CN110068561A - A kind of bisphenol-A fluorescence detection method based on atom transition free radical polymerization reaction and truncation aptamers - Google Patents
A kind of bisphenol-A fluorescence detection method based on atom transition free radical polymerization reaction and truncation aptamers Download PDFInfo
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Abstract
The invention discloses a kind of based on atom transition free radical polymerization reaction and truncates the bisphenol-A fluorescence detection method of aptamers, the testing principle of this method is, under bisphenol-A existence condition, the hair clip DNA expansion being fixed on magnetic bead, hair clip DNA after expansion can connect upper ATRP reaction initiator PBIB by click-reaction, a large amount of fluorescent monomers are aggregated on the hair clip DNA of expansion by ATRP reaction forming, the fluorescence intensity finally measured through Fluorescence Spectrometer is positively correlated with bisphenol A concentration, is successfully realized the sensitive and accurate measurement to bisphenol A concentration.The result shows that the method for the present invention has higher sensitivity, selectivity, anti-interference and accuracy to the detection of bisphenol-A, there is huge practical application value in food safety and environmental monitoring.
Description
Technical field
The present invention relates to a kind of based on atom transition free radical polymerization reaction and truncates the bisphenol-A fluorescence detection side of aptamers
Method belongs to bioassay technique field.
Background technique
Bisphenol-A (BPA), full name 2, bis- (4- hydroxy phenyl) propane of 2-, are the important synthesis of polycarbonate and epoxy resin
The manufacture raw material of raw material and many common daily necessities such as packaging material for food, tableware, feeding bottle etc..BPA can pass through food packet
Packaging container or plastic film penetrate into food or beverage, the Reusability of these articles or are exposed to high-heat environment and will lead to BPA
Leaching, and then taken in by human body.Further, since containing in the landfill of house refuse, discarding and manufacturing works in daily life
There is a discharge of bisphenol-A waste water, there is also a large amount of bisphenol-As in environment.Many studies have shown that bisphenol-A has stronger endocrine
Interference activity, and basic physiology course can be destroyed.Bisphenol-A and many diseases such as thyroid function, metabolic disease, nerve are imitated
Answer, cancer etc. it is related.The traditional sensing techniques of bisphenol-A have high performance liquid chromatography (HPLC), gas chromatography combined with mass spectrometry (GC-
MS), liquid chromatography mass combination (LC-MS) etc., but there are sample pretreatment process complexity, expensive equipment, special for these methods
The deficiencies of operator of industry.Therefore it is very necessary for developing new highly selective, highly sensitive bisphenol-A detection method.
Aptamer is obtained by index ligand enrichment evolution technology (SELEX) screening to (small point of predetermined substance
Sub- compound, protein, cell, ion etc.) one section of single stranded DNA or RNA with high affinity.Relative to Immune discrimination
Detection technique, aptamers because have be easily-synthesized, chemical property is stable, be easy to modify, exists without the advantages such as prototype, cheap are immunized
Bio-sensing field is rapidly developed.There is high sensitivity, specificity based on the bio-sensing detection architecture that aptamers are established
The advantages that strong, since bisphenol-A aptamers screen to obtain, the bio-sensing detection method of various bisphenol-As continues to bring out out.
At the same time, in order to improve detection performance, various signal amplification techniques, such as hybridization chain reaction, rolling circle amplification are anti-
It answers, nano material, polymerization reaction etc. are applied to bio-sensing field.Wherein, atom transition free radical polymerization reaction (ATRP)
Have many advantages, such as that reaction process is controllable, reaction monomers are extensive, has been obtained since the nineties is suggested in fields such as material, synthesis
It widely applies, the application in bio-sensing also obtains the favor of researcher.
Summary of the invention
In view of the deficiencies of the prior art, the object of the present invention is to provide one kind based on atom transition free radical polymerization reaction and
The bisphenol-A fluorescence detection method for truncating aptamers, has the characteristics that highly selective, highly sensitive.
To achieve the goals above, the technical scheme is that
A kind of bisphenol-A fluorescence detection method based on atom transition free radical polymerization reaction and truncation aptamers, including with
Lower step:
(1) pre-treatment of hair clip DNA:
1. being cooled to room temperature after being heated to 95 DEG C by hair clip DNA solution and keep 15min；
2. hair clip DNA solution (100 μM) is mixed with TCEP solution (10 μM) with the ratio of volume ratio 1:1, and will mixing
Liquid is protected from light 3h under the conditions of 37 DEG C of constant-temperature table, and -20 DEG C save backup；In use, hair clip DNA solution is diluted to 1 μM；
(2) amino Fe3O4The modification of magnetic bead
1. measuring a certain amount of amino Fe3O4Magnetic bead stores liquid (10mg/ml), and Magnetic Isolation is removed supernatant, buffered with PBS
Liquid washing, Magnetic Isolation remove supernatant, and PBS buffer solution is added and is resuspended and restores to the magnetic bead storing liquid product of measurement；
2. measuring 20 μ L magnetic bead re-suspension liquids, 20 μ L Sulfo-SMCC solution (100 μM) and 160 μ L PBS buffer solution, mix
Afterwards, 2h is protected from light under the conditions of 37 DEG C of constant-temperature table；
3. by step 2. gained mixed liquor Magnetic Isolation, remove supernatant, washed with PBS buffer solution, Magnetic Isolation removes supernatant
Liquid；
4. 180 μ L PBS buffer solution and 20 μ L hair clip DNA solutions (1 μM) are added, after mixing, in 37 DEG C of conditions of constant-temperature table
Under be protected from light overnight；
(3) nucleic acid hybridizes
1. removing supernatant, being washed with TE buffer reaction solution Magnetic Isolation obtained by step (2)；
2. the target liquid and 180 μ L TE buffers of 20 μ L is added, after mixing, it is protected from light under the conditions of 37 DEG C of constant-temperature table anti-
Answer 2h；
(4) click-reaction
1. removing supernatant, being washed with PBS buffer solution, Magnetic Isolation is gone reaction solution Magnetic Isolation obtained by step (3)
Clear liquid；
2. 140 μ L PBS buffer solution, 20 μ L CuSO are added4Solution (1mM), 20 μ L PBIB solution (1mM), 20 μ L AA
Solution (2mM) after mixing, is protected from light 2h under the conditions of 37 DEG C of constant-temperature table；
(5) atom transfer radical polymerization (ATRP) is reacted
1. removing supernatant, being washed with PBS buffer solution, Magnetic Isolation is gone reaction solution Magnetic Isolation obtained by step (4)
Clear liquid；
2. 20 μ L fluorescent monomer solution (1mM), 140 μ L ultrapure waters and 20 μ L complex Cu are addedIIBr/Me6TREN solution
(1mM), 20 μ L AA solution (2mM) after mixing, are protected from light 2h under the conditions of 37 DEG C of constant-temperature table；
(6) fluorescence detection
1. reaction solution Magnetic Isolation obtained by step (5) is removed into supernatant, it is successively slow with 30% (v/v) DMF solution and PBS
Fliud flushing washing；
2. PBS buffer solution is added, after mixing, mixed liquor carries out fluorescence detection as sample on sepectrophotofluorometer.
The sequence of hair clip DNA is 5 '-SH- (CH2)6-CCACGCCGGTGGGTGGA ACGTGG-N3-3’。
Target liquid the preparation method comprises the following steps:
1. ssDNA-A (100 μM) and ssDNA-B (100 μM) is mixed with the ratio of volume ratio 1:1, and mixed liquor is added
Heat is to 95 DEG C and keeps 15min, is cooled to room temperature, and -20 DEG C save backup；
2. the mixed liquor of 10 μ L steps 1. is mixed with a certain amount of sample to be tested, then 500 μ L are diluted to, in constant-temperature table
In be protected from light under the conditions of 37 DEG C overnight to get target liquid, -20 DEG C save backup.
The sequence of ssDNA-A is 5 '-CCGGTGGGTGGA A-3 '.
The sequence of ssDNA-B is 5 '-TTCCACCCACCGG-3 '.
Step (3) -2. in be 0.5mM containing concentration in TE buffer MgCl2。
PBS buffer solution concentration is 0.1M, pH 7.4.
The excitation wavelength of fluorescence detection is 489nm, slit width 4nm in step (6).
The schematic illustration of detection method is as shown in Figure 1.
Beneficial effects of the present invention:
1, relative to traditional bisphenol-A aptamers, bisphenol-A aptamers is truncated and (refer to Highly sensitive
detection of bisphenol a by NanoAptamer assay with truncated aptamer.ACS Appl
The aptamers sequence reported in Mater Interfaces 9 (17): 14889-14898 documents, particular sequence 5 '-
CCGGTGGGTGGAA-3 ') and object between combine steric hindrance reduce, thus the two have more high-affinity, to improve
The selectivity and sensitivity of BPA detection.
2, use atom transfer radical polymerization to amplify strategy as signal, avoid nanometer in current convectional signals strategy
The use of material and biological enzyme (influencing vulnerable to external environment such as pH and temperature), signal obtain amplification at double, and sensitivity is very big
While raising, stability and reproducibility are relatively higher.
3, under bisphenol-A existence condition, the hair clip DNA expansion being fixed on magnetic bead, the hair clip DNA after expansion can pass through a little
ATRP reaction initiator PBIB on reaction forming is hit, a large amount of fluorescent monomers are aggregated in the hair clip of expansion by ATRP reaction forming
On DNA, finally, the fluorescence intensity measured through Fluorescence Spectrometer is positively correlated with bisphenol A concentration, is successfully realized dense to bisphenol-A
The sensitive and accurate measurement of degree.The result shows that under optimum experimental condition, in the concentration range of 100fM to 100nM, bisphenol A concentration
Logarithm and fluorescence intensity it is in a linear relationship, linear equation be F=3384LogC+11153 (R2=0.9985), detection limit is low
To 0.51fM.The sample containing 100nM bisphenol-A analog BPB, BPC, BPF, DES, E3 is examined using method of the invention
It surveys, measured fluorescence intensity is that 1nM BPA generates the 11.90%, 30.10%, 21.90% of signal strength respectively,
19.20%, 15.80%, it is substantially suitable with blank background signal, illustrate that the method for the present invention has very high selectivity.In addition, adopting
With the method for the present invention detection, the drinking water containing 1pM, 100pM, 10000pM BPA, the rate of recovery are respectively 102.0% respectively,
91.1%, 122.5%.In conclusion the method for the present invention has higher sensitivity, selectivity, anti-interference to the detection of bisphenol-A
And accuracy, there is huge practical application value in food safety and environmental monitoring.
Detailed description of the invention
Fig. 1 is the schematic illustration of detection method.
Fig. 2 is to be modified with the magnetic bead (A) of SSMCC/Hairpins/dsDNA-BPA/PBIB/P (FA) and be modified with SSMCC/
The laser confocal fluorescence microscope picture of the magnetic bead (B) of Hairpins/PBIB/P (FA).
Fig. 3 is the fluorescence spectrum of the magnetic bead of different modifying state.
Fig. 4 is the optimization in ATRP reaction time.
Fig. 5 is the optimization of fluorescent monomer concentration.
Fig. 6 is the fluorescence spectra (A) and bisphenol A concentration and corresponding fluorescence intensity of the bisphenol-A system containing various concentration
Relational graph (B).Figure A in bisphenol-A concentration be followed successively by from top to bottom 100nM, 10nM, 1nM, 100pM, 10pM, 1pM,
100fM。
Fig. 7 is that 100nM bisphenol-A analog BPB, BPF, BPC, DES, E3 and 1nM bisphenol-A is glimmering under the conditions of same detection
Luminous intensity comparison diagram.
Specific embodiment
Specific embodiments of the present invention will be described in further detail with reference to embodiments.
Amino Fe3O4Magnetic bead is purchased from Pu Ruimai lattice Biotechnology Co., Ltd (Xiamen).
The sequence of hair clip DNA are as follows: 5 '-SH- (CH2)6-CCACGCCGGTGGGTGGAACGTGG-N3-3’(SEQ ID
NO.1)。
The sequence of ssDNA-A are as follows: 5 '-CCGGTGGGTGGAA-3 ' (SEQ ID NO.2).
The sequence of ssDNA-B are as follows: 5 '-TTCCACCCACCGG-3 ' (SEQ ID NO.3).
Embodiment 1: the building of detection method
A kind of bisphenol-A fluorescence detection method based on atom transition free radical polymerization reaction and truncation aptamers, including with
Lower step:
(1) pre-treatment of hair clip DNA:
1. being slowly cooled to room temperature after being heated to 95 DEG C by hair clip DNA solution and keep 15min；
2. by hair clip DNA solution (100 μM) and three (2- carboxyethyl) phosphonium salt hydrochlorate (TCEP) solution (10 μM) with volume ratio
The ratio of 1:1 mixes, and mixed liquor is protected from light 3h under the conditions of 37 DEG C of constant-temperature table, and -20 DEG C save backup；In use,
DdH is added2Hair clip DNA solution is diluted to 1 μM by O；
(2) amino Fe3O4The modification of magnetic bead
1. measuring the amino Fe of 50 μ L3O4Magnetic bead stores liquid (10mg/ml), and Magnetic Isolation removes supernatant, uses PBS buffer solution
Wash (0.1M, pH 7.4) 3 times, Magnetic Isolation removes supernatant, and PBS buffer solution is added and is resuspended and restores to 50 μ L；
2. measuring 20 μ L magnetic bead re-suspension liquids, 20 μ L Sulfo-SMCC solution (100 μM) and 160 μ L PBS buffer solution, mix
Afterwards, it is protected from light 2h (suspended state of period holding magnetic bead) under the conditions of 37 DEG C of constant-temperature table, makes crosslinking aid S ulfo-SMCC
With amino magnetic bead with amido bond in conjunction with；
3. by step 2. gained mixed liquor Magnetic Isolation, remove supernatant, washed with PBS buffer solution, Magnetic Isolation removes supernatant
Liquid；
4. 180 μ L PBS buffer solution and 20 μ L hair clip DNA solutions (1 μM) are added, after mixing, in 37 DEG C of conditions of constant-temperature table
Under be protected from light overnight the suspended state of magnetic bead (period holding), so that hair clip DNA be made to be bonded with crosslinking aid S ulfo-SMCC；
(3) nucleic acid hybridizes
1. removing supernatant, being washed 1 time with 200 μ L TE buffers (pH 8) reaction solution Magnetic Isolation obtained by step (2)；
2. the target liquid and 180 μ L TE buffer (pH 8, MgCl containing 0.5mM of 20 μ L is added2), after mixing, in constant temperature
2h (suspended state of period holding magnetic bead) is protected from light under the conditions of 37 DEG C of shaking table；
Wherein, target liquid is the mixture of sample to be tested and dsDNA, it is specific the preparation method comprises the following steps:
1. ssDNA-A (100 μM) and ssDNA-B (100 μM) is mixed with the ratio of volume ratio 1:1, and mixed liquor is added
Heat is to 95 DEG C and keeps 15min, is slowly cooled to room temperature, and -20 DEG C save backup (dsDNA)；
2. the mixed liquor of 10 μ L steps 1. is mixed with 5 μ L samples to be tested, and TE buffer is added and is diluted to 500 μ L,
It is protected from light under the conditions of 37 DEG C in constant-temperature table overnight to get target liquid, -20 DEG C save backup；
(4) click-reaction
1. reaction solution Magnetic Isolation obtained by step (3) is removed supernatant, washed with 200 μ L PBS buffer solution, magnetism point
From removing supernatant；
2. 140 μ L PBS buffer solution, 20 μ L CuSO are added4Solution (1mM), 20 μ L bromo acid propynyl esters (PBIB)
Solution (1mM), 20 μ L ascorbic acid (AA) solution (2mM), after mixing, are protected from light 2h under the conditions of 37 DEG C of constant-temperature table；
(5) atom transfer radical polymerization (ATRP) is reacted
1. removing supernatant, being washed with PBS buffer solution, Magnetic Isolation is gone reaction solution Magnetic Isolation obtained by step (4)
Clear liquid；
2. 20 μ L fluorescent monomer solution (1mM), 140 μ L ultrapure waters and 20 μ L complex Cu are addedIIBr/Me6TREN solution
(1mM), 20 μ L AA solution (2mM), after mixing, 2h is protected from light under the conditions of 37 DEG C of constant-temperature table, and (period keeps the outstanding of magnetic bead
Floating state)；
(6) fluorescence detection
1. reaction solution Magnetic Isolation obtained by step (5) is removed into supernatant, it is successively slow with 30% (v/v) DMF solution and PBS
Fliud flushing is washed 2 times respectively；
2. 3000 μ L PBS buffer solution are added, after mixing, mixed liquor carries out on sepectrophotofluorometer glimmering as sample
Light detection (excitation wavelength 489nm, slit width 4nm).
Resulting fluorescence intensity is positively correlated with the bisphenol A concentration after dilution, after dilution is calculated using linear equation
Bisphenol A concentration, the concentration of bisphenol-A in sample to be tested can be obtained using volume conversion.
Under the conditions of blue light excitation module, when in target liquid there are when BPA, resulting final product SSMCC/
A large amount of dotted greens can be observed in the magnetic bead of Hairpins/dsDNA-BPA/PBIB/P (FA) modification under Laser Scanning Confocal Microscope
Fluorescence spot；On the contrary, resulting final product SSMCC/Hairpins/PBIB/P (FA) is repaired when BPA is not present in target liquid
The magnetic bead of decorations does not observe obvious fluorescence phenomenon (Fig. 2) under Laser Scanning Confocal Microscope, it was demonstrated that detection method of the invention is feasible.
Embodiment 2: feasibility verifying
Test the fluorescence spectrum of the magnetic bead of different modifying state respectively first.As shown in figure 3, firstly, being added without fluorescence list
Body FA (fluorescent monomer-O- acrylate), SSMCC/Hairpins/dsDNA-BPA/PBIB modify magnetic bead, and (curve b) is not examined
Measure any fluorescence intensity.On the contrary, by repeatedly washing still it can be observed that apparent fluorescence emission peak, this is after FA is added
Because a large amount of FA is introduced on magnetic bead by ATRP, great fluorescence signal (curve a) is produced.This not only shows BPA
In conjunction with dsDNA and release one can with hair clip DNA circle portion sequence complementary pairing and can by foldable structure open single stranded DNA,
And show to test signal-to-noise ratio with higher (S/N) and sensitivity.By comparative experiments, sufficiently demonstrate based on truncation bis-phenol
The signal amplification strategy of the ATRP of A aptamers is feasible applied to the detection of BPA.
Embodiment 3: testing conditions optimization
(1) optimization in reaction time
By detecting the fluorescence intensity of the magnetic bead in different ATRP reaction time, reaction time and the fluorescence for having studied ATRP are strong
The relationship of degree.As a result as shown in figure 4, fluorescence intensity is gradually increased with the extension of reaction time and has reached steady in 120min
It is fixed.Therefore, the ATRP reaction time is selected as 120min.
(2) optimization of fluorescent monomer concentration
Fluorescent monomer concentration can influence the length of polymer chain and then influence detection performance.When fluorescent monomer content in system
When insufficient, maximum is not achieved in fluorescence intensity.Respectively measure ATRP system in fluorescent monomer concentration be 0.025mM, 0.05mM,
0.1mM, 0.2mM, 0.3mM, bisphenol A concentration are the fluorescence intensity of the magnetic bead of 100nM, as a result as shown in figure 5, when fluorescent monomer is dense
The fluorescence intensity of magnetic bead reaches maximum when degree is 0.1mM, hereafter continues the concentration for increasing fluorescent monomer, and fluorescence intensity is simultaneously not apparent from
Increase, therefore 0.1mM fluorescent monomer concentration is selected to be applied to following experiment.
(3) optimization of pH
PH value is to CuIIBr/Me6TREN and CuI/Me6TREN (restores Cu by AAIIBr/Me6TREN is obtained) composition catalysis
System has a great impact.In acid condition, be conducive to lead to Cu containing the protonation of a variety of amine ligandsIIBr/Me6TREN and
CuI/Me6The dissociation of TREN.In the case where not stablizing ligand, from CuI/Me6The Cu come is dissociateed in TREN1+More easily go out
Now it is disproportionated.Under alkaline condition, CuIIBr/Me6TREN can be converted to CuIIOH/Me6TREN, this is because OH-Compare Br-Have stronger
Coordination ability.Generally speaking, alkaline condition and acid condition are all unfavorable for the stability of catalyst system, to ATRP and unfavorable
's.Therefore, select pH 7 as optimal parameter.
In conclusion ATRP optimum optimizing condition are as follows: the ATRP reaction time is 120min, and fluorescent monomer concentration is 0.1mM,
PH is 7.
Embodiment 4: sensitivity experiment
According to the optimum experimental condition that 3 condition optimizing of embodiment is obtained, the bisphenol-A of various concentration in target liquid is detected
(100fM, 1pM, 10pM, 100pM, 1nM, 10nM, 100nM) generate fluorescence intensity size, come study the method for the present invention for
The detection performance of bisphenol-A.As shown in fig. 6, the fluorescence intensity of magnetic bead increases as the concentration of bisphenol-A increases, this is because double
Phenol A concentration is bigger, and after dsDNA reaction bonded, the free ssDNA-B number of generation is more, and then leads to the hair clip DNA of expansion
Number is bigger, to keep the fluorescent monomer for introducing magnetic bead more, the fluorescence intensity of generation is stronger.Fluorescence emission maximum peak intensity with
Good linear relationship, linear equation F=3384LogC+ is presented in 100fM to 100nM range in the logarithm of bisphenol A concentration
11153(R2=0.9985), detection is limited to 0.51fM, the results showed that the method for the present invention detection sensitivity with higher, it is lower
Detection limit, have excellent performance.
Embodiment 5: selectivity experiment
According to the optimum experimental condition that 3 condition optimizing of embodiment is obtained, bisphenol-A analog 2, (the 4- hydroxyl of 2- bis- are studied
Phenyl) butane (BPB), 4,4 ' dihydroxydiphenyl methane (BPF), 2,2- bis- (4- hydroxyl -3- tolyl) (BPC), hexene be female
The fluorescence intensity that phenol (DES), estriol (E3) use the method for the present invention to generate.Bisphenol-A particular by comparison 100nM is similar
The signal strength gap that the bisphenol-A of object and 1nM generate.As shown in fig. 7, the fluorescence intensity of BPB, BPC, BPF, DES, E3 are respectively
11.90%, 30.10%, 21.90%, 19.20%, the 15.80% of BPA, bisphenol-A analog generate fluorescence signal relative to
Bisphenol-A is smaller, substantially suitable with blank background signal, it was demonstrated that the method for the present invention selectivity with higher, this is because bisphenol-A
Aptamers for bisphenol-A high affinity, to bisphenol-A analog recognition capability with higher.
Embodiment 6: the detection performance in actual sample
According to the optimum experimental condition that 3 condition optimizing of embodiment is obtained, by quantitative concentrations (final concentration 1pM, 100pM,
Bisphenol-A 10000pM) is dissolved in drinking water, probes into the content of bisphenol A detected using the method for the present invention, studies the method for the present invention
Detection performance in actual sample.The results are shown in Table 1, be quantitatively adding respectively into drinking water final concentration 1pM, 100pM,
The bisphenol-A of 10000pM, is detected using the method for the present invention, and the rate of recovery is respectively 102.0%, 91.1%, 122.5%, shows this hair
Bright method has preferable reliability and accuracy in actual sample, has strong anti-interference ability, can be used for practical sample
The detection of product.
Table 1: the bisphenol-A various concentration rate of recovery
Sequence table
<110>Henan university of TCM
<120>a kind of bisphenol-A fluorescence detection method based on atom transition free radical polymerization reaction and truncation aptamers
<160> 3
<170> SIPOSequenceListing 1.0
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ccacgccggt gggtggaacg tgg 23
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ccggtgggtg gaa 13
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ttccacccac cgg 13
Claims (7)
1. a kind of bisphenol-A fluorescence detection method based on atom transition free radical polymerization reaction and truncation aptamers, feature exist
In, comprising the following steps:
(1) pre-treatment of hair clip DNA:
1. being cooled to room temperature after being heated to 95 DEG C by hair clip DNA solution and keep 15min；
2. hair clip DNA solution (100 μM) is mixed with TCEP solution (10 μM) with the ratio of volume ratio 1:1, and mixed liquor is existed
It is protected from light 3h under the conditions of 37 DEG C of constant-temperature table, -20 DEG C save backup；In use, hair clip DNA solution is diluted to 1 μM；
(2) amino Fe3O4The modification of magnetic bead
1. measuring a certain amount of amino Fe3O4Magnetic bead stores liquid (10mg/ml), and Magnetic Isolation is removed supernatant, washed with PBS buffer solution
It washs, Magnetic Isolation, removes supernatant, PBS buffer solution is added and is resuspended and restores to the magnetic bead storing liquid product of measurement；
2. 20 μ L magnetic bead re-suspension liquids, 20 μ L Sulfo-SMCC solution (100 μM) and 160 μ L PBS buffer solution are measured, after mixing,
2h is protected from light under the conditions of 37 DEG C of constant-temperature table；
3. by step 2. gained mixed liquor Magnetic Isolation, remove supernatant, washed with PBS buffer solution, Magnetic Isolation removes supernatant；
4. 180 μ L PBS buffer solution and 20 μ L hair clip DNA solutions (1 μM) are added, after mixing, kept away under the conditions of 37 DEG C of constant-temperature table
Light reaction is stayed overnight；
(3) nucleic acid hybridizes
1. removing supernatant, being washed with TE buffer reaction solution Magnetic Isolation obtained by step (2)；
2. the target liquid and 180 μ L TE buffers of 20 μ L is added, after mixing, 2h is protected from light under the conditions of 37 DEG C of constant-temperature table；
(4) click-reaction
1. removing supernatant, being washed with PBS buffer solution, Magnetic Isolation removes supernatant reaction solution Magnetic Isolation obtained by step (3)；
2. 140 μ L PBS buffer solution, 20 μ L CuSO are added4Solution (1mM), 20 μ L PBIB solution (1mM), 20 μ L AA solution
(2mM) after mixing, is protected from light 2h under the conditions of 37 DEG C of constant-temperature table；
(5) atom transfer radical polymerization (ATRP) is reacted
1. removing supernatant, being washed with PBS buffer solution, Magnetic Isolation removes supernatant reaction solution Magnetic Isolation obtained by step (4)；
2. 20 μ L fluorescent monomer solution (1mM), 140 μ L ultrapure waters and 20 μ L complex Cu are addedIIBr/Me6TREN solution
(1mM), 20 μ L AA solution (2mM) after mixing, are protected from light 2h under the conditions of 37 DEG C of constant-temperature table；
(6) fluorescence detection
1. reaction solution Magnetic Isolation obtained by step (5) is removed supernatant, successively with 30% (v/v) DMF solution and PBS buffer solution
Washing；
2. PBS buffer solution is added, after mixing, mixed liquor carries out fluorescence detection as sample on sepectrophotofluorometer.
2. the bisphenol-A fluorescence inspection according to claim 1 based on atom transition free radical polymerization reaction and truncation aptamers
Survey method, which is characterized in that the sequence of the hair clip DNA is 5 '-SH- (CH2)6-CCACGCCGGTGGGTGGAACGTGG-N3-
3’。
3. the bisphenol-A fluorescence inspection according to claim 1 based on atom transition free radical polymerization reaction and truncation aptamers
Survey method, which is characterized in that the target liquid the preparation method comprises the following steps:
1. ssDNA-A (100 μM) and ssDNA-B (100 μM) is mixed with the ratio of volume ratio 1:1, and mixed liquor is heated to
95 DEG C and 15min is kept, is cooled to room temperature, -20 DEG C save backup；
2. the mixed liquor of 10 μ L steps 1. is mixed with a certain amount of sample to be tested, then 500 μ L are diluted to, 37 in constant-temperature table
It is protected from light under the conditions of DEG C overnight to get target liquid, -20 DEG C save backup.
4. the bisphenol-A fluorescence inspection according to claim 3 based on atom transition free radical polymerization reaction and truncation aptamers
Survey method, which is characterized in that the sequence of the ssDNA-A is 5 '-CCGGTGGGTGGA A-3 '；The sequence of ssDNA-B is 5 '-
TTCCACCCACCGG-3’。
5. the bisphenol-A fluorescence inspection according to claim 1 based on atom transition free radical polymerization reaction and truncation aptamers
Survey method, which is characterized in that step (3) -2. in be 0.5mM containing concentration in TE buffer MgCl2。
6. the bisphenol-A fluorescence inspection according to claim 1 based on atom transition free radical polymerization reaction and truncation aptamers
Survey method, which is characterized in that the PBS buffer solution concentration is 0.1M, pH 7.4.
7. the bisphenol-A fluorescence inspection according to claim 1 based on atom transition free radical polymerization reaction and truncation aptamers
Survey method, which is characterized in that the excitation wavelength of fluorescence detection is 489nm, slit width 4nm in step (6).
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111239094A (en) * | 2020-03-13 | 2020-06-05 | 河南中医药大学 | Sensitive detection method of alkaline phosphatase |
| CN114410749A (en) * | 2022-01-25 | 2022-04-29 | 河南中医药大学 | Electrochemiluminescence detection kit and method based on click chemistry and ARGET-ATRP amplification strategy |
| CN115232862A (en) * | 2022-07-18 | 2022-10-25 | 四川大学华西医院 | Method for detecting bisphenol A by gold nanoparticle-DNA enzyme motor triggered double-color DNA tweezers fluorescence amplification |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101627307A (en) * | 2006-12-04 | 2010-01-13 | 通用电气公司 | Composition, device and correlation technique |
| CN103439296A (en) * | 2013-07-31 | 2013-12-11 | 南昌大学 | Construction and application of aptamer sensor based on Au NPs and DNA circulation double amplification technique |
| CN104655759A (en) * | 2015-02-13 | 2015-05-27 | 王利兵 | Method for detecting recovery rate of [2,2-bis(4-hydroxyphenol)propane] absorbed by using aptamer functional magnetic nanomaterial |
| CN108796041A (en) * | 2018-06-27 | 2018-11-13 | 中南民族大学 | A kind of amplification of signal system and its detection method based on bioluminescence resonance energy transfer |
-
2019
- 2019-04-29 CN CN201910352884.XA patent/CN110068561B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101627307A (en) * | 2006-12-04 | 2010-01-13 | 通用电气公司 | Composition, device and correlation technique |
| CN103439296A (en) * | 2013-07-31 | 2013-12-11 | 南昌大学 | Construction and application of aptamer sensor based on Au NPs and DNA circulation double amplification technique |
| CN104655759A (en) * | 2015-02-13 | 2015-05-27 | 王利兵 | Method for detecting recovery rate of [2,2-bis(4-hydroxyphenol)propane] absorbed by using aptamer functional magnetic nanomaterial |
| CN108796041A (en) * | 2018-06-27 | 2018-11-13 | 中南民族大学 | A kind of amplification of signal system and its detection method based on bioluminescence resonance energy transfer |
Non-Patent Citations (3)
| Title |
|---|
| EUN-HEE LEE 等: "Highly Sensitive Detection of Bisphenol A by NanoAptamer Assay with Truncated Aptamer", 《ACS APPL. MATER. INTERFACES》 * |
| HAOBO SUN 等: "Ultrasensitive DNA biosensor based on electrochemical atom transfer radical polymerization", 《BIOSENSORS AND BIOELECTRONICS》 * |
| ZACHARY T. ALLEN 等: "Fluorogenic atom transfer radical polymerization in aqueous media as a strategy for detection", 《CHEMICAL SCIENCE》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111239094A (en) * | 2020-03-13 | 2020-06-05 | 河南中医药大学 | Sensitive detection method of alkaline phosphatase |
| CN111239094B (en) * | 2020-03-13 | 2022-08-26 | 河南中医药大学 | Sensitive detection method of alkaline phosphatase |
| CN114410749A (en) * | 2022-01-25 | 2022-04-29 | 河南中医药大学 | Electrochemiluminescence detection kit and method based on click chemistry and ARGET-ATRP amplification strategy |
| CN114410749B (en) * | 2022-01-25 | 2023-10-24 | 河南中医药大学 | Electrochemiluminescence detection kit and method based on click chemistry and ARGET-ATRP amplification strategy |
| CN115232862A (en) * | 2022-07-18 | 2022-10-25 | 四川大学华西医院 | Method for detecting bisphenol A by gold nanoparticle-DNA enzyme motor triggered double-color DNA tweezers fluorescence amplification |
| CN115232862B (en) * | 2022-07-18 | 2023-03-24 | 四川大学华西医院 | Method for detecting bisphenol A by gold nanoparticle-DNA enzyme motor triggered double-color DNA tweezers fluorescence amplification |
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