CN104155278A - Method of detecting bisphenol A (BPA) based on fluorescence resonance energy transfer - Google Patents
Method of detecting bisphenol A (BPA) based on fluorescence resonance energy transfer Download PDFInfo
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- CN104155278A CN104155278A CN201410430253.2A CN201410430253A CN104155278A CN 104155278 A CN104155278 A CN 104155278A CN 201410430253 A CN201410430253 A CN 201410430253A CN 104155278 A CN104155278 A CN 104155278A
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Abstract
The invention discloses a method of detecting BPA in environmental hormone on the basis of fluorescence resonance energy transfer (FRET) and belongs to the field of analytic chemistry. An organic fluorescent dye FAM is employed as a donor molecule of the FRET and TAMRA is employed as an acceptor molecule of the FRET. A principle of a specific combination between a BPA aptamer and the BPA is employed and the content of the BPA is detected through the FRET. The method has an excellent linearity with a concentration of the BPA ranging from 1.0*10<-10> mol/L to 1.0*10<-8> mol/L and a lowest detection limit being 7.0*10<-11> mol/L. The detection limit is far lower than a requirement of a dissolving-out quantity of phenol in carbonic ester resin and a moulded product in a national health standard. Through the method, the content of the BPA is rubber and plastic products such as kettles, cups, feeding-bottles, food containers and the like.
Description
Technical field
The invention belongs to analytical chemistry field, be specifically related to a kind of method that detects BPA based on FRET (fluorescence resonance energy transfer).
Background technology
FRET (fluorescence resonance energy transfer) (Fluorescence resonance energy transfer is called for short FRET) is the non-radiative process that the donor molecule in excited state is transferred to self-ability by long-range dipole effect the in-plant acceptor molecule in ground state.Acceptor minute must be able to absorb the energy of donor transmitted wave strong point, therefore the excitation wavelength of acceptor molecule must be overlapping with the emission wavelength of donor molecule.From F rster(F rster T. Annals of Physics, 1948,2:55) set forth first the mechanism of FRET since, FRET is widely used in the fields such as nucleic acids research, protein research, cell research, immunoassays.
Aptamer (aptamer), derives from Latin " aptus ", is called for short aptamers, claims again aptamer or nucleic acid aptamer, is one section of oligonucleotide fragment by 25-80 RNA or DNA base composition.Aptamers can be folded and be combined by adaptability with object, forms the special three-dimensional structures such as hair fastener (hairpin), false knot (pseudoknot), bulge loop (bulge), G-tetra-serobilas (G-quartet).Since (Tuerk C. such as nineteen ninety Tuerk, Gold L. Science 1990,249,505), since the in-vitro screening technology (SELEX) that utilization index enrichment system is evolved first filters out the RNA type aptamers of bacteriophage T4 archaeal dna polymerase, the research work of aptamers is developed rapidly.Only 1999, NeXstar Pharmaceuticals company and Colorado university just filtered out the aptamers that exceedes 100 kinds.Ellington group has created an aptamers database, the details that the inside has comprised aptamers.
Bisphenol-A (Bisphenol A, be called for short BPA) full name 2,2-bis-(4-hydroxy phenyl) propane, for the synthesis of the material such as polycarbonate and epoxy resin, also the stabilizing agent that can be used as phenolics, plasticity polyester, antioxidant and Polyvinylchloride, is widely used in manufacturing baby bottles, kitchen utensils and Medical Devices.There are some researches show, BPA has the estrogenic effect of simulation, is a kind of environmental hormone, can enter in Food & Drink by wrappage, cause human endocrine imbalance, threaten fetus and children's health, also may be relevant with the obesity that cancer and metabolic disturbance cause.Therefore it is very necessary, setting up a kind of easy and simple to handle, highly sensitive BPA detection method.The present invention utilizes FRET (fluorescence resonance energy transfer) technology, adopt organic fluorescent dye hydroxyl fluorescein (FAM) as the donor molecule of FRET, carboxyl tetramethyl rhodamine (TAMRA) acceptor molecule as FRET, utilize the principle of BPA aptamers and BPA specific binding, can Accurate Determining sample in the content of BPA.
Summary of the invention
The object of the present invention is to provide a kind of method that detects BPA based on FRET (fluorescence resonance energy transfer), this detection method is high to the detection sensitivity of BPA, specificity good, 1.0 × 10
-10-1.0 × 10
-8in mol/L concentration range, the detection of BPA is presented to good linearity, lowest detection is limited to 7.0 × 10
-11mol/L, can realize BPA fast, Accurate Determining.
For achieving the above object, the present invention adopts following technical scheme:
A method that detects BPA based on FRET (fluorescence resonance energy transfer), is the principle based on BPA aptamers and BPA specific binding, utilizes FRET (fluorescence resonance energy transfer) to detect the content of BPA;
Comprise the following steps:
1) DNA, after centrifugal 5 min of rotating speed with 8000 r/min, is added to abundant vibration 5-10 min in the BR damping fluid that 50 mmol/L, pH value are 7.4, put into 4 DEG C of refrigerators for subsequent use;
2) DNA in step 1) is joined in the BR damping fluid that 50 mmol/L, pH value are 7.4, then add D1, T1 and T2, hybridization reaction 2 h;
3) detecting step 2) the reaction fluorescence signal of solution afterwards, be designated as initial fluorescence signal value F
0;
4) to step 3) detect after solution in add solution to be measured, specific binding 10 h;
5) detecting step 4) the reaction fluorescence signal of solution afterwards, be designated as reaction fluorescence signal value F;
6) calculate the concentration of BPA according to the linear equation of BPA concentration and fluorescence intensity change value, and then draw the content of BPA in solution to be measured.
Step 2) add after DNA, in BR damping fluid, the concentration of DNA is 1.0 × 10
-7mol/L.
Step 2) described D1 is BPA aptamers probe, its sequence is: 5 ’ – TGGTCGTTGGTCGTTCGCGTTTCTGGATTTTTTATTTCTGGGGTTCAGTTCTTTTT TGT-3 ' (SEQ ID NO:1).
Step 2) described T1 is BPA aptamers complementary strand 1 probe, its sequence is: 5 ’ – FAM-ATCCAGAAACGCGAACGA-3 ' (SEQ ID NO:2).
Step 2) described T2 is BPA aptamers complementary strand 2 probes, its sequence is: 5 '-TAMRA-AACTGAACCCCAGAAAT-3 ' (SEQ ID NO:3).
The setup parameter that fluorescence signal detects Fluorescence spectrophotometer used is: excitation wavelength is 485 nm, and emission wavelength is 500 – 650 nm, and entrance slit is 10 nm, and transmitting slit is 10 nm, and photomultiplier transit tube voltage is 700 V.
remarkable advantage of the present invention is:
(1) simple, the high specificity of operation steps of the present invention, highly sensitive, cost is low, practical, and the lowest detection of BPA is limited to 7.0 × 10
-11mol/L, is significantly less than the requirement to phenol stripping quantity in carbonate resin and products formed in state health standards, can Accurate Determining sample in the content of BPA.
(2) the present invention foundation detects the fluorescence analysis detection method of BPA based on FRET (fluorescence resonance energy transfer), easy and simple to handle, detectability is low, can realize the online detection of BPA in the products of rubber and plastic such as kettle, water tumbler, feeding bottle, food containers, and detection material is not damaged.
Brief description of the drawings
Fig. 1 is the schematic diagram that the present invention is based on FRET (fluorescence resonance energy transfer) detection bisphenol-A.
Fig. 2 is the each reaction solution fluorogram of the present invention.
Fig. 3 is the working curve of embodiment of the present invention standard solution.
Embodiment
In order to make content of the present invention more be convenient to understand, below in conjunction with embodiment, technical solutions according to the invention are described further, but the present invention is not limited only to this.
The required instrument of the present embodiment and reagent solution are as follows: Fluorescence spectrophotometer(model specification: cary eclipse; Device numbering: EL02065703, the Varian U.S.), precision acidity meter (Shanghai great Pu Instrument Ltd.), three hole electric heating constant temperature tanks (Shanghai Yiheng Scientific Instruments Co., Ltd), quartz colorimetric utensil (Xin Hang instrument plant of Community of Jin Tan County city); BPA, DNA(Shanghai Sangon Biological Engineering Technology And Service Co., Ltd).
The sequence of BPA aptamers probe D1: 5 ’ – TGGTCGTTGGTCGTTCGCGTTTCTGGATTTTTTATTTCTGGGGTTCAGTTCTTTTT TGT-3 ';
The sequence of BPA aptamers complementary strand 1 probe T1: 5 ’ – FAM-ATCCAGAAACGCGAACGA-3;
The sequence of BPA aptamers complementary strand 2 probe T2: 5 '-TAMRA-AACTGAACCCCAGAAAT-3 '.
A method that detects BPA based on FRET (fluorescence resonance energy transfer), is the principle based on BPA aptamers and BPA specific binding, utilizes FRET (fluorescence resonance energy transfer) to detect the content of BPA; Comprise the following steps:
1) by DNA after centrifugal 5 min of rotating speed with 8000 r/min, slowly open pipe lid, cover again upper tube cap after adding the BR buffer solution that 50 mmol/L, pH value are 7.4, fully vibrate after 10 min, put into 4 DEG C of refrigerators for subsequent use;
2) by 5 μ L 6.0 × 10
-6dNA in mol/L step 1) joins in the BR damping fluid that 300 μ L 50 mmol/L, pH value are 7.4, then adds respectively 5 μ L 6.0 × 10
-6t1, T2 and the D1 of mol/L, hybridization reaction 2 h;
3) detecting step 2) the reaction fluorescence signal of solution afterwards, be designated as initial fluorescence signal value F
0;
4) be 1.0 × 10 with absolute ethyl alcohol compound concentration
-5the BPA mother liquor of mol/L, then obtain concentration with intermediate water dilution mother liquor and be followed successively by 1.0 × 10
-10, 5.0 × 10
-10, 1.0 × 10
-9, 5.0 × 10
-9, 1.0 × 10
-8the BPA standard solution of mol/L;
5) what in the solution after detecting to step 3), add respectively that step 4) prepares is 1.0 × 10 containing BPA concentration
-10, 5.0 × 10
-10, 1.0 × 10
-9, 5.0 × 10
-9, 1.0 × 10
-8the standard solution of mol/L, specific binding 10 h;
6) distinguish detecting step 5) the reaction fluorescence signal of solution afterwards, be designated as reaction fluorescence signal value F;
7) according to BPA concentration and fluorescence intensity change value drawing standard solution working curve, draw linear equation;
8) testing sample is cleaned, dried, by 2ml/cm
2ratio add distilled water, 100 DEG C are soaked after 30min and are cooled to room temperature, moisturizing to former immersion volume as solution to be measured; Measure after the fluorescence signal value of solution to be measured, the BPA concentration logarithm value drawing according to step 7) and the linear equation of fluorescence intensity change value calculate the concentration of BPA in solution to be measured, and then draw the content of BPA in testing sample.
The setup parameter that fluorescence signal detects Fluorescence spectrophotometer used is: excitation wavelength is 485 nm, and emission wavelength is 500 nm, and entrance slit is 10 nm, and transmitting slit is 10 nm, and photomultiplier transit tube voltage is 700 V.
As can be seen from Figure 2, after BPA aptamers probe D1 is combined with BPA aptamers complementary strand probe T1, T2, solution fluorescence intensity is lower; When BPA and BPA aptamers probe specificity association reaction completely after, BPA aptamers complementary strand probe T1, T2 are free out significantly strengthens fluorescence intensity, can its fluorescence signal value of Accurate Determining by fluorospectrophotometer.
As can be seen from Figure 3, the linear equation of BPA concentration and fluorescence intensity change value is: △ F=0.2493+0.0203X, R=0.9986;
Wherein △ F=F-F
0: F
0for the ratio of the fluorescence intensity under the fluorescence intensity under 580 nm after T1, T2 and D1 hybridization and 520 nm, F adds BPA to react the ratio of the fluorescence intensity under the fluorescence intensity under 580 nm and 520 nm after 10 h, X is the logarithm value of BPA concentration, and concentration unit is mol/L.
Utilize the detection method of the present embodiment to measure the content of BPA in feeding bottle and PC plastic bottle, the results are shown in Table 1.
The measurement result of BPA content in the different samples of table 1
As seen from Table 1, detection method of the present invention can be accurately, the content of BPA in working sample easily, and it is easy and simple to handle, destructive test sample not, is applicable to the fast detecting of BPA content in sample.
The foregoing is only preferred embodiment of the present invention, all equalizations of doing according to the present patent application the scope of the claims change and modify, and all should belong to covering scope of the present invention.
SEQUENCE LISTING
<110> Inspection & Quarantine Technology Center of Fujian Entry-Exit Inspection & Quar
Mono-kind of <120> detects the method for bisphenol-A based on FRET (fluorescence resonance energy transfer)
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 59
<212> DNA
<213> Artificial
<400> 1
tggtcgttgg tcgttcgcgt ttctggattt tttatttctg gggttcagtt cttttttgt 59
<210> 2
<211> 18
<212> DNA
<213> Artificial
<400> 2
atccagaaac gcgaacga 18
<210> 3
<211> 17
<212> DNA
<213> Artificial
<400> 3
aactgaaccc cagaaat 17
Claims (6)
1. a method that detects BPA based on FRET (fluorescence resonance energy transfer), is the principle based on BPA aptamers and BPA specific binding, utilizes FRET (fluorescence resonance energy transfer) to detect the content of BPA; Comprise the following steps:
1) DNA, after centrifugal 5 min of rotating speed with 8000 r/min, is added to abundant vibration 5-10 min in the BR damping fluid that 50 mmol/L, pH value are 7.4, put into 4 DEG C of refrigerators for subsequent use;
2) DNA in step 1) is joined in the BR damping fluid that 50 mmol/L, pH value are 7.4, then add D1, T1 and T2, hybridization reaction 2 h;
3) detecting step 2) the reaction fluorescence signal of solution afterwards, be designated as initial fluorescence signal value F
0;
4) to step 3) detect after solution in add solution to be measured, specific binding 10 h;
5) detecting step 4) the reaction fluorescence signal of solution afterwards, be designated as reaction fluorescence signal value F;
6) calculate the concentration of BPA according to the linear equation of BPA concentration and fluorescence intensity change value, and then draw the content of BPA in solution to be measured.
2. the method that detects according to claim 1 BPA based on FRET (fluorescence resonance energy transfer), is characterized in that: step 2) add after DNA, in BR damping fluid, the concentration of DNA is 1.0 × 10
-7mol/L.
3. detect according to claim 1 the method for BPA based on FRET (fluorescence resonance energy transfer), it is characterized in that: step 2) described D1 is BPA aptamers probe, its sequence is: 5 ’ – TGGTCGTTGGTCGTTCGCGTTTCTGGATTT TTTATTTCTGGGGTTCAGTTCTTTTTTGT-3 '.
4. detecting the method for BPA based on FRET (fluorescence resonance energy transfer) according to claim 1, it is characterized in that: step 2) described T1 is BPA aptamers complementary strand 1 probe, its sequence is: 5 ’ – FAM-ATCCAGAAACGCGAACGA-3.
5. detecting the method for BPA based on FRET (fluorescence resonance energy transfer) according to claim 1, it is characterized in that: step 2) described T2 is BPA aptamers complementary strand 2 probes, its sequence is: 5 '-TAMRA-AACTGAACCCCAGAAAT-3 '.
6. detect according to claim 1 the method for BPA based on FRET (fluorescence resonance energy transfer), it is characterized in that: the setup parameter that fluorescence signal detects Fluorescence spectrophotometer used is: excitation wavelength is 485 nm, emission wavelength is 500 – 650 nm, entrance slit is 10 nm, transmitting slit is 10 nm, and photomultiplier transit tube voltage is 700 V.
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|---|---|---|---|
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107727624A (en) * | 2017-10-16 | 2018-02-23 | 太原理工大学 | A kind of antibiotic detection method based on fit sensing fluorescence resonance energy transfer |
| CN108956991A (en) * | 2018-07-25 | 2018-12-07 | 南通大学 | A kind of fluorescence resonance energy transfer biosensor and its application |
| CN112834469A (en) * | 2020-12-28 | 2021-05-25 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Detection method and application of environmental estrogen |
-
2014
- 2014-08-28 CN CN201410430253.2A patent/CN104155278A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| 吴燕平: "新型适配体核酸探针在双酚A检测中的应用研究", 《CNKI中国优秀硕士学位论文全文数据库》 * |
| 唐熙等: "气相色谱法测定塑料奶瓶中迁移出的双酚A", 《福建分析测试》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107727624A (en) * | 2017-10-16 | 2018-02-23 | 太原理工大学 | A kind of antibiotic detection method based on fit sensing fluorescence resonance energy transfer |
| CN107727624B (en) * | 2017-10-16 | 2020-12-22 | 太原理工大学 | Antibiotic detection method based on aptamer sensing fluorescence energy resonance transfer |
| CN108956991A (en) * | 2018-07-25 | 2018-12-07 | 南通大学 | A kind of fluorescence resonance energy transfer biosensor and its application |
| CN108956991B (en) * | 2018-07-25 | 2021-04-27 | 南通大学 | Fluorescence resonance energy transfer biosensor and application thereof |
| CN112834469A (en) * | 2020-12-28 | 2021-05-25 | 军事科学院军事医学研究院环境医学与作业医学研究所 | Detection method and application of environmental estrogen |
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