CN107723347A - A kind of hypersensitive colorimetric sensing detection method for determining ochratoxin A - Google Patents
A kind of hypersensitive colorimetric sensing detection method for determining ochratoxin A Download PDFInfo
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Abstract
A kind of hypersensitive colorimetric sensing detection method for determining ochratoxin A, belongs to field of biological detection.The present invention includes：Magnetic nanometer modifies ochratoxin A aptamers, and the aptamers of magnetic nanometer modification form the DNA double chain structure of partial hybridization, the preparation of the gold nanoparticle probe of DNA modification, the structure of ochratoxin A colorimetric sensor with partial complementarity DNA molecular.The present invention is by means of ochratoxin A aptamers to ochratoxin A molecular specificity identification function, under the conditions of existing for various concentrations ochratoxin A, it is different by the assembling degree of ligase chain reaction golden nanometer particle, so as to form different color gradients, by determine react after ultraviolet absorption value change so as to realizing the detection to target ochratoxin A.The inventive method detection sensitivity is high, test limit is low, specificity is good, testing result is easy to observe, and a kind of effective method is provided for the detection of trace ochratoxin A.
Description
Technical field
A kind of hypersensitive colorimetric sensing detection method for determining ochratoxin A, belongs to field of biological detection.
Background technology
Ochratoxin is mycetogenetic one group of analogue, mainly there is tetra- kinds of compounds of A, B, C, D, its Poisoning
It is maximum, with human health most closely, production poison amount highest, the pollution to crops is most heavy, distribution is most wide is Aspergillus ochraceus poison
Plain A.Ochratoxin A is the toxic metabolic products as caused by aspergillus and Penicillium, mainly by pure green cyan is mould, Aspergillus ochraceus and carbon
Aspergillus niger produces, and is distributed widely in nature, is produced in Grain and its product, animal feed, animal product, cocoa and chocolate etc.
Pollution rate is higher in product, has strong Toxicity of Kidney and hepatotoxicity, and has teratogenesis, mutagenesis and carcinogenesis, therefore right
Human health forms and the huge potential threat of animal husbandry development, therefore it is outstanding to develop overdelicate Determination Methods for Ochratoxin A
To be important.
The instrumental method of detection ochratoxin A has liquid chromatogram, gas-chromatography, mass spectrum and capillary electrophoresis at present
Deng although these method sensitivity are higher, as a result stablizing, required instrument and equipment is expensive, and time-consuming for preparation of samples, is unsuitable for big
The screening of sample is measured, limits the large-scale application of such method.Immunological method based on antigen-antibody reaction is without costliness
The limitation of instrument, but the Antibody preparation of high sensitivity high selectivity needs longer cycle, while the stability of antibody also pole
It is vulnerable to the limitation of local environment, therefore the stability of Detection results is had a very big impact.Aptamers are rich by index
The Fas lignand system evolution technology of collection（SELEX）One kind of screening can identify the one section of single stranded DNA or RNA molecule of target molecule,
Identification of this quasi-molecule to target molecule has a high-affinity, and with stability it is good, it is easily prepared, reuse, be easy to
The advantages that modification, therefore aptamers are widely used in biological detection as identification molecule.
Golden nanometer particle has unique optical characteristics, including：Surface plasma resonance, higher absorption and extinction coefficient
And dependent on particle diameter and the properties of distance so that golden nanometer particle is widely used to basis as bioprobe and should
With research, the color change shown especially in accordance with the change of golden nanometer particle aggregation extent, it can develop and pass through naked eyes
The colorimetric sensor that can be identified so that the detection of biomolecule is more convenient.Ligase chain reaction is in heat-staple DNA
In the presence of ligase, it is possible to achieve the connection of shorter double chain DNA molecule otch, therefore can be incited somebody to action under the conditions of existing for template
The two segment DNA probe molecules and template Complementary hybridization, the otch of formation of golden nanometer particle modification are attached shape by ligase
Into a complete DNA, then the golden nanometer particle single strand dna newly to be formed is divided as new template by adding probe
Son forms starting template molecule again, and so as to realize the amplification of template strand and golden nanometer particle single strand dna, starting template contains
Amount is more, and the golden nanometer particle single strand dna amplified is more, and golden nanometer particle color is deeper.
The present invention is by means of ochratoxin A aptamers specific recognition ochratoxin A molecule, so that with being adapted to
The DNA molecular of body portion base complementrity disintegrates down, using the DNA molecular under dissociation as template, and with the Jenner complementary with template
The two kinds of DNA moleculars and be probe with two kinds of DNA moleculars of template sequence identical that rice corpuscles is modified, pass through ligase chain reaction
Expanded, under conditions of various concentrations ochratoxin A, with the increase of ochratoxin A concentration, be used under dissociating
The template DNA molecule of ligase chain reaction is more, then the assembling degree of the golden nanometer particle of DNA probe modification is bigger, Jenner
Grain of rice sub-color is deeper, according to the concentration of ochratoxin A and UV absorption ratio A630/A521 corresponding relation, so as to right
The content of ochratoxin A is detected.
The content of the invention
Technical problems to be solved：Because traditional instrument detection method equipment is expensive, time-consuming for preparation of samples, is unsuitable for big
The screening of sample is measured, limits the large-scale application of such method；Immunological method based on antigen-antibody relies on the sensitive of antibody
Degree and specificity, therefore the quality of antibody limits sensitivity and the accuracy of immunological method.
Technical scheme：The invention discloses a kind of hypersensitive colorimetric sensing detection method for determining ochratoxin A, including
Following specific steps：
（1）Magnetic nanometer modifies ochratoxin A aptamers
It is that 200-400nm concentration is 1mg mL by the particle diameter newly synthesized-1Carboxyl modified magnetic nanometer, with pH7.4's
0.01M phosphate buffer, which is diluted in 10 times, then the magnetic nanometer diluted to 1mL, adds 0.068 mg carbodiimides
With 0.15mg N- hydroxy thiosuccinimides, put it to and vibrate activation 20min on shaking table at ambient temperature, then will
The ochratoxin A aptamers DNA fragmentation that the 3 ' Amino End Groups that 50 μ L concentration are 10 μM are modified adds the magnetic nanometer activated above
In particle, under room temperature condition after oscillating reactions 3h, make it in centrifuge tube congregate with magnetic frame absorption magnetic nanometer, simultaneously
The free aptamers DNA molecular that supernatant is not coupled to remove is removed, then adds 1mL 0.01M pH8.0 TE buffer solutions
（Tris and EDTA）Magnetic nanometer is resuspended；
Ochratoxin A aptamers: 5’-GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGAAA
AA-NH2-3’。
（2）The aptamers of magnetic nanometer modification form part hybrid dna duplex structure with partial complementarity DNA molecular
Step will be added with the DNA fragmentation of ochratoxin A aptamers partial complementarity（1）Make in the middle magnetic nanometer modified
Its final concentration of 1 μM, then put it into 37 DEG C of thermostat water baths and be incubated 2h so that complementary DNA fragmentation fully hybridizes, then
Magnetic nanometer is eluted with magnetic frame, the DNA molecular to be dissociated with removing, then received magnetic with pH7.0 combination buffer
Rice corpuscles is resuspended, so as to obtain the partial hybridization DNA double chain structure of magnetic nanometer modification.
With ochratoxin A aptamers partial complementarity DNA：5’-CCACACCCGATCGGTAACCGACCG-3’.
（3）The preparation of the gold nanoparticle probe of DNA modification
The 4mL 15-25nm newly synthesized golden nanometer particle is centrifuged under conditions of 9000r/min and is concentrated 10 times, is used in combination
PH7.4 0.01M PBS is resuspended, and measures its final concentration of 25nM, then by the golden nanometer particle concentrated point
Every μ L of pipe 200 two pipes are dressed up, two kinds of DNA probe molecules of sulfydryl modification are added separately to the golden nanometer particle concentrated
In, the final concentration for making its DNA probe molecule is 100nM, after being incubated overnight at ambient temperature, by it in 10000r/min bars
Centrifuged under part, to remove uncombined DNA probe molecule, be then resuspended, obtained with pH7.4 0.01M phosphate buffer
To the gold nanoparticle probe of DNA modification；Two kinds of DNA probe molecules of sulfydryl modification can be with step（2）In with Aspergillus ochraceus poison
It is capable of the DNA molecular of complete complementary in the DNA of plain A aptamers partial complementarity both ends.
Probe 1：5 '-GATCGGGTGTGGAAAAA-SH-3 ',
Probe 2：5’-SH-AAAAACGGTCGGTTACC-3’.
（4）The structure of ochratoxin A colorimetric sensor
By step（2）The partial hybridization DNA double chain structure of the magnetic nanometer modification of middle preparation is dispensed into PCR pipe the often μ of pipe 100
L, it is then 100000 pg mL by initial concentration-110 times of the ochratoxin A standard solution concentration of gradient dilution 8, by it
The often μ L of pipe 10 are added separately in PCR pipe, are then incubated 20-50min under the conditions of 45 DEG C；By the reaction system magnetic after incubation
Power frame is separated, and collects the supernatant containing the lower DNA of dissociation respectively.
Every kind of supernatant takes 10 μ L to be separately added into different PCR pipes respectively, is then separately added into step in each PCR pipe
（3）Each 5 μ L of gold nanoparticle probe, the 5 μ L 50U DNA ligase of two kinds of DNA modifications of middle preparation（Purchased from the great life in Shanghai
Thing Technology Co., Ltd.）, 10 μ L 5 × PCR buffer solutions（Purchased from Shanghai Sheng Gong bioengineering limited company）And in addition
Each 5 μ L of two kinds of DNA molecular probes so that total reaction system is 45 μ L, puts it into progress ligase chain type in PCR amplification instrument
Reaction, reaction condition are：First in 90 DEG C of pre-degeneration 30s, then 60 DEG C of reaction 2min, 90 DEG C of denaturation 30s, are total up to 30 and follow
Ring, wherein other two kinds of DNA molecular probes are the both ends of template DNA molecule independence；Golden nanometer particle is determined after reaction respectively to exist
630nm and 521nm ultraviolet absorption value, and obtain A630/A521 UV absorption ratio, establish ochratoxin A concentration with
The standard curves of A630/A521 between the two, so as to realize the quantitative detection to ochratoxin A.
Probe 3：5 '-CCACACCCGATC-3 ',
Probe 4：5’-GGTAACCGACCG-3’.
The magnetic of carboxyl modified used in the hypersensitive colorimetric sensing detection method of measure ochratoxin A of the present invention
The particle diameter of nano-particle is 300nm.
The combination of pH7.0 used in the hypersensitive colorimetric sensing detection method of measure ochratoxin A of the present invention
The composition of buffer solution includes：0.01M Tris, 0.1M NaCl, 0.005M KCl and 0.02 mM CaCl2。
Golden nanometer particle used in the hypersensitive colorimetric sensing detection method of measure ochratoxin A of the present invention
Particle diameter is 18nm.
The hypersensitive colorimetric sensing detection method step of measure ochratoxin A of the present invention（4）Middle Aspergillus ochraceus poison
The incubation time of plain A standard items is 30min.
The magnetic nanometer of 300nm of the present invention carboxyl modified is synthesized by coprecipitation and to its surface
Carboxylated modification is carried out, synthesis step is：By 4gFeCl3·6H2O is weighed in three-necked flask, is added in 70mL deionized water
Dissolving, 70 DEG C are heated with stirring to, weigh 2gFeCl2·4H2O is dissolved in 5mL deionized waters, and 4mL is added in there-necked flask, fast
10mL25% concentrated ammonia liquor is rapidly added in the state of speed stirring, 3g oleic acid is added dropwise after reacting 1min, continues stirring reaction 1h
Afterwards, magnetic nanometer is separated by externally-applied magnetic field, and washed repeatedly with ethanol to remove unnecessary oleic acid, then used
Deionized water is washed to pH7.0, then adds the KMnO that 80mL concentration is 10mg/mL4Solution, sonic oscillation 8h, obtains carboxyl
The magnetic nanometer of modification.
The method that 18nm of the present invention golden nanometer particle reduces gold chloride by trisodium citrate is synthesized, and is closed
Into step：By there-necked flask chloroazotic acid soaked overnight, then cleaned up with ultra-pure water, add 95mL's in the there-necked flask of cleaning
Ultra-pure water, the gold chloride that 2.5mL mass concentrations are 0.4%, magnetic agitation and ebuillition of heated are added, 2 mL are added after 7-8min
Mass concentration is 1% trisodium citrate, and solution is changed into stopping heating after red, continues to stir 15min, that is, obtain from colourless
18nm golden nanometer particles.
Beneficial effect：The present invention by means of ochratoxin A aptamers specific recognition ochratoxin A molecule, so as to
So that the DNA molecular complementary with aptamers number of base disintegrates down, and ochratoxin A concentration is higher, the use under dissociating
It is more in the template DNA molecule of ligase chain reaction, using the DNA molecular under dissociation as template, with the Jenner complementary with template
The two kinds of DNA moleculars and be probe with two kinds of DNA moleculars of template sequence identical that rice corpuscles is modified, pass through ligase chain reaction
Expanded, different degrees of assembling occurs for the golden nanometer particle of DNA probe modification, golden after the higher amplification of initial template concentration
Nano-particle color is deeper, i.e. ochratoxin A concentration is higher, and golden nanometer particle color is deeper, according to the dense of ochratoxin A
The corresponding relation of degree and UV absorption ratio A630/A521 establishes standard curve, so as to realize the inspection to ochratoxin A content
Survey.
Brief description of the drawings
The TEM figures of Fig. 1 300nm magnetic nanometers.
The TEM figures of Fig. 2 18nm golden nanometer particles.
The change of ultra-violet absorption spectrum before and after the addition of Fig. 3 ochratoxin As.
The standard curve of Fig. 4 ochratoxin As detection.
Embodiment
Embodiment 1
A kind of hypersensitive colorimetric sensing detection method for determining ochratoxin A, is comprised the following specific steps that：
（1）Magnetic nanometer modifies ochratoxin A aptamers
It is that 300nm concentration is 1mg mL by the particle diameter newly synthesized-1Carboxyl modified magnetic nanometer, with pH7.4 0.01M
Phosphate buffer dilute 10 times, then the magnetic nanometer diluted to 1mL in add 0.068 mg carbodiimides with
0.15mg N- hydroxy thiosuccinimides, put it to and vibrate activation 20min on shaking table at ambient temperature, then by 50 μ
The ochratoxin A aptamers DNA fragmentation that the 3 ' Amino End Groups that L concentration is 10 μM are modified adds the magnetic nanometer activated above
In, under room temperature condition after oscillating reactions 3h, it is removed simultaneously in centrifuge tube congregate with magnetic frame absorption magnetic nanometer
The free aptamers DNA molecular that supernatant is not coupled to remove, 1mL 0.01M pH8.0 TE buffer solutions are then added by magnetic
Nano-particle is resuspended；
Ochratoxin A aptamers: 5’-GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGAAA
AA-NH2-3’。
（2）The aptamers of magnetic nanometer modification form part hybrid dna duplex structure with partial complementarity DNA molecular
Step will be added with the DNA fragmentation of ochratoxin A aptamers partial complementarity（1）Make in the middle magnetic nanometer modified
Its final concentration of 1 μM, then put it into 37 DEG C of thermostat water baths and be incubated 2h so that complementary DNA fragmentation fully hybridizes, then
Magnetic nanometer is eluted with magnetic frame, the DNA molecular to be dissociated with removing, then received magnetic with pH7.0 combination buffer
Rice corpuscles is resuspended, so as to obtain the partial hybridization DNA double chain structure of magnetic nanometer modification；
With ochratoxin A aptamers partial complementarity DNA：5’-CCACACCCGATCGGTAACCGACCG-3’.
（3）The preparation of the gold nanoparticle probe of DNA modification
The 4mL 18nm newly synthesized golden nanometer particle is centrifuged under conditions of 9000r/min and is concentrated 10 times, is used in combination
PH7.4 0.01M PBS is resuspended, and measures its final concentration of 25nM, then by the golden nanometer particle concentrated point
Every μ L of pipe 200 two pipes are dressed up, two kinds of DNA probe molecules of sulfydryl modification are added separately to the golden nanometer particle concentrated
In, the final concentration for making its DNA probe molecule is 100nM, after being incubated overnight at ambient temperature, by it in 10000r/min bars
Centrifuged under part, to remove uncombined DNA probe molecule, be then resuspended, obtained with pH7.4 0.01M phosphate buffer
To the gold nanoparticle probe of DNA modification；Two kinds of DNA probe molecules of sulfydryl modification can be with step（2）In with Aspergillus ochraceus poison
It is capable of the DNA molecular of complete complementary in the DNA of plain A aptamers partial complementarity both ends；
Probe 1：5 '-GATCGGGTGTGGAAAAA-SH-3 ',
Probe 2：5’-SH-AAAAACGGTCGGTTACC-3’.
（4）The structure of ochratoxin A colorimetric sensor and the research of detection sensitivity
By step（2）The partial hybridization DNA double chain structure of the magnetic nanometer modification of middle preparation is dispensed into PCR pipe the often μ of pipe 100
L, it is then 100000 pg mL-1 ochratoxin As 10 times of the standard solution concentration of gradient dilution 8 by initial concentration, will
It is added separately in PCR pipe the often μ L of pipe 10, and specific concentration is 0.01 pg mL-1、0.1 pg mL-1、1 pg mL-1、10pg
mL-1、100 pg mL-1、1000 pg mL-1、10000 pg mL-1、100000 pg mL-1, then it is incubated under the conditions of 45 DEG C
30min；Reaction system after incubation is separated with magnetic frame, and collects the supernatant containing the lower DNA of dissociation respectively.
Every kind of supernatant takes 10 μ L to be separately added into different PCR pipes respectively, is then separately added into step in each PCR pipe
（3）Each 5 μ L of gold nanoparticle probe of two kinds of DNA modifications of middle preparation, 5 μ L 50U DNA ligase, 10 5 × PCR of μ L delay
Fliud flushing and other each 5 μ L of two kinds of DNA molecular probes so that total reaction system is 45 μ L, is put it into PCR amplification instrument
PCR is attached, reaction condition is：First in 90 DEG C of pre-degeneration 30s, then 60 DEG C of reaction 2min, 90 DEG C of denaturation
30s, it is total up to 30 circulations；Determine the UV absorption of 630nm and 521nm in each reaction tube respectively with ultraviolet specrophotometer
Value, A630nm/A521nm ratio is then obtained, and in 0.1 pg mL-1-10000 pg mL-1In the range of, with Aspergillus ochraceus
Toxin A concentration is abscissa, a standard curve is made by ordinate of A630nm/A521nm ratio, according to standard curve
Calculate detection and be limited to 0.06 pg mL-1。
Probe 3：5 '-CCACACCCGATC-3 ',
Probe 4：5’-GGTAACCGACCG-3’.
（5）Special Journal of Sex Research
By four kinds of other biotoxins（The red enzyme ketenes of aflatoxin B1, fumonisins, vomitoxin, corn）As target
Molecule, the specificity of detection checking the method is carried out with the method.In 1ng mL-1Detectable concentration under, this several biotoxin
The reacted golden nanometer particle color of detecting system is no compared with before reacting to occur obvious change, so as to draw Aspergillus ochraceus
Toxin A is not identified with these four biotoxins, therefore the method goes out good spy to the detected representation of ochratoxin A
The opposite sex.
（6）Add recovery experiment
In negative corn extracts sample, the ochratoxin A measure addition recovery result of various concentrations is added respectively, 0.5,
5、20、50、150、200、500pg mL-1Addition concentration under, the recovery scope of ochratoxin A between 91%-105%, from
And illustrate the method and can be used for the detection of ochratoxin A in actual sample.
Claims (5)
1. a kind of hypersensitive colorimetric sensing detection method for determining ochratoxin A, it is characterised in that comprise the following specific steps that：
（1）Magnetic nanometer modifies ochratoxin A aptamers
It is that 200-400nm concentration is 1mg mL by the particle diameter newly synthesized-1Carboxyl modified magnetic nanometer, with pH7.4's
0.01M phosphate buffer dilute added in 10 times, then the magnetic nanometer diluted to 1mL 0.068mg carbodiimides with
0.15mg N- hydroxy thiosuccinimides, put it to and vibrate activation 20min on shaking table at ambient temperature, then by 50 μ
The ochratoxin A aptamers DNA fragmentation that the 3 ' Amino End Groups that L concentration is 10 μM are modified adds the magnetic nanometer activated above
In, under room temperature condition after oscillating reactions 3h, it is removed simultaneously in centrifuge tube congregate with magnetic frame absorption magnetic nanometer
The free aptamers DNA molecular that supernatant is not coupled to remove, 1mL 0.01M pH8.0 TE buffer solutions are then added by magnetic
Nano-particle is resuspended；
Ochratoxin A aptamers：5’-GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGAAAAA-
NH2-3’；
（2）The aptamers of magnetic nanometer modification form part hybrid dna duplex structure with partial complementarity DNA molecular
Step will be added with the DNA fragmentation of ochratoxin A aptamers partial complementarity（1）Make in the middle magnetic nanometer modified
Its final concentration of 1 μM, then put it into 37 DEG C of thermostat water baths and be incubated 2h so that complementary DNA fragmentation fully hybridizes, then
Magnetic nanometer is eluted with magnetic frame, the DNA molecular to be dissociated with removing, then received magnetic with pH7.0 combination buffer
Rice corpuscles is resuspended, so as to obtain the partial hybridization DNA double chain structure of magnetic nanometer modification；
With ochratoxin A aptamers partial complementarity DNA：5’-CCACACCCGATCGGTAACCGACCG-3’；
（3）The preparation of the gold nanoparticle probe of DNA modification
The 4mL 15-25nm newly synthesized golden nanometer particle is centrifuged under conditions of 9000r/min and is concentrated 10 times, is used in combination
PH7.4 0.01M PBS is resuspended, and measures its final concentration of 25nM, then by the golden nanometer particle concentrated point
Every μ L of pipe 200 two pipes are dressed up, two kinds of DNA probe molecules of sulfydryl modification are added separately to the golden nanometer particle concentrated
In, the final concentration for making its DNA probe molecule is 100nM, after being incubated overnight at ambient temperature, by it in 10000r/min bars
Centrifuged under part, to remove uncombined DNA probe molecule, be then resuspended, obtained with pH7.4 0.01M phosphate buffer
To the gold nanoparticle probe of DNA modification；Two kinds of DNA probe molecules of sulfydryl modification can be with step（2）In with Aspergillus ochraceus poison
It is capable of the DNA molecular of complete complementary in the DNA of plain A aptamers partial complementarity both ends；
Probe 1：5 '-GATCGGGTGTGGAAAAA-SH-3 ',
Probe 2：5’-SH-AAAAACGGTCGGTTACC-3’；
（4）The structure of ochratoxin A colorimetric sensor
By step（2）The partial hybridization DNA double chain structure of the magnetic nanometer modification of middle preparation is dispensed into PCR pipe the often μ of pipe 100
L, it is then 100000 pg mL by initial concentration-110 times of the ochratoxin A standard solution concentration of gradient dilution 8, by it
The often μ L of pipe 10 are added separately in PCR pipe, are then incubated 20-50min under the conditions of 45 DEG C；By the reaction system magnetic after incubation
Power frame is separated, and collects the supernatant containing the lower DNA of dissociation respectively；
Every kind of supernatant takes 10 μ L to be separately added into different PCR pipes respectively, is then separately added into step in each PCR pipe（3）
Each 5 μ L of gold nanoparticle probe of two kinds of DNA modifications of middle preparation, 5 μ L 50U DNA ligase, 10 5 × PCR of μ L bufferings
Liquid and other each 5 μ L of two kinds of DNA molecular probes so that total reaction system is 45 μ L, and it is enterprising to put it into PCR amplification instrument
Row ligase chain reaction, reaction condition are：First in 90 DEG C of pre-degeneration 30s, then 60 DEG C are reacted 2min, 90 DEG C of denaturation 30s,
30 circulations are total up to, wherein other two kinds of DNA molecular probes are the both ends of template DNA molecule independence；Determined respectively after reaction
Golden nanometer particle and obtains A630/A521 UV absorption ratio, establishes Aspergillus ochraceus in 630nm and 521nm ultraviolet absorption value
Toxin A concentration and the standard curves of A630/A521 between the two, so as to realize the quantitative detection to ochratoxin A；
Probe 3：5 '-CCACACCCGATC-3 ',
Probe 4：5’-GGTAACCGACCG-3’.
2. a kind of hypersensitive colorimetric sensing detection method for determining ochratoxin A according to claim 1, its feature exist
In the particle diameter of the magnetic nanometer of described carboxyl modified be 300nm.
3. a kind of hypersensitive colorimetric sensing detection method for determining ochratoxin A according to claim 1, its feature exist
Include in the composition of described pH7.0 combination buffer：0.01M Tris, 0.1M NaCl, 0.005M KCl and 0.02 mM
CaCl2。
4. a kind of hypersensitive colorimetric sensing detection method for determining ochratoxin A according to claim 1, its feature exist
In the particle diameter of described golden nanometer particle be 18nm.
5. a kind of hypersensitive colorimetric sensing detection method for determining ochratoxin A according to claim 1, its feature exist
In described step（4）The incubation time of middle ochratoxin A standard items is 30min.
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| CN109813703A (en) * | 2019-01-11 | 2019-05-28 | 东南大学 | The method of electrochemical luminescence aptamer sensor detection ochratoxin A based on the building of DNA walking robot |
| CN113670891A (en) * | 2021-08-04 | 2021-11-19 | 南京师范大学 | SERS sensor based on photonic crystal microspheres and preparation method and application thereof |
| CN113720794A (en) * | 2021-06-11 | 2021-11-30 | 海南大学 | Method for sensing and detecting mycotoxin in rice by using gold nanoparticle-based colorimetric aptamer |
| CN114354591A (en) * | 2022-01-10 | 2022-04-15 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Colorimetric/fluorescent dual-mode biosensing detection method for rapidly detecting aflatoxin B1 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102912020A (en) * | 2012-10-20 | 2013-02-06 | 江南大学 | Construction method of aptamer sensor for measuring ochratoxin A |
| US8680017B2 (en) * | 2010-02-13 | 2014-03-25 | Gregory Francis Lopreato | Lariat aptamer: aptamer candidate exclusion by nuclease digestion |
| CN105301085A (en) * | 2015-11-09 | 2016-02-03 | 江南大学 | Method for simultaneously detecting three antibiotic residues including streptomycin, chlorampenicol and tetracycline based on nucleic acid aptamer and quantum dots |
-
2017
- 2017-08-14 CN CN201710689444.4A patent/CN107723347A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8680017B2 (en) * | 2010-02-13 | 2014-03-25 | Gregory Francis Lopreato | Lariat aptamer: aptamer candidate exclusion by nuclease digestion |
| CN102912020A (en) * | 2012-10-20 | 2013-02-06 | 江南大学 | Construction method of aptamer sensor for measuring ochratoxin A |
| CN105301085A (en) * | 2015-11-09 | 2016-02-03 | 江南大学 | Method for simultaneously detecting three antibiotic residues including streptomycin, chlorampenicol and tetracycline based on nucleic acid aptamer and quantum dots |
Non-Patent Citations (4)
| Title |
|---|
| RICHA SHARMA等: "Recent advances in nanoparticle based aptasensors for food contaminants", 《BIOSENSORS AND BIOELECTRONICS》 * |
| SHUNBI XIE等: "Development of an electrochemical method for Ochratoxin A detection based on aptamer and loop-mediated isothermal amplification", 《BIOSENSORS AND BIOELECTRONICS》 * |
| 吕建新等: "《分子诊断学》", 28 February 2010, 中国医药科技出版社 * |
| 陈枢青等: "《精准医疗》", 31 July 2016, 天津科学技术出版社 * |
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|---|---|---|---|---|
| CN109342732A (en) * | 2018-10-31 | 2019-02-15 | 中国农业科学院油料作物研究所 | A kind of zygotic induction DNA assembling fluorescence detection device and the method using its detection small molecule content |
| CN109342732B (en) * | 2018-10-31 | 2022-04-15 | 中国农业科学院油料作物研究所 | Fluorescence detection device assembled by combining induced DNA and method for detecting content of small molecules by using fluorescence detection device |
| CN109632910A (en) * | 2018-12-20 | 2019-04-16 | 温州大学 | Functionalization Fe3O4The preparation method of/Au nanocomposite and its application detected in mycotoxin |
| CN109813703A (en) * | 2019-01-11 | 2019-05-28 | 东南大学 | The method of electrochemical luminescence aptamer sensor detection ochratoxin A based on the building of DNA walking robot |
| CN113720794A (en) * | 2021-06-11 | 2021-11-30 | 海南大学 | Method for sensing and detecting mycotoxin in rice by using gold nanoparticle-based colorimetric aptamer |
| CN113670891A (en) * | 2021-08-04 | 2021-11-19 | 南京师范大学 | SERS sensor based on photonic crystal microspheres and preparation method and application thereof |
| CN114354591A (en) * | 2022-01-10 | 2022-04-15 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Colorimetric/fluorescent dual-mode biosensing detection method for rapidly detecting aflatoxin B1 |
| CN114354591B (en) * | 2022-01-10 | 2023-06-02 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Colorimetric/fluorescent double-mode biosensing detection method for rapidly detecting aflatoxin B1 |
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