CN108152256B - Sensitive high-selectivity good method for detecting BPA in water body - Google Patents
Sensitive high-selectivity good method for detecting BPA in water body Download PDFInfo
- Publication number
- CN108152256B CN108152256B CN201711309153.4A CN201711309153A CN108152256B CN 108152256 B CN108152256 B CN 108152256B CN 201711309153 A CN201711309153 A CN 201711309153A CN 108152256 B CN108152256 B CN 108152256B
- Authority
- CN
- China
- Prior art keywords
- bpa
- water
- capture probe
- detecting
- aptamer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
Landscapes
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Optics & Photonics (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
The invention discloses a method for detecting BPA in water with high sensitivity and good selectivity, when target object BPA exists in the water, an aptamer for marking fluorescent dye AHN is released from a double-helix structure which is formed by complementing with a capture probe, is specifically combined with the BPA and wraps the BPAWrapping, wherein fluorescence can be detected in supernatant after magnetic separation; in the absence of BPA, the AHN-labeled aptamer remained hybridized to the capture probe and there was no fluorescent signal in the supernatant after magnetic separation. According to the method, under the excitation wavelength of 450nm, the fluorescence intensity of the induction system changes along with the change of the concentration of BPA, the linear range of BPA detection is 0-8.00 ng/mL, the detection limit is 0.047ng/mL, the anti-interference capability is very strong, the method can be used for detecting low-concentration BPA, and the sensitivity is high. Furthermore, NH2‑Fe3O4Can be recycled and reused, and can save cost.
Description
Technical Field
The invention mainly relates to the technical field of environmental monitoring, in particular to a method for high-sensitivity and selective detection of bisphenol A (BPA) in water by using a magnetic separation fluorescent aptamer sensor.
Technical Field
Today, environmental endocrine hormones have become human-hazardous pollutants, with BPA playing an important role. The structure of BPA is similar to that of endocrine hormones, and can bind to estrogen receptors due to the presence of a phenol group. BPA is mainly used for producing high polymer materials such as polycarbonate, epoxy resin, polyphenyl ether resin, unsaturated polyester resin and the like; and can be widely applied to fine chemical products such as plasticizers, flame retardants, antioxidants, heat stabilizers, rubber antioxidants, pesticides, coatings and the like. Although bisphenol a has many uses in production and life, bisphenol a is still highly harmful to the human body, may cause endocrine disorders, threatens the health of fetuses and children, and obesity caused by cancer and metabolic disorders is also considered to be associated therewith.
Due to these hazards, efficient detection of BPA is highly desirable. Traditional analysis techniques for BPA include high performance liquid chromatography, liquid chromatography-mass spectrometry, gas chromatography-mass spectrometry, enzyme-linked immunosorbent assay, immunoassays and biochemical analysis. Although there are a number of methods for detecting BPA, there is still a need to find new, simple, mild-reacting and highly sensitive monitoring methods.
Fluorescence analysis is an attractive assay. The most important characteristics of the fluorescence method are high sensitivity, good selectivity and convenient use. More recently, magnetic nanomaterials have been concerned, in particular magnetic graphene oxide or/and modified Fe3O4Fluorescent methods of nanoparticles have been used for biochemical analytical studies. Superparamagnetic materials are widely used in biochemical analysis and medicine due to their special magnetic properties and low toxicity. Magnetic separation and concentration during detection is an important approach to improve the sensitivity of analytical methods. It not only increases the concentration of the target monitoring substance, but also can separate the target from complex environment or biological sample, and reduces the concentration of interfering substances. Nowadays, the development of new sensing and biosensing technologies has been the focus of attention, and aptamer technologies established simultaneously therewith have attracted much attention. The aptamer is a short single-stranded DNA or RNA sequence that can be screened in vitro and that can bind with high affinity and specificity to the corresponding ligand. Furthermore, the method based on the aptamer strategy can effectively provide simple and rapid detection with low cost and satisfactory selectivity. Aptamers have been widely used for detecting various targets, as compared to antibodies. Thus, aptamer-based sensing systems have been widely used for molecular analysis and medical diagnostics. In view of and combined with the advantages of the magnetic nanoparticles and the aptamers, the invention constructs a magnetic separation fluorescence method for detecting bisphenol A with high sensitivity.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the defects in the prior art, the invention aims to provide a method for detecting BPA in water with high sensitivity, high selectivity and high selectivity, which has high sensitivity and selectivity and can be used for detecting BPA with low concentration, and the used materials can be recycled.
The technical scheme is as follows: in order to achieve the purpose of the invention, the invention adopts the technical scheme that:
a sensitive high-selectivity good method for detecting BPA in water, when there is target object BPA in the water, the aptamer of the labeled fluorochrome AHN is released from the double helix structure formed by complementing with the capture probe, and is combined with BPA specificity and wraps it, and fluorescence can be detected in the supernatant after magnetic separation; in the absence of BPA in the water, the AHN-labeled aptamer remained hybridized to the capture probe and there was no fluorescent signal in the supernatant after magnetic separation.
The fluorescent dye AHN has good water solubility and thermal stability and strong fluorescence, and the molecular formula is as follows: c16H17O3N3The structural formula is as follows:
the fluorochrome AHN was labeled at either end of the aptamer via an amide bond.
The capture probe is designed according to the base complementary pairing principle of BPA aptamer.
The capture probe is connected with the magnetic nanoparticles with amino groups through amide bonds.
The aptamer is more prone to specifically bind the target contaminant BPA than is complementary to the capture probe hybridization.
The magnetic separation is to utilize the magnetic adsorption of a magnet to magnetic nano particles, thereby achieving the enrichment effect and improving the sensitivity of the method.
Has the advantages that: compared with the prior art, the method for detecting BPA in water with high sensitivity and good selectivity has the advantages that the adopted aptamer sensor has strong anti-interference capability, can be used for detecting low-concentration B PA and also has high sensitivity. In addition, the magnetic nanoparticles with amino groups can be recycled, which can save cost.
Drawings
FIG. 1 is a schematic diagram of a sensitive, highly selective method for detecting BPA in a body of water;
FIG. 2 is a fluorescence emission spectrum of a sensor detecting different concentrations of BPA;
FIG. 3 is a graph of BPA concentration and fluorescence intensity;
fig. 4 is a graph of the results of the interference rejection capability for the method of the present invention.
Detailed Description
The invention will now be further illustrated by reference to specific examples
Example 1
Preparation of capture probe-nanoparticle complexes: 1nmol capture probe solution (DNA fragment, purchased from Shanghai Producer, capture probe sequence: 5 '-COOH-TGGTGCGAACCCGTGATGCG-3') was mixed with 0.2mL of 5. mu.M EDAC in a 5mL centrifuge tube for 5 minutes, 0.2mL of 5. mu.M NHS was added and mixed for 2 minutes, and finally 2mL of 1mg/mL amino ferroferric oxide nanoparticle solution was added to the centrifuge tube, keeping the pH of the solution at 7.2. The centrifuge tubes were incubated in a constant temperature shaking chamber for 2 hours. It was then transferred to a refrigerator at 4 ℃ overnight to deactivate the unreacted EDAC. Finally, the capture probe-nanoparticle complex was obtained.
Preparation of AHN-labeled aptamer: 2nmol of aptamer solution (DNA fragment, from Shanghai Producer, aptamer sequence: 5 '-COOH-CCGGTGGGTGGTCAGGTGGGATAGCGTTCCGCGT ATGGCCCAGCGCATCACGGGTTCGCACCA-3') was mixed with 10. mu.M EDAC in a 5mL centrifuge tube for 5 min, 10. mu.M NHS was added, mixing was performed for 2 min, and finally 2.5. mu.M AHN solution in PBS was added, maintaining the solution pH at 7.2. The centrifuge tubes were incubated in a constant temperature shaking chamber for 2 hours. It was then transferred to a refrigerator at 4 ℃ overnight to deactivate the unreacted EDAC. Finally, the solution was dialyzed several times to remove unattached AHN. This labeled AHN with good fluorescent properties on BPA aptamers.
The method for detecting BPA in water with high sensitivity and good selectivity comprises the following steps:
for BPA detection, 300 μ L of PBS solution is added into a centrifuge tube, then 50 μ L of capture probe-nanoparticle complex and 50 μ L of AHN labeled BPA aptamer are added, the mixed solution is shaken up, then the mixture is incubated at room temperature for 20 minutes and then taken out, magnetic separation is carried out by a magnet, and the supernatant is poured off; add 320. mu.L of PBS solution to the centrifuge tube, resuspend the capture probe-nanoparticles in solution, then add 80. mu.L of 100ng/mL BPA to the solution and incubate the mixture at room temperature for 10 min. The centrifuge tube was again placed on the magnet to separate the capture probe-nanoparticles from the supernatant. Finally, the fluorescence intensity of the supernatant was measured.
As shown in FIG. 2, the BPA concentration gradually increased from top to bottom and was 0.20, 0.60, 1.00, 1.50, 2.00, 4.00, 6.00, 8.00, 10.0, 20.0, 30.0, 40.0, 50.0, 60.0, and 100ng/mL, respectively. The width of the slit of the fluorometer is set to 10 nm.
As shown in FIG. 3, the higher the concentration of BPA, the higher the fluorescence intensity detected, and when the concentration of BPA is in the range of 0-8.00 ng/mL, the BPA concentration and the fluorescence intensity show good linear relationship.
Detection limit according to formula
Calculated, where S is the standard deviation of the blank, K is the slope of the regression equation,
b is the intercept of the linear regression equation, which is the average of the background fluorescence intensity. The linear regression equation of the method is that Y is 212716X +1041206, R20.9984. Finally, the detection limit is calculated to be 0.047 ng/mL.
Example 2
The detection method has strong selectivity and anti-interference capability, can be used for detecting BPA with low concentration, and also has high sensitivity. First, BPA and benzidine, benzophenone, hydroquinone, resorcinol and BPB chemicals (which are structurally very similar to BPA and all contain two benzene rings) were added separately to a centrifuge tube and the fluorescence intensity was recorded. As shown in FIG. 4, only the supernatant from the centrifuge tube in which BPA was present could detect a strong fluorescence signal, while benzidine, benzophenone, hydroquinone, resorcinol and BPB could detect only a weak fluorescence signal. This indicates that BPA has excellent selectivity relative to the other chemical analogs. On the other hand, when BPA and these several chemical analogs were added to the same centrifuge tube and the fluorescence intensity was measured, the results showed that no significant fluorescence signal could be detected in the presence of interfering chemicals alone, but in the presence of both BPA and interfering chemicals. This shows that the sensor of the present invention has strong selectivity and anti-interference capability.
Example 3
BPA was added in an amount of 0, 0.40, 0.80 and 1.20ng/mL to tap water, river water and lake water, respectively, wherein large particles were previously removed from the river water and the lake water with a needle filter, and three parallel samples were set for each type of water sample, respectively. The fluorescence intensity in the water sample is measured by using the sensor method provided by the invention, and then the corresponding concentration value is obtained according to a linear regression equation. To verify that the method was applicable to the real sample detection, the same water sample was detected using high performance liquid chromatography (H PLC), and the results are shown in table 1. The result shows that the sensor can be effectively used for detecting the actual water sample.
TABLE 1 comparative test results
Note: the concentration units are ng/mL.
Claims (5)
1. A sensitive high-selectivity good method for detecting BPA in water is characterized in that when a target object BPA exists in the water, an aptamer of a labeled fluorescent dye AHN is released from a double-helix structure which is formed by complementing a capture probe, specifically combined with the BPA and wrapped, and fluorescence can be detected in supernatant after magnetic separation; in the absence of BPA, the AHN-labeled aptamer remained hybridized to the capture probe, and there was no fluorescent signal in the supernatant after magnetic separation; the aptamer is more prone to specifically bind and encapsulate the target contaminant BPA than it is complementary to the capture probe; the magnetic separation is to utilize the magnetic adsorption of the magnet to the magnetic nano particles, thereby achieving the effect of enrichment and being easy to separate.
2. The method for detecting BPA in water with high sensitivity and good selectivity according to claim 1, wherein the molecular formula of the fluorescent dye AHN is as follows: c16H17O3N3The structural formula is as follows:
3. the method for detecting BPA in a water body with high sensitivity and selectivity as claimed in claim 1, wherein the fluorescent dye AHN is labeled at either end of the aptamer through an amide bond.
4. The method for detecting BPA in water with high sensitivity and selectivity according to claim 1, wherein the capture probe is designed according to the base complementary pairing principle of BPA aptamers.
5. The method for detecting BPA in water with high sensitivity and selectivity according to claim 1, wherein the capture probe is connected with the magnetic nanoparticles with amino groups through amide bonds.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711309153.4A CN108152256B (en) | 2017-12-11 | 2017-12-11 | Sensitive high-selectivity good method for detecting BPA in water body |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711309153.4A CN108152256B (en) | 2017-12-11 | 2017-12-11 | Sensitive high-selectivity good method for detecting BPA in water body |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108152256A CN108152256A (en) | 2018-06-12 |
| CN108152256B true CN108152256B (en) | 2020-06-12 |
Family
ID=62466861
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711309153.4A Active CN108152256B (en) | 2017-12-11 | 2017-12-11 | Sensitive high-selectivity good method for detecting BPA in water body |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108152256B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109187507B (en) * | 2018-06-26 | 2021-01-12 | 宁波大学 | Electrochemiluminescence sensor for detecting bisphenol A and preparation method and application thereof |
| CN108828239B (en) * | 2018-08-24 | 2020-03-10 | 清华大学 | Biosensing analysis method for detecting estrogen binding activity of water sample |
| CN113049562A (en) * | 2021-03-19 | 2021-06-29 | 苏州健雄职业技术学院 | Fluorescence method for rapidly and quantitatively detecting BPA |
| 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 |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104807791B (en) * | 2015-04-20 | 2017-11-21 | 南京农业大学 | A kind of method detected based on quantum dot gold nano assembling superstructure to bisphenol-A |
| CN106908598B (en) * | 2017-03-21 | 2018-11-06 | 南方医科大学南海医院 | A kind of aptamer based on single-walled carbon nanotube-suspending chip magnetic detection microballoon and its preparation and detection method |
-
2017
- 2017-12-11 CN CN201711309153.4A patent/CN108152256B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN108152256A (en) | 2018-06-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108152256B (en) | Sensitive high-selectivity good method for detecting BPA in water body | |
| Yue et al. | Simultaneous detection of Ochratoxin A and fumonisin B1 in cereal samples using an aptamer–photonic crystal encoded suspension Array | |
| Kim et al. | A novel colorimetric aptasensor using gold nanoparticle for a highly sensitive and specific detection of oxytetracycline | |
| Bai et al. | Gold nanoparticle–based colorimetric aptasensor for rapid detection of six organophosphorous pesticides | |
| Xie et al. | Magnetic nanoparticles-based immunoassay for aflatoxin B1 using porous g-C3N4 nanosheets as fluorescence probes | |
| Bamrungsap et al. | Detection of lysozyme magnetic relaxation switches based on aptamer-functionalized superparamagnetic nanoparticles | |
| CN103674935B (en) | A kind of method of measuring gibberellin based on hybridization chain reaction signal amplification technique | |
| Xu et al. | A colorimetric aptasensor for the antibiotics oxytetracycline and kanamycin based on the use of magnetic beads and gold nanoparticles | |
| CN107841527B (en) | Fluorescence detection method for detecting thrombin by using aptamer and magnetic material | |
| Fu et al. | A tetrahedral DNA nanostructure functionalized paper-based platform for ultrasensitive colorimetric mercury detection | |
| Wu et al. | Fluorescent molecularly imprinted nanoparticles for selective and rapid detection of ciprofloxacin in aquaculture water | |
| Bian et al. | Fluorescent probe for detection of Cu2+ using core‐shell CdTe/ZnS quantum dots | |
| Gu et al. | Nuclease-assisted target recycling signal amplification strategy for graphene quantum dot-based fluorescent detection of marine biotoxins | |
| Qi et al. | Graphite nanoparticle as nanoquencher for 17β-estradiol detection using shortened aptamer sequence | |
| Kong et al. | Photoluminescent nanosensors capped with quantum dots for high-throughput determination of trace contaminants: strategies for enhancing analytical performance | |
| Zhao et al. | Magnetic solid-phase extraction based on multi-walled carbon nanotubes combined ferroferric oxide nanoparticles for the determination of five heavy metal ions in water samples by inductively coupled plasma mass spectrometry | |
| CN103695540A (en) | Method for detecting lead ions based on combination of fluorescence quantitative PCR (polymerase chain reaction) and nuclease GR-5 | |
| Jia et al. | Immunosensor of nitrofuran antibiotics and their metabolites in animal-derived foods: A review | |
| Zeng et al. | Determination of aflatoxin B1 in Pixian Douban based on aptamer magnetic solid-phase extraction | |
| Liu et al. | Dual-modal fluorescence and light-scattering sensor based on water-soluble carbon dots for silver ions detection | |
| Li et al. | Highly selective and sensitive determination of doxycycline integrating enrichment with thermosensitive magnetic molecular imprinting nanomaterial and carbon dots based fluorescence probe | |
| Jia et al. | The role of suspension array technology in rapid detection of foodborne pollutants: Applications and future challenges | |
| CN108107124B (en) | Method for detecting benzo (a) pyrene in large-volume water sample | |
| Liu et al. | Construction of a fluorescence biosensor for ochratoxin A based on magnetic beads and exonuclease III-assisted DNA cycling signal amplification | |
| Kong et al. | Fluorescence detection of three types of pollutants based on fluorescence resonance energy transfer and its comparison with colorimetric detection |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |