CN104975079A - 17beta-estradiol visualization detection method based on DNA nano-structure, and 17beta-estradiol visualization detection kit based on DNA nano-structure - Google Patents
17beta-estradiol visualization detection method based on DNA nano-structure, and 17beta-estradiol visualization detection kit based on DNA nano-structure Download PDFInfo
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Abstract
The present invention discloses a 17beta-estradiol visualization detection method based on DNA nano-structure, and a 17beta-estradiol visualization detection kit based on DNA nano-structure. The working principle is that 17b-estradiol interacts with aptamer, a strand displacement reaction is started, three groups of stem-loop DNA probes are constantly opened to form DNA nano-structures, a G tetramer having catalysis activity is formed on the terminals of the three DNA arms in the DNA nano-structures, the G tetramer is bound with hemin so as to form a compound having catalysis activity and similar to HRP, TMB is subjected to catalytic oxidation, and a blue substrate is produced, wherein the result is visible, and the concentration of 17b-estradiol is directly related to the blue shade. According to the present invention, the operation is simple, the whole reaction can be completed at the room temperature, the high sensitivity is provided, the detection limit on 17b-estradiol is 100 fM, the good specificity is provided, the detection result is directly visible without any detection equipment, and the method and the kit can be used for the on-site rapid detection of 17b-estradiol.
Description
Technical field
The invention belongs to analytical chemistry field, relate to a kind of DNA nanostructure for 17-estradiol visible detection method and detection kit.
Background technology
17 beta estradiols (17 β-estradiol, E2) are that a class effect is the strongest, the endocrine disrupter of most potential hazard, are extensively present in river, soil, water source, air and agricultural-food.It is mainly derived from sterilant, waste gas, foodstuff additive, people and animals' movement and sanitary sewage, even if also can produce significantly impact to organism under extremely low concentration, its contents level and prostate cancer, mammary cancer, ovarian cancer, uterus carcinoma and male genetic obstacle have significant correlation.17 traditional beta estradiol detection methods mainly contain high performance liquid chromatography, gas chromatography mass spectrometry method, Liquid Chromatography/Mass Spectrometry etc., these methods need loaded down with trivial details sample pre-treatments and expensive instrument, high, the time-consuming effort of testing cost, needs veteran operator to be difficult to realize the on-the-spot real-time analysis of sample in enormous quantities.Another kind method detects 17 beta estradiols with antibody, but the preparation process of antibody is loaded down with trivial details, preserves trouble, and will relate to repeatedly washing and sepn process, thus limits their widespread use.
Summary of the invention
In order to solve deficiency existing in prior art, the object of the present invention is to provide a kind of 17 beta estradiol visible detection method and detection kit based on DNA nanostructure.
The technical solution used in the present invention is:
Based on 17 beta estradiol Visual retrieval test kits of DNA nanostructure, it comprises following component:
(1) 5' or the 3 ' end of the aptamer of DNA1:17 beta estradiol extends at least 24 bases, and form DNA1, it from 5 ' to 3 ' comprises Unit 5,2* unit, Unit 5,1* unit and 17 beta estradiol aptamer unit successively;
(2) the 1* unit of DNA2:DNA2 and DNA1 and part 17 beta estradiol aptamer unit complementary;
(3) stem circular DNA probe A: from 5 ' to 3 ' comprises Unit 4, Unit 1,5* unit, Unit 2,5* unit, Unit 5,3* unit, Unit 5,2* unit, Unit 5 successively;
(4) stem circular DNA probe B: stem circular DNA probe A from 5 ' to 3 ' comprises Unit 4, Unit 2,5* unit, Unit 3,5* unit, Unit 5,1* unit, Unit 5,3* unit, Unit 5 successively;
(5) stem circular DNA probe C: stem circular DNA probe C from 5 ' to 3 ' comprises Unit 4, Unit 3,5* unit, Unit 1,5* unit, Unit 5,2* unit, Unit 5,1* unit, Unit 5 successively;
In stem circular DNA probe A-C, Unit 4 and Unit 5 of head and the tail cut into two-part G tetramer nucleotide sequence, and the two is in separated position, cannot form complete G tetramer structure, catalytically inactive;
In above DNA molecular, Unit 1,2,3,5 are complementary with 1*, 2*, 3*, 5* unit respectively;
(6) teichmann's crystals
(7)H
2O
2
(8) TMB or ABTS or O-Phenylene Diamine.
As preferably, the complementary base radix of described DNA2 and DNA117 beta estradiol aptamer unit is 9-24; The base number of the Unit 1 in described DNA molecular, 1* unit, Unit 2,2* unit, Unit 3,3* unit, Unit 5,5* unit is 6-9.
As preferably, containing 3 groups of GGG bases in Unit 4 of described stem circular DNA probe A, stem circular DNA probe B or stem circular DNA probe C, containing 1 group of GGG base in Unit 5.
As preferably, in described DNA1, the sequence of 17 beta estradiol aptamers is as follows:
5'-GCTTCCAGCTTATTGAATTACACGCAGAGGGTAGCGGCTCTGCGCATTCAATTGCTGCGCGCTGAAGCGCGGAAGC-3'(SEQ ID NO.6)。
As preferably, the sequence of each DNA molecular is as follows:
DNA1：
5'-ATGGGTCTCACTATGGGTTCAACGGCTTCCAGCTTATTGAATTACACGCAGAGGGTAGCGGCTCTGCGCATTCAATTGCTGCGCGCTGAAGCGCGGAAGC-3'(SEQ ID NO.1)；
DNA2：5'-ATTCAATAAGCTGGAAGCCGTTGA-3'(SEQ ID NO.2)；
Stem circular DNA probe A:
5'-TGGGTAGGGCGGGTCGTTGAACCCATAGTGAGACCCATATGGGTCAAGACATGGGTCTCACTATGGGT-3'(SEQ ID NO.3)；
Stem circular DNA probe B:
5'-TGGGTAGGGCGGGTAGTGAGACCCATGTCTTGACCCATATGGGTTCAACGATGGGTCAAGACATGGGT-3'(SEQ ID NO.4)；
Stem circular DNA probe C:
5'-TGGGTAGGGCGGGTGTCTTGACCCATCGTTGAACCCATATGGGTCTCACTATGGGTTCAACGATGGGT-3'(SEQ ID NO.5)。
Also containing Tris-HCl damping fluid and colorbuffer in described test kit.
Based on 17 beta estradiol visible detection methods of DNA nanostructure, comprise the steps:
(1) DNA1, DNA2 and stem circular DNA probe are dissolved in damping fluid respectively;
(2) DNA1 and DNA2 solution is mixed, room temperature reaction;
(3) measuring samples is added in the reaction solution of step (2), mixing, room temperature reaction;
(4) in the reaction solution of step (3), stem circular DNA probe A, stem circular DNA probe B and stem circular DNA probe C is added, mixing, room temperature reaction;
(5) in step (4) reaction solution, teichmann's crystals is added, mixing, room temperature reaction;
(6) reaction solution getting step (5) adds containing TMB and H
2o
2colorbuffer in, mixing, room temperature reaction, observes colour-change, if produce blue substrate in reaction solution, then shows in measuring samples containing 17 beta estradiols;
Wherein, the sequence composition of DNA1, DNA2, stem circular DNA probe A, stem circular DNA probe B and stem circular DNA probe C as mentioned above.
As preferably, step (1) described damping fluid is Tris-HCl damping fluid, and pH is 7.4, containing 200mMNaCl and 50mMKCl.
As preferably, step (6) described colorbuffer contains 26.6mM citric acid, 51.4mM Sodium phosphate dibasic, 25mMKCl, the TMB of 10 μ L 0.5%, the H of 20 μ L 30%
2o
2, pH=5.0.
The invention has the beneficial effects as follows:
(1) detection method of the present invention and detection kit have higher sensitivity, are limited to 100fM to the detection of 17 beta estradiols, and detected result naked eyes are visible, are suitable for on-the-spot real-time analysis;
(2) detection method of 17 beta estradiols of the present invention has good specificity, and other common chaff interferences do not have an impact to detection;
(3) taking aptamer as molecular recognition elements, by forming DNA nanostructure, reaching the object that cascade signal amplifies.Using the G tetramer as signal reporter molecules, the direct naked eyes of detected result are visible, without the need to using any detecting instrument, without the need to using antibody, without the need to washing sepn process, simple to operate, are suitable for field quick detection.
Accompanying drawing explanation
Fig. 1 is the schematic diagram of detection method of the present invention;
Fig. 2 is the result figure detected 17 beta estradiols of different concns;
Fig. 3 is specificity experiments result figure.
Embodiment
Below in conjunction with embodiment, the present invention is further illustrated, but be not limited thereto.
Embodiment 1
Based on 17 beta estradiol Visual retrieval test kits of DNA nanostructure, it comprises following component:
(1)DNA1：
52*51*17 beta estradiol aptamer
5'-ATGGGT—CTCACT--ATGGGT--TCAACG—GCTTCCAGCTTATTGAATTACACGCAGAGGGTAGCGGCTCTGCGCATTCAATTGCTGCGCGCTGAAGCGCGGAAGC-3'(SEQ ID NO.1)
The 5' of the aptamer of 17 beta estradiols or 3 ' end extends, and form DNA1, it comprises Unit 5,2* unit, Unit 5,1* unit and 17 beta estradiol aptamer regions.
(2)DNA2：
5'-ATTCAATAAGCTGGAAGCCGTTGA-3'(SEQ ID NO.2)
The 1* region of DNA2 and DNA1 and part 17 beta estradiol aptamer regional complementarity.
(3) stem circular DNA probe A:
415*25*53*
5'-TGGGTAGGGCGGGT--CGTTGA—ACCCAT--AGTGAG—ACCCAT—ATGGGT--CAAGAC--ATGGGT--CTCACT--ATGGGT-3'(SEQ ID NO.3)
52*5
Stem circular DNA probe A from 5 ' to 3 ' comprises Unit 4, Unit 1,5* unit, Unit 2,5* unit, Unit 5,3* unit, Unit 5,2* unit and Unit 5 successively, and wherein, 5*-2-5* unit and the complementation of 5-2*-5 unit form loop-stem structure.
(4) stem circular DNA probe B:
425*35*51*
5'-TGGGTAGGGCGGGT--AGTGAG—ACCCAT—GTCTTG—ACCCAT—ATGGGT--TCAACG--ATGGGT--CAAGAC--ATGGGT-3'(SEQ ID NO.4)
53*5
Stem circular DNA probe A from 5 ' to 3 ' comprises Unit 4, Unit 2,5* unit, Unit 3,5* unit, Unit 5,1* unit, Unit 5,3* unit and Unit 5 successively, and wherein, 5*-3-5* region and 5-3*-5 regional complementarity form loop-stem structure.
(5) stem circular DNA probe C:
435*15*52*
5'-TGGGTAGGGCGGGT—GTCTTG--ACCCAT—CGTTGA—ACCCAT—ATGGGT—CTCACT--ATGGGT--TCAACG--ATGGGT-3'(SEQ ID NO.5)
51*5
Stem circular DNA probe C from 5 ' to 3 ' comprises Unit 4, Unit 3,5* unit, Unit 1,5* unit, Unit 5,2* unit, Unit 5,1* unit and Unit 5 successively, and wherein, 5*-1-5* region and 5-1*-5 regional complementarity form loop-stem structure.
In stem circular DNA probe A-C, Unit 4 and Unit 5 of head and the tail cut into two-part G tetramer nucleotide sequence, and the two is in separated position, cannot form complete G tetramer structure, catalytically inactive.
In above DNA molecular, Unit 1,2,3,5 are complementary with 1*, 2*, 3*, 5* unit respectively.
(6) teichmann's crystals;
(7)H
2O
2；
(8) TMB or ABTS or O-Phenylene Diamine;
(9) Tris-HCl damping fluid, containing 200mMNaCl and 50mMKCl.
(10) colorbuffer, comprises containing 26.6mM citric acid, 51.4mM Sodium phosphate dibasic, 25mMKCl, 10 μ L
The TMB of 0.5%, the H of 20 μ L 30%
2o
2, pH=5.0.
The principle of work of this test kit is:
(1) DNA1 and DNA2 is reacted for some time in buffer system, form DNA1-DNA2 mixture, in DNA1-DNA2 mixture, the 1* region in DNA1 is closed by DNA2.
(2) measuring samples is joined in DNA1-DNA2 reaction system, if containing 17 beta estradiols in measuring samples, 17 beta estradiols by with 17 beta estradiol aptamer regional interactions in DNA1, thus DNA2 to be replaced, is come out in the 1* region in DNA1.
(3) the 1* region of DNA1 exposure and 1 regional complementarity of stem circular DNA probe A are hybridized, and start strand replacement reaction, open loop-stem structure, form IA mixture, thus the original 2* region closed in stem circular DNA probe A is opened.
(4) 2 regional complementarities in the 2* region in IA mixture and stem circular DNA probe B are hybridized, and start strand replacement reaction, open loop-stem structure, form IAB mixture, thus the original 3* region closed in stem circular DNA probe B is opened.
(5) 3 regional complementarities in the 3* region in IAB mixture and stem circular DNA probe C are hybridized, and start strand replacement reaction, open loop-stem structure, form IABC mixture.This mixture itself is unstable, DNA1 can be replaced, and forms ABC mixture (DNA nanostructure).The DNA1 replaced can restart again the amplification of signal process of next round, thus constantly forms a large amount of DNA nanostructure.
(6) in ABC mixture, the end of every bar DNA arm all can further 4 regions and 5 regions, forms complete G tetramer sequences, after adding teichmann's crystals, can form the G tetramer with catalytic activity at every bar DNA arm end.They can catalyzed oxidation TMB-H
2o
2(tetramethyl benzidine-hydrogen peroxide) detection system, produce blue substrate, result naked eyes are visible, thus reach the object of detection 17 beta estradiol.
Embodiment 2
The test kit utilizing embodiment 1 to set up detects 17 beta estradiols, and step is as follows:
(1) Tris-HCl damping fluid (20mM, pH are 7.4, containing 200mMNaCl and 50mMKCl) dissolving DNA 1, DNA2 and stem circular DNA probe respectively is first used;
(2) DNA2 of DNA1 and the 400nM of 100nM is mixed, room temperature reaction 20 minutes, form DNA1-DNA2 mixture;
(3) measuring samples is joined in DNA1-DNA2 mixture, room temperature reaction 45 minutes;
(4) stem circular DNA probe A, B, C of 1 μM is added again, room temperature reaction 60 minutes;
(5) hemin (teichmann's crystals) of 0.3 μM is added, room temperature reaction 30 minutes;
(6) 50 μ L reaction solutions are taken out, to join in 950 μ L colorbuffer (containing 26.6mM citric acid, 51.4mM Sodium phosphate dibasic, 25mMKCl, the TMB of 10 μ L 0.5%, the H of 20 μ L 30%
2o
2, pH=5.0), room temperature reaction 15 minutes, observes colour-change;
If produce blue substrate in reaction solution, then show in measuring samples containing 17 beta estradiols.
Embodiment 3
Detection to different concns 17 beta estradiol:
Prepare 17 beta estradiol standardized solution, concentration is respectively 100fM, 1pM, 10pM, 100pM and 1nM, room temperature preservation.
Be added in the reaction system described in embodiment 1 respectively by 17 beta estradiol solution of different concns, fully observation experiment result after reaction, as shown in Figure 2,17 beta estradiols of 100fM can produce significantly blue change, illustrate that its detection is limited to 100fM.Along with the increase of 17 beta estradiol concentration, color also increases, and is tending towards saturated gradually.
Embodiment 4
Specificity experiments:
Compound concentration is the disturbance thing standardized solution of 10nM, is trihydroxy-oestrin, dihydroxyphenyl propane, progesterone, SevinCarbaryl, U-10149, mitomycin respectively.
The disturbance thing standardized solution of 10nM and 10pM17 beta estradiol standardized solution are added in the reaction system described in embodiment 1 respectively, colour-change is observed after abundant reaction, as shown in Figure 3, the trihydroxy-oestrin of 10nM, dihydroxyphenyl propane, progesterone, SevinCarbaryl, U-10149 and mitomycin do not produce colour-change, do not have an impact to detection.Only have and just can produce blueness after adding 17 beta estradiols, this proves that the detection of the method to 17 beta estradiols has good specificity.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from spirit of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
<110> Guangdong Prov. Inst. of Ecological Environment & Soil Science
<120> is based on 17 beta estradiol visible detection method and detection kit of DNA nanostructure
<130>
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 100
<212> DNA
<213> artificial sequence
<400> 1
atgggtctca ctatgggttc aacggcttcc agcttattga attacacgca gagggtagcg 60
gctctgcgca ttcaattgct gcgcgctgaa gcgcggaagc 100
<210> 2
<211> 24
<212> DNA
<213> artificial sequence
<400> 2
attcaataag ctggaagccg ttga 24
<210> 3
<211> 68
<212> DNA
<213> artificial sequence
<400> 3
tgggtagggc gggtcgttga acccatagtg agacccatat gggtcaagac atgggtctca 60
ctatgggt 68
<210> 4
<211> 68
<212> DNA
<213> artificial sequence
<400> 4
tgggtagggc gggtagtgag acccatgtct tgacccatat gggttcaacg atgggtcaag 60
acatgggt 68
<210> 5
<211> 68
<212> DNA
<213> artificial sequence
<400> 5
tgggtagggc gggtgtcttg acccatcgtt gaacccatat gggtctcact atgggttcaa 60
cgatgggt 68
<210> 6
<211> 76
<212> DNA
<213> artificial sequence
<400> 6
gcttccagct tattgaatta cacgcagagg gtagcggctc tgcgcattca attgctgcgc 60
gctgaagcgc ggaagc 76
Claims (10)
1., based on 17 beta estradiol Visual retrieval test kits of DNA nanostructure, it comprises following component:
(1) 5' or the 3 ' end of the aptamer of DNA1:17b-estradiol extends at least 24 bases, and form DNA1, it from 5 ' to 3 ' comprises Unit 5,2* unit, Unit 5,1* unit and 17b-estradiol aptamer unit successively;
(2) the 1* unit of DNA2:DNA2 and DNA1 and part 17b-estradiol aptamer unit complementary;
(3) stem circular DNA probe A: from 5 ' to 3 ' comprises Unit 4, Unit 1,5* unit, Unit 2,5* unit, Unit 5,3* unit, Unit 5,2* unit, Unit 5 successively;
(4) stem circular DNA probe B: stem circular DNA probe A from 5 ' to 3 ' comprises Unit 4, Unit 2,5* unit, Unit 3,5* unit, Unit 5,1* unit, Unit 5,3* unit, Unit 5 successively;
(5) stem circular DNA probe C: stem circular DNA probe C from 5 ' to 3 ' comprises Unit 4, Unit 3,5* unit, Unit 1,5* unit, Unit 5,2* unit, Unit 5,1* unit, Unit 5 successively;
In stem circular DNA probe A-C, Unit 4 and Unit 5 of head and the tail cut into two-part G tetramer nucleotide sequence, and the two is in separated position, cannot form complete G tetramer structure, catalytically inactive;
In above DNA molecular, Unit 1,2,3,5 are complementary with 1*, 2*, 3*, 5* unit respectively;
(6) teichmann's crystals;
（7）H
2O
2；
(8) TMB or ABTS or O-Phenylene Diamine.
2. 17 beta estradiol Visual retrieval test kits according to claim 1, is characterized in that, the complementary base radix of described DNA2 and DNA117b-estradiol aptamer unit is 9-24; The base number of the Unit 1 in described DNA molecular, 1* unit, Unit 2,2* unit, Unit 3,3* unit, Unit 5,5* unit is 6-9.
3. 17 beta estradiol Visual retrieval test kits according to claim 1, is characterized in that, containing 3 groups of GGG bases in Unit 4 of described stem circular DNA probe A, stem circular DNA probe B or stem circular DNA probe C, containing 1 group of GGG base in Unit 5.
4. 17 beta estradiol Visual retrieval test kits according to claim 1, is characterized in that, in described DNA1, the sequence of 17b-estradiol aptamer is as follows:
5'-GCTTCCAGCTTATTGAATTACACGCAGAGGGTAGCGGCTCTGCGCATTCAATTGCTGCGCGCTGAAGCGCGGAAGC-3'。
5. require 17 beta estradiol Visual retrieval test kits shown in any one of 1-4 according to power, it is characterized in that, the sequence of each DNA molecular is as follows:
DNA1：5'-ATGGGTCTCACTATGGGTTCAACGGCTTCCAGCTTATTGAATTACACGCAGAGGGTAGCGGCTCTGCGCATTCAATTGCTGCGCGCTGAAGCGCGGAAGC-3'；
DNA2：5'-ATTCAATAAGCTGGAAGCCGTTGA-3'；
Stem circular DNA probe A:5'-TGGGTAGGGCGGGTCGTTGAACCCATAGTGAGACCCATATGGGTCAAGACA TGGGTCTCACTATGGGT-3';
Stem circular DNA probe B:5'-TGGGTAGGGCGGGTAGTGAGACCCATGTCTTGACCCATATGGGTTCAACGA TGGGTCAAGACATGGGT-3';
Stem circular DNA probe C:5'-TGGGTAGGGCGGGTGTCTTGACCCATCGTTGAACCCATATGGGTCTCACTA TGGGTTCAACGATGGGT-3'.
6. 17 beta estradiol Visual retrieval test kits according to claim 1, is characterized in that, also containing Tris-HCl damping fluid and colorbuffer in described test kit.
7., based on 17 beta estradiol visible detection methods of DNA nanostructure, comprise the steps:
(1) DNA1, DNA2 and stem circular DNA probe are dissolved in damping fluid respectively;
(2) DNA1 and DNA2 solution is mixed, room temperature reaction;
(3) measuring samples is added in the reaction solution of step (2), mixing, room temperature reaction;
(4) in the reaction solution of step (3), stem circular DNA probe A, stem circular DNA probe B and stem circular DNA probe C is added, mixing, room temperature reaction;
(5) in step (4) reaction solution, teichmann's crystals is added, mixing, room temperature reaction;
(6) reaction solution getting step (5) adds containing TMB and H
2o
2colorbuffer in, mixing, room temperature reaction, observes colour-change, if produce blue substrate in reaction solution, then shows in measuring samples containing 17 beta estradiols;
Wherein, the sequence composition of DNA1, DNA2, stem circular DNA probe A, stem circular DNA probe B and stem circular DNA probe C is as shown in any one of claim 1-5.
8. 17 beta estradiol visible detection methods according to claim 7, is characterized in that, step (1) described damping fluid is Tris-HCl damping fluid, and pH is 7.4, containing 200 mMNaCl and 50 mMKCl.
9. 17 beta estradiol visible detection methods according to claim 7, is characterized in that, step (6) described colorbuffer contains 26.6 mM citric acids, 51.4 mM Sodium phosphate dibasics, 25 mMKCl, the TMB of 10 mL 0.5%, the H of 20 mL 30%
2o
2, pH=5.0.
The aptamer of 10.17b-estradiol, its for DNA molecular shown in following nucleotide sequence or with the DNA molecular shown in the nucleotide sequence of its complementation:
5'-GCTTCCAGCTTATTGAATTACACGCAGAGGGTAGCGGCTCTGCGCATTCAATTGCTGCGCGCTGAAGCGCGGAAGC-3'。
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