe design is simple, application cost is low, easy to operate, and for detection limit down to 0.08 μ g/L, high specificity can be wide
It is general to be applied to ideal buffer solution system and actual sample.
Another object of the present invention is to provide application of the above-mentioned detection probe in detection aflatoxin AFB1.
Another object of the present invention is to provide the detection methods of aflatoxin AFB1 a kind of.
For achieving the above object, the present invention adopts the following technical scheme:
It is a kind of for detecting the detection probe of aflatoxin AFB1, the probe includes having the sequence as shown in SEQ ID NO:1
The aptamer of column and the aggregation-induced emission molecule AIE as shown in formula (I)；
。
Specifically, the sequence of the aptamer are as follows:
5’-GTT CGG CAC GTG TTG TCT CTC TGT GTC TCG TGC CCT TCG CTA GGC CCA CA-3’。
Probe provided by the invention is based on the aggregation-induced emission characteristic of tetraphenyl ethylene salt (TPE-Z), by the nucleic acid of AFB1
Aptamers and TPE-Z by electrostatic force in conjunction with, form stable compound.In the presence of no AFB1, TPE-Z is in dispersion
State, fluorescent quenching；In the presence of having AFB1, the aptamer of AFB1 forms duplex structure, at this time TPE- in conjunction with AFB1
Z is in coherent condition, and fluorescence restores.Changed by monitoring fluorescence signal, the content of AFB1 in judgement sample.
Detection probe provided by the invention is label-free probe, and probe design is simple, application cost is low, easy to operate, inspection
Limit is surveyed down to 0.08 μ g/L, high specificity can be widely applied to ideal buffer solution system and actual sample (grape wine, coffee
Coffee, milk).
Application of the above-mentioned detection probe in detection aflatoxin AFB1 is also within the scope of the present invention.
Preferably, the application is that detection probe detects aflatoxin AFB1 in grape wine, coffee, milk sample.
A kind of detection method of aflatoxin, includes the following steps:
S1: aptamer, AIE and buffer solution described in claim 1 are added into prepare liquid and obtains reaction solution；The reaction
The concentration of liquid amplifying nucleic acid aptamers is not less than 5.0 × 10-7mol/L；The concentration of the aptamer and AIE ratio are not less than 1:
50。
S2: for the reaction solution after 25 ~ 37 DEG C of reactions, fluorescence intensity when according to fluorescence detection can measure aspergillus flavus poison
The content of plain AFB1.
Method provided by the invention can realize the quick detection (detection time is about 60min) of aflatoxin AFB1, can
It is at low cost by property height.
Preferably, the concentration of the reaction solution amplifying nucleic acid aptamers is 5.0 × 10-7mol/L；The concentration of the AIE is
2.5×10-5 mol/L。
Preferably, the buffer solution is phosphoric acid (PBS) buffer solution.
Preferably, the pH of the buffer solution is 7.47 ± 0.5.
It is further preferable that the pH of the PBS solution is 7.47, including 2.5 mM MgCl2With 0.5 mM CaCl2。
Preferably, prepare liquid and the detection probe further include the detection probe at 90 ~ 95 DEG C before mixing in S1
The step of heating 3 ~ 5 min.
Preferably, the temperature reacted in S1 is 30 DEG C.
Preferably, further include the aptamer before S1 step at 90 ~ 95 DEG C, heat 3 ~ 5 min, cooling step
Suddenly.
It is further preferable that the temperature of the heating is 95 DEG C, time 4min.
Preferably, the excitation wavelength of the fluorescence detection is 350 nm, 400 ~ 650nm of launch wavelength scanning range.
Compared with prior art, the invention has the following beneficial effects:
Detection probe provided by the invention is label-free probe, and probe design is simple, application cost is low, easy to operate, detection limit
Down to 0.08 μ g/L, high specificity can be widely applied to ideal buffer solution system and actual sample (grape wine, coffee, ox
Milk).
Detailed description of the invention
Fig. 1 is the schematic diagram for the probe in detecting aflatoxin AFB1 that embodiment 1 provides；
Fig. 2 is influence diagram of the aflatoxin AFB1 to solution fluorescence intensity；
Fig. 3 is the glimmering light intensity and zero moment fluorescence intensity ratio figure of differential responses time；
Fig. 4 is the aflatoxin AFB1 fluorescence intensity testing result of different temperatures；
Fig. 5 is the aflatoxin AFB1 fluorescence intensity testing result of various concentration；
Fig. 6 is aflatoxin AFB1 fluorescence intensity figure in the probe in detecting grape wine solution of the offer of embodiment 1；
Fig. 7 is aflatoxin AFB1 fluorescence intensity figure in the probe in detecting infusion of the offer of embodiment 1；
Fig. 8 is aflatoxin AFB1 fluorescence intensity figure in the probe in detecting baby milk powder of the offer of embodiment 1.
Specific embodiment
Below with reference to embodiment, the present invention is further explained.These embodiments are merely to illustrate the present invention rather than limitation
The scope of the present invention.Test method without specific conditions in lower example embodiment usually according to this field normal condition or is pressed
The condition suggested according to manufacturer；Used raw material, reagent etc., unless otherwise specified, being can be from the business such as conventional market
The raw materials and reagents that approach obtains.The variation for any unsubstantiality that those skilled in the art is done on the basis of the present invention
And replacement belongs to scope of the present invention.
Embodiment 1
The present embodiment provides a kind of for detecting the detection probe of aflatoxin AFB1, including with following sequence: GTT CGG
Aptamer (the aspergillus flavus of CAC GTG TTG TCT CTC TGT GTC TCG TGC CCT TCG CTA GGC CCA C
The aptamer of toxin AFB1) and aggregation-induced emission molecule AIE.
The testing conditions that detection probe provided in this embodiment is used to detect aflatoxin are explored as follows:
(1) influence of aflatoxin AFB1 is explored
Using commercially available aflatoxin AFB1 as determinand, 1g/L solution is prepared.2 1.5 mL centrifuge tubes are taken, to the 1st
In centrifuge tube, the aptamer (AFB1 aptamer) and PBS of tetraphenyl ethylene salt (TPE-Z), aflatoxin AFB1 is added
Buffer solution；Into the 2nd centrifuge tube, the aptamer of tetraphenyl ethylene salt (TPE-Z), aflatoxin AFB1 is added
Aflatoxin AFB1 and the PBS buffer solution of (AFB1 aptamer), 8 μ g/L.React 90 points for (25 ~ 35 DEG C) at room temperature
Fluorescent value is measured after clock immediately.Reaction total volume in 2 centrifuge tubes is 200 μ L, and the end of tetraphenyl ethylene salt (TPE-Z) is dense
Degree is 2.5 × 10-5 The final concentration of the aptamer of mol/L, aflatoxin AFB1 is 5.0 × 10-7 mol/L.Its
Fluorescence spectral measuring condition are as follows: voltage is 600 v, and excitation wavelength is 350 nm, emits scanning range are as follows: 400 ~ 650 nm.
Fig. 1 is the schematic diagram for the method detection aflatoxin AFB1 that embodiment 1 provides.
Fig. 2 be in embodiment 1 aflatoxin AFB1 to the influence diagram of solution fluorescence intensity.It can be seen from the figure that
After introducing aflatoxin AFB1, fluorescence intensity substantially enhances, and illustrates that this method can be used for quantitative detection aflatoxin
AFB1。
(2) influence in reaction time is explored
9 centrifuge tubes are taken, tetraphenyl ethylene salt is separately added into, four root canals are all separately added into the nucleic acid adaptation of aflatoxin AFB1
Aflatoxin AFB1 and the PBS buffer solution of body, 8 μ g/L.It is 500 in revolving speed under (25 ~ 35 DEG C) at room temperature
After reacting 0 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 45 min, 60 min under rpm/min,
Survey its fluorescence.Reaction total volume in 9 centrifuge tubes is 200 μ L, and the final concentration of tetraphenyl ethylene salt (TPE-Z) is 2.5 ×
10-5The final concentration of the aptamer of mol/L, aflatoxin AFB1 is 5.0 × 10-7mol/L.Its fluorescence spectrum is surveyed
Amount condition are as follows: voltage is 600 v, and excitation wavelength is 350 nm, emits scanning range are as follows: 400 ~ 650 nm.
Fig. 3 is the influence diagram that the reaction time detects aflatoxin AFB1 in embodiment 1.It can be seen from the figure that with
The increase in reaction time, the fluorescence intensity of solution gradually increase, when reacted between be 60 min when, the fluorescence intensity of solution is most
Greatly and tend towards stability.When illustrating this method for quantitative detection aflatoxin AFB1,60 min are optimum reacting time.
(3) influence of reaction temperature is explored
4 centrifuge tubes are taken, tetraphenyl ethylene salt, the aptamer of aflatoxin AFB1,1.6 × 10 is added-6 The Huang of mol/L
Aspertoxin AFB1 and PBS buffer solution.It is individually placed to 4 DEG C, 16 DEG C, 25 DEG C, 30 DEG C, 37 DEG C and is protected from light place, reaction 60
After min, its fluorescence is detected.Reaction total volume in 4 centrifuge tubes is 200 μ L, the final concentration of tetraphenyl ethylene salt (TPE-Z)
It is 2.5 × 10-5The final concentration of the aptamer of mol/L, aflatoxin AFB1 is 5.0 × 10-7 mol/L.Its is glimmering
Light spectral measurement condition are as follows: voltage is 600 v, and excitation wavelength is 350 nm, emits scanning range are as follows: 400 ~ 650 nm.
Fig. 4 is the influence diagram that reaction temperature detects aflatoxin AFB1 in embodiment 1.It can be seen from the figure that with
The increase of reaction temperature, the fluorescence intensity of solution gradually increase, when reaction temperature be 30 DEG C when, the fluorescence intensity of solution is most
Greatly.When illustrating this method for quantitative detection aflatoxin AFB1,30 DEG C are optimal reaction temperature.
When can be seen that this method applied to aflatoxin AFB1 detection from above-mentioned test, optimal detection condition are as follows: anti-
Answering 30 DEG C of temperature, 60 min of reaction time, voltage is 600 v, and excitation wavelength is 350 nm, emits scanning range are as follows: 400 ~
65 0 nm。
(5) sensitivity that this method detects AFB1 is explored
6 1.5mL centrifuge tubes are taken, the aptamer and tetraphenyl ethylene salt of isoconcentration aflatoxin AFB1 are added thereto,
(concentration of aflatoxin AFB1 is by low for the aflatoxin AFB1 solution and PBS buffer solution for adding various concentration gradient
It is respectively as follows: 0 μ g/L, 0.08 μ g/L, 0.4 μ g/L, 0.8 μ g/L, 4 μ g/L, 8 μ g/L to height), the end of tetraphenyl ethylene salt is dense
Degree is 2.5 × 10-5 The final concentration of the aptamer of mol/L, aflatoxin AFB1 is 5.0 × 10-7 Mol/L).Instead
Answer condition and test condition identical as reaction condition in step 1 and test condition.
Fig. 5 is the aflatoxin AFB1 fluorescence intensity testing result of various concentration.It can be seen from the figure that with yellow bent
The increase of mould toxin AFB1 concentration, the fluorescence intensity of tetraphenyl ethylene salt gradually enhance.This method detects aflatoxin AFB1's
Detection is limited to 0.08 μ g/L.
Embodiment 2
It can detect aflatoxin AFB1 in grape wine, and the best inspection obtained by embodiment 1 using the probe that embodiment 1 provides
Survey condition is tested.It is specific as follows.
Step 1: 5 mL of grape wine for taking supermarket to buy, 4 DEG C of 1 weeks of standing.Supernatant liquor is taken to be centrifuged (10000 rpm/
Min), centrifugation after five minutes, takes supernatant liquor, and filtered with 13 μm of filter.
Step 2: taking grape wine in 5 parts of step 1, adds 5 μ L(0 μ g/L, 2.5 μ g/L, 5 μ g/L respectively) Huang it is bent
Mould toxin AFB1, adds PBS buffer solution.Reaction condition and survey in reaction condition and test condition and 1 step 1 of embodiment
Strip part is identical.
Fig. 6 is the aflatoxin AFB1 fluorescence intensity testing result that various concentration is added into grape wine solution.
Embodiment 3
It can detect aflatoxin AFB1 in coffee using the probe that embodiment 1 provides.It is specific as follows.
Step 1: the coffee for taking supermarket to buy takes 100 mg to be dissolved in 100 mL ultrapure waters, 4 DEG C of 1 weeks of standing.It takes
Layer clear liquid centrifugation (10000 rpm/min), centrifugation after five minutes, take supernatant liquor, and filtered with 13 μm of filter.
Step 2: taking infusion in 5 parts of step 1, adds the Huang of 5 μ L (0 μ g/L, 2.5 μ g/L, 5 μ g/L) respectively
Aspertoxin AFB1 adds PBS buffer solution.In reaction condition and test condition and 1 step 1 of embodiment reaction condition and
Test condition is identical.
Fig. 7 is the aflatoxin AFB1 fluorescence intensity testing result that various concentration is added into infusion.
Embodiment 4
It can detect aflatoxin AFB1 in milk powder using the probe that embodiment 1 provides.It is specific as follows.
Step 1: the baby milk powder for taking supermarket to buy takes 100 mg to be dissolved in 100 mL ultrapure waters, 4 DEG C of 1 stars of standing
Phase.Supernatant liquor is taken to be centrifuged (10000 rpm/min), centrifugation after five minutes, takes supernatant liquor, and filtered with 13 μm of filter.
Step 2: taking milk soln in 5 parts of step 1, adds 5 μ L's (0 μ g/L, 2.5 μ g/L, 5 μ g/L) respectively
Aflatoxin AFB1 adds PBS buffer solution.Reaction condition in reaction condition and test condition and 1 step 1 of embodiment
It is identical with test condition.
Fig. 8 is the aflatoxin AFB1 fluorescence intensity testing result that various concentration is added into baby milk powder.
Sequence table
<110>south of the Five Ridges college of education
<120>a kind of detection method of detection probe and aflatoxin for detecting aflatoxin AFB1
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 50
<212> DNA
<213> 1(1)
<400> 1
gttcggcacg tgttgtctct ctgtgtctcg tgcccttcgc taggcccaca 50
Claims (9)
1. a kind of for detecting the detection probe of aflatoxin AFB1, which is characterized in that the probe includes having such as SEQ
The aptamer of sequence shown in ID NO:1 and the aggregation-induced emission molecule AIE as shown in formula (I)；
。
2. application of the detection probe described in claim 1 in detection aflatoxin AFB1.
3. applying according to claim 2, which is characterized in that the application be detection probe white wine, grape wine, beer,
Aflatoxin AFB1 is detected in coffee, milk or grain sample.
4. a kind of detection method of aflatoxin AFB1, which comprises the steps of:
S1: aptamer, AIE and buffer solution described in claim 1 are added into prepare liquid and obtains reaction solution；The reaction
The concentration of liquid amplifying nucleic acid aptamers is not less than 5.0 × 10-7mol/L；The concentration of the aptamer and AIE ratio are not less than 1:
50；
S2: for the reaction solution after 25 ~ 37 DEG C of reactions, fluorescence intensity when according to fluorescence detection can measure aflatoxin
The content of AFB1.
5. detection method according to claim 4, which is characterized in that the concentration of the reaction solution amplifying nucleic acid aptamers is 5.0
×10-7mol/L；The concentration of the AIE is 2.5 × 10-5 mol/L。
6. detection method according to claim 4, which is characterized in that the buffer solution is phosphate buffer.
7. detection method according to claim 4, which is characterized in that the pH of the buffer solution is 7.47 ± 0.5.
8. detection method according to claim 7, which is characterized in that further include the aptamer before S1 step in 90 ~
At 95 DEG C, 3 ~ 5 min, cooling step are heated.
9. detection method according to claim 7, which is characterized in that the excitation wavelength of the fluorescence detection is 350 nm, hair
Penetrate 400 ~ 650 nm of wavelength scanning range.
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| CN113702370A (en) * | 2021-09-16 | 2021-11-26 | 盐城工学院 | Method for detecting aflatoxin B1 by using glucose-gold nanoparticles |
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| CN113866405A (en) * | 2021-10-15 | 2021-12-31 | 河南工业大学 | Preparation method of fluorescent aptamer sensor for simultaneously detecting ochratoxin A and aflatoxin B1 |
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Cited By (9)
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| CN111443066A (en) * | 2019-01-16 | 2020-07-24 | 香港科技大学 | Biological probe and application |
| CN111443066B (en) * | 2019-01-16 | 2023-02-10 | 香港科技大学 | Biological probe and application |
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| CN109946274B (en) * | 2019-03-15 | 2021-04-09 | 四川大学 | Method for detecting aflatoxin B1 based on aptamer inherent conformation induction |
| CN112326950A (en) * | 2020-10-20 | 2021-02-05 | 甘肃农业大学 | Detection method of T-2 toxin |
| CN113702367A (en) * | 2021-08-17 | 2021-11-26 | 川北医学院 | Method for detecting aflatoxin by naked eye visual detection probe |
| CN113702367B (en) * | 2021-08-17 | 2023-06-23 | 川北医学院 | Method for detecting aflatoxin by naked eye visual detection probe |
| CN113702370A (en) * | 2021-09-16 | 2021-11-26 | 盐城工学院 | Method for detecting aflatoxin B1 by using glucose-gold nanoparticles |
| CN113866405A (en) * | 2021-10-15 | 2021-12-31 | 河南工业大学 | Preparation method of fluorescent aptamer sensor for simultaneously detecting ochratoxin A and aflatoxin B1 |
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| CN109030442B (en) | 2019-06-21 |
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