CN114235762A - Biosensor for detecting bisphenol A (BPA) - Google Patents
Biosensor for detecting bisphenol A (BPA) Download PDFInfo
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- CN114235762A CN114235762A CN202111468649.2A CN202111468649A CN114235762A CN 114235762 A CN114235762 A CN 114235762A CN 202111468649 A CN202111468649 A CN 202111468649A CN 114235762 A CN114235762 A CN 114235762A
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- 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
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
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- G—PHYSICS
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- 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
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- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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Abstract
The invention belongs to the technical field of sensors, and provides a biosensor for detecting BPA, which comprises an aptamer Apt with a nucleotide sequence shown in SEQ ID NO 1-4, a complementary strand T, A chain, a B chain, KF polymerase, dNTP without dCTP and ThT dye. The invention is based on the specific combination between the aptamer and the target object, and realizes the target cyclic amplification by using the strand displacement, thereby realizing the biosensor for quickly and sensitively detecting the target object. The sensor has the advantages of high detection speed, simple operation, high sensitivity, low detection limit and the like, can make up for the defects of the existing detection method of bisphenol A (BPA), and realizes the rapid and accurate quantitative detection of the bisphenol A (BPA).
Description
Technical Field
The invention belongs to the technical field of sensors, and relates to a nucleic acid aptamer biosensor for detecting BPA (bisphenol A) and a preparation method thereof.
Background
Bisphenol a (BPA) is a commonly used synthetic material, BPA is ubiquitous from mineral water bottles, medical devices, to food packaging. Studies have shown that BPA not only affects the central nervous system, immune system and reproductive system, but also induces apoptosis of spermatogenic cells through extracellular molecular signaling patterns. When BPA is mainly used for producing polycarbonate, epoxy resin and other high molecular materials, and the materials are heated, unstable lipophilic compound BPA can be released and migrated into water and food through food contact materials, and can also enter the environment through waste water discharge of plastic manufacturing plants, thereby seriously harming human health. Therefore, sensitive detection of BPA is critical for food safety and public health. In view of the current situation of BPA pollution and potential health risks, the development of a rapid, simple, ultrasensitive and highly selective BPA detection technology is necessary, which is of great significance in the aspects of biomedical diagnosis, environmental monitoring, food safety and the like.
Disclosure of Invention
In order to realize the detection of BPA with higher speed, ultra-sensitivity, high selectivity and low cost, the application provides a biosensor for detecting bisphenol A (BPA) based on a nucleic acid aptamer with strand displacement.
A biosensor for detecting BPA, comprising:
a) aptamer Apt, complementary strand T, A strand, B strand, and the nucleotide sequences of which are shown in SEQ ID NOS: 1-4
b) KF polymerase, dNTP without dCTP, ThT dye.
Preferably, the aptamer Apt and the complementary strand T are replaced by an arcuate probe Q hybridized therewith. More preferably, the method for preparing the arcuate probe Q is as follows: and (3) heating the aptamer Apt and the complementary strand T in equimolar mode in a water bath kettle at the temperature of 95 ℃ for 5min, and naturally cooling to room temperature to obtain the arch-shaped probe Q.
Preferably, the A chain and the B chain are replaced by hairpin H formed by hybridization of the A chain and the B chain. More preferably, the preparation method of the hairpin H is as follows: heating equimolar A chain and B chain in water bath at 95 deg.C for 5min, and naturally cooling to room temperature to obtain hairpin H.
Preferably, BPA standards and/or buffer solutions are also included in the sensor.
A kit for detecting BPA, which is prepared by the biosensor.
The above biosensor and kit can be used to detect BPA in a solution.
The detection comprises the following steps:
(1) hybridizing an aptamer Apt and a complementary strand T to obtain an arch probe Q;
(2) hybridizing the A chain and the B chain to obtain hairpin H;
(3) adding the arched probe Q, the hairpin H, a sample to be detected, dNTP without dCTP, KF polymerase and ThT dye into a buffer solution, uniformly mixing, and then incubating at 37 ℃ to obtain a reaction solution;
(4) and (4) carrying out fluorescence detection on the reaction solution.
Preferably, step (3) further comprises the step of preparing a standard curve of the target.
Preferably, the excitation wavelength of the fluorescence detection in the step (4) is 425 nm, and the detection wavelength band is 450-600 nm.
The working principle of the biosensor is as follows:
the biosensor used the following sequence in detecting BPA:
Apt：5’-CCGGTGGGTGGTCAGGTGGGATAGCGTTCCGCGTATGGCCCAGCGCATCAC GGGTTCGCACCA-3’
T：5’-AGAGGTCATACGCGGAAG-3’
A：5’-TTAGGGCCCGGTTTTTTGGGGGCCCTAACCCTTCCGCG-3’
B：5’-GGTTAGGGCCCCCTTTTTTCCGGGCCCTAACCCTAACC-3’。
as shown in FIG. 1, when bisphenol A (BPA) which is a target substance exists in the reaction system, the bisphenol A (BPA) can react with Apt chains in the arch-shaped probe Q to take away aptamer chains and release T chains. Adding H, KF hairpin polymerase and dNTP without dCTP, forming repeated human telomerase sequence TTAGGG through continuous strand displacement reaction of T strand and hairpin H, and finally generating strong fluorescence by embedding thioflavin ThT into the repeated TTAGGG sequence.
The invention has the following advantages:
the invention is based on the specific combination between the aptamer and the target object, and realizes the target cyclic amplification by using the strand displacement, thereby realizing the biosensor for quickly and sensitively detecting the target object. The sensor has the advantages of high detection speed, simple operation, high sensitivity, low detection limit and the like, can make up for the defects of the existing detection method of bisphenol A (BPA), and realizes the rapid and accurate quantitative detection of the bisphenol A (BPA).
The construction of the biosensor only needs one step, effectively avoids the complexity of a plurality of steps and possible pollution caused by the complexity, and has the advantages of simple operation and higher reaction speed; the sensor has mild reaction conditions and high reaction speed; the main process of the detection principle is realized in a homogeneous solution, the specific combination of bisphenol A and an aptamer is utilized, the cyclic amplification of a target object is realized through chain displacement, and the detection sensitivity is improved; the preparation process has low process cost and is suitable for the requirement of low price in industrialization.
Drawings
FIG. 1 is a schematic diagram of the operation of the present invention;
FIG. 2 is a graph showing the results of hairpin H concentration screening;
FIG. 3 is a graph showing the results of the reaction time screening;
FIG. 4 is a ThT concentration screening result chart;
FIG. 5 is a graph of the operation of the biosensor to detect BPA.
Detailed Description
The present invention will be further described with reference to the following examples and drawings, but the present invention is not limited to the following examples.
Example 1 hairpin H concentration screening
(1) Respectively synthesizing an aptamer Apt and a complementary chain T according to a nucleotide sequence shown as SEQ ID NO: 1-2, adding water to dilute the aptamer Apt and the complementary chain T into 100 mu M, and then preparing an arch probe Q according to the following method:
adding 21 muL of sterilized water, 3 muL of NEB Buffer, 3 muL of aptamer Apt and 3 muL of complementary chain T into a sterilized centrifuge tube, oscillating for 30s, placing the centrifuge tube into a water bath kettle at 95 ℃ for 5min, cooling to room temperature to obtain a solution of the arch probe Q, and storing the solution at 4 ℃ for later use.
(2) Respectively synthesizing an A chain and a B chain according to a nucleotide sequence shown as SEQ ID NO: 3-4, adding water to dilute the A chain and the B chain into 100 mu M, and then preparing hairpin H according to the following method:
adding 21 mu L of sterilized water, 3 mu L of NEB Buffer, 3 mu L A chains and 3 mu L B chains into a sterilized centrifuge tube, oscillating for 30s, placing the centrifuge tube into a water bath kettle at 95 ℃ for 5min, cooling to room temperature to obtain a solution of the hairpin H, and storing the solution at 4 ℃ for later use.
(3) Sequentially adding 9 mu L of sterilizing water, 3 mu L of arched probes Q (1 mu M), 3 mu L of hairpin H (final concentration is 0.2 mu M, 0.6 mu M, 1.0 mu M, 1.2 mu M, 1.5 mu M, 1.8 mu M, 2.0 mu M), 3 mu L of BPA solution (10 ng/mL), 3 mu L of dNTP (10 mM) without dCTP, 3 mu L of KF polymerase (1U/mu L), 3 mu L of ThT (final concentration is 15 mu M) and 3 mu L of NEffer 2 (1 x) into a sterilized EP tube, shaking for 30s, and putting the tube into a constant-temperature water bath at 37 ℃ for 90 min to obtain a reaction solution;
(4) and (3) diluting the reaction solution to 100 mu L, and reading the fluorescence intensity detected at 480 nm by exciting the fluorescence with the wavelength of 425 nm and the detection range of 450-600 nm. The results are shown in FIG. 2, and it can be seen from the figure that the fluorescence intensity of the system is continuously enhanced with the increase of the concentration of the hairpin H, and the fluorescence intensity is basically unchanged after the concentration of the hairpin H reaches 1.5 μ M, which indicates that the optimal concentration of the hairpin H is 1.5 μ M.
Example 2 optimization of reaction time
Arch probe Q and hairpin H were prepared according to steps (1) and (2) in example 1, and then according to the following steps (3), (4):
(3) sequentially adding 9 mu L of sterilizing water, 3 mu L of arch-shaped probes Q (1 mu M), 3 mu L of hairpin H (final concentration of 1.5 mu M), 3 mu L of BPA solution (10 ng/mL), 3 mu L of dNTP (10 mM) without dCTP, 3 mu L of KF polymerase (1U/mu L), 3 mu L of ThT (final concentration of 15 mu M) and 3 mu L of NEBuffer 2 (1 x) into a sterilized EP tube, shaking for 30s, uniformly mixing, putting into a constant-temperature water bath at 37 ℃ for incubation for 30 min, 45 min, 60 min, 75 min, 90 min, 105 min and 120 min, and obtaining a reaction solution;
(4) and (3) diluting the reaction solution to 100 mu L, and reading the fluorescence intensity detected at 480 nm by exciting the fluorescence with the wavelength of 425 nm and the detection range of 450-600 nm. The results are shown in FIG. 3, from which it can be seen that the fluorescence intensity increases with the increase of the incubation time, and after the reaction time reaches 90 min, the fluorescence intensity is basically unchanged, indicating that the optimal reaction time is 90 min.
Example 3 optimization of ThT concentration
Arch probe Q and hairpin H were prepared according to steps (1) and (2) in example 1, and then according to the following steps (3), (4):
(3) sequentially adding 9 mu L of sterilizing water, 3 mu L of arch-shaped probe Q (1 mu M), 3 mu L of hairpin H (final concentration is 1.5 mu M), 3 mu L of BPA solution (10 ng/mL), 3 mu L of dNTP (10 mM) without dCTP, 3 mu L of KF polymerase (1U/mu L) and 3 mu L of ThT (final concentrations are 5 mu M, 10 mu M, 12 mu M, 15 mu M, 20 mu M, 25 mu M and 30 mu M respectively) and 3 mu L of NEBuffer 2 (1 x) into a sterilized EP tube, shaking for 30s, uniformly mixing, and putting into a constant-temperature water bath kettle at 37 ℃ for 90 min to obtain a reaction solution;
(4) and (3) diluting the reaction solution to 100 mu L, and reading the fluorescence intensity detected at 480 nm by exciting the fluorescence with the wavelength of 425 nm and the detection range of 450-600 nm. The results are shown in FIG. 4, from which it can be seen that the fluorescence intensity increases with the increase in the concentration of ThT, and after the concentration of ThT reached 15. mu.M, the fluorescence intensity was substantially unchanged, indicating that the optimum concentration of ThT reaction was 15. mu.M.
Detection of bisphenol A (BPA) Using example 1 biosensor
(1) Respectively synthesizing an aptamer Apt and a complementary chain T according to a nucleotide sequence shown as SEQ ID NO: 1-2, adding water to dilute the aptamer Apt and the complementary chain T into 100 mu M, and then preparing an arch probe Q according to the following method:
adding 21 muL of sterilized water, 3 muL of NEB Buffer, 3 muL of aptamer Apt and 3 muL of complementary chain T into a sterilized centrifuge tube, oscillating for 30s, placing the centrifuge tube into a water bath kettle at 95 ℃ for 5min, cooling to room temperature to obtain a solution of the arch probe Q, and storing the solution at 4 ℃ for later use.
(2) Respectively synthesizing an A chain and a B chain according to a nucleotide sequence shown as SEQ ID NO: 3-4, adding water to dilute the A chain and the B chain into 100 mu M, and then preparing hairpin H according to the following method:
adding 21 mu L of sterilized water, 3 mu L of NEB Buffer, 3 mu L A chains and 3 mu L B chains into a sterilized centrifuge tube, oscillating for 30s, placing the centrifuge tube into a water bath kettle at 95 ℃ for 5min, cooling to room temperature to obtain a solution of the hairpin H, and storing the solution at 4 ℃ for later use.
(3) Sequentially adding 9 muL of sterilizing water, 3 muL of arch-shaped probes Q (1 muM), 3 muL of hairpin H (final concentration is 1.5 muM), 3 muL of BPA solution (concentrations are 0pM, 1 pM, 5 pM, 10 pM, 50pM, 100 pM, 500pM, 1000pM and 5000 pM respectively), 3 muL of dNTP (10 mM) without dCTP, 3 muL of KF polymerase (1U/muL) and 3 muL of ThT (final concentration is 15 muM) into a sterilized EP tube, shaking for 30s, uniformly mixing, putting into a constant-temperature water bath kettle at 37 ℃ and incubating for 90 min to obtain a reaction solution;
(4) and (3) diluting the reaction solution to 100 mu L, and reading the fluorescence intensity detected at 480 nm by exciting the fluorescence with the wavelength of 425 nm and the detection range of 450-600 nm. The results are shown in FIG. 5, from which it can be seen that the fluorescence intensity increases with the increase in the concentration of bisphenol A as the target, and after the concentration of bisphenol A reaches 1000pM, the fluorescence intensity is substantially unchanged, so that the maximum detection concentration of bisphenol A (BPA) is 1000 pM. The logarithm of the concentration of BPA in the range of 0.1-10 ng/mL is in direct proportion to the magnitude of the fluorescence intensity value, and the linear equation is：F=0.01601+0.43109×log C，R2And 0.994, wherein F is the characteristic absorption peak of the fluorescence signal at 485nm and C is the concentration of the target BPA (ng/mL), and the lowest detection limit is 0.051 ng/mL calculated according to the triple standard deviation plus the blank response.
Sequence listing
<110> university of Jinan
<120> a biosensor for detecting bisphenol A (BPA)
<130> 20211124
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 63
<212> DNA
<213> Artificial Sequence
<220>
<223> Apt
<400> 1
ccggtgggtg gtcaggtggg atagcgttcc gcgtatggcc cagcgcatca cgggttcgca 60
cca 63
<210> 2
<211> 18
<212> DNA
<213> Artificial Sequence
<220>
<223> T
<400> 2
agaggtcata cgcggaag 18
<210> 3
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> A
<400> 3
ttagggcccg gttttttggg ggccctaacc cttccgcg 38
<210> 4
<211> 38
<212> DNA
<213> Artificial Sequence
<220>
<223> B
<400> 4
ggttagggcc cccttttttc cgggccctaa ccctaacc 38
Claims (9)
1. A biosensor for detecting BPA, comprising:
a) aptamer Apt, complementary strand T, A strand, B strand, and the nucleotide sequences of which are shown in SEQ ID NOS: 1-4
b) KF polymerase, dNTP without dCTP, ThT dye.
2. The biosensor in accordance with claim 1, wherein said aptamer Apt and complementary strand T are replaced by an arcuate probe Q hybridized therewith; more preferably, the method for preparing the arcuate probe Q is as follows: and (3) heating the aptamer Apt and the complementary strand T in equimolar mode in a water bath kettle at the temperature of 95 ℃ for 5min, and naturally cooling to room temperature to obtain the arch-shaped probe Q.
3. The biosensor in accordance with claim 1, wherein said A strand and B strand are replaced by hairpin H formed by hybridization thereof; more preferably, the preparation method of the hairpin H is as follows: heating equimolar A chain and B chain in water bath at 95 deg.C for 5min, and naturally cooling to room temperature to obtain hairpin H.
4. The biosensor of claim 1, further comprising a BPA standard and/or a buffer solution.
5. A kit for detecting BPA prepared by the biosensor of any of claims 1-4.
6. Use of a biosensor according to any of claims 1-4 or a kit according to claim 5 for detecting BPA in a solution.
7. Use according to claim 6, characterized in that it comprises the following steps:
(1) hybridizing an aptamer Apt and a complementary strand T to obtain an arch probe Q;
(2) hybridizing the A chain and the B chain to obtain hairpin H;
(3) adding the arched probe Q, the hairpin H, a sample to be detected, dNTP without dCTP, KF polymerase and ThT dye into a buffer solution, uniformly mixing, and then incubating at 37 ℃ to obtain a reaction solution;
(4) and (4) carrying out fluorescence detection on the reaction solution.
8. The use of claim 7, wherein step (3) further comprises the step of preparing a standard curve of the target.
9. The use according to claim 7, wherein the excitation wavelength of the fluorescence detection in step (4) is 425 nm, and the detection wavelength band is 450-600 nm.
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| CN202111468649.2A CN114235762A (en) | 2021-12-03 | 2021-12-03 | Biosensor for detecting bisphenol A (BPA) |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104962608A (en) * | 2015-05-28 | 2015-10-07 | 广东省生态环境与土壤研究所 | Bisphenol A detecting method and detecting kit based on dual-quenching-group nucleic acid self-assembling technology |
| CN109596592A (en) * | 2019-01-30 | 2019-04-09 | 济南大学 | Biosensor and its detection method based on aptamer detection salmonella |
| CN112501260A (en) * | 2020-11-27 | 2021-03-16 | 广东省科学院生态环境与土壤研究所 | Bisphenol A detection method, fluorescence detection kit and application thereof |
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- 2021-12-03 CN CN202111468649.2A patent/CN114235762A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104962608A (en) * | 2015-05-28 | 2015-10-07 | 广东省生态环境与土壤研究所 | Bisphenol A detecting method and detecting kit based on dual-quenching-group nucleic acid self-assembling technology |
| CN109596592A (en) * | 2019-01-30 | 2019-04-09 | 济南大学 | Biosensor and its detection method based on aptamer detection salmonella |
| CN112501260A (en) * | 2020-11-27 | 2021-03-16 | 广东省科学院生态环境与土壤研究所 | Bisphenol A detection method, fluorescence detection kit and application thereof |
Non-Patent Citations (1)
| Title |
|---|
| JOCELYN Y. KISHI 等: "Programmable autonomous synthesis of single-stranded DNA", NATURE CHEMISTRY * |
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