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A Fluorescence Detection Method for Bisphenol A Based on Atom Transfer Radical Polymerization and Truncated Aptamers

A polymerization reaction and atom transfer technology, applied in the field of bioanalysis, can solve the problems of expensive instruments, professional operators, complicated processing process, etc., and achieve the effects of increased sensitivity, high affinity, stability and high reproducibility

Active Publication Date: 2021-10-01
HENAN UNIV OF CHINESE MEDICINE
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Traditional detection techniques for bisphenol A include high-performance liquid chromatography (HPLC), gas chromatography-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), etc., but these methods have complex sample pretreatment processes. , expensive equipment, professional operators, etc.

Method used

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  • A Fluorescence Detection Method for Bisphenol A Based on Atom Transfer Radical Polymerization and Truncated Aptamers
  • A Fluorescence Detection Method for Bisphenol A Based on Atom Transfer Radical Polymerization and Truncated Aptamers
  • A Fluorescence Detection Method for Bisphenol A Based on Atom Transfer Radical Polymerization and Truncated Aptamers

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Embodiment 1

[0054] Embodiment 1: Construction of detection method

[0055] A bisphenol A fluorescence detection method based on atom transfer radical polymerization and truncated aptamers, comprising the following steps:

[0056] (1) Pretreatment of hairpin DNA:

[0057] ①Heat the hairpin DNA solution to 95°C and keep it for 15 minutes, then slowly cool to room temperature;

[0058] ②Mix the hairpin DNA solution (100 μM) and tris(2-carboxyethyl)phosphine hydrochloride (TCEP) solution (10 μM) at a volume ratio of 1:1, and place the mixture on a constant temperature shaker at 37°C React in the dark for 3 hours, store at -20°C for later use; add ddH when in use 2 O dilute the hairpin DNA solution to 1 μM;

[0059] (2) Amino Fe 3 o 4 Modification of Magnetic Beads

[0060] ①Measure 50 μL of amino Fe 3 o 4 Magnetic beads storage solution (10mg / ml), magnetic separation, supernatant removed, washed with PBS buffer (0.1M, pH 7.4) 3 times, magnetic separation, supernatant removed, resuspen...

Embodiment 2

[0081] Example 2: Feasibility Verification

[0082] Firstly, the fluorescence spectra of the magnetic beads in different modification states were tested respectively. Such as image 3 As shown, first, without adding the fluorescent monomer FA (fluorescent monomer-O-acrylate), SSMCC / Hairpins / dsDNA-BPA / PBIB modified magnetic beads (curve b) did not detect any fluorescence intensity. On the contrary, obvious fluorescence emission peaks could still be observed after multiple washes after adding FA, because a large amount of FA was introduced to the magnetic beads by ATRP, resulting in a large fluorescence signal (curve a). This not only indicates that BPA binds to dsDNA and releases a single-stranded DNA that can complementarily pair with the loop sequence of the hairpin DNA and open the folded structure, but also indicates that the experiment has a high signal-to-noise ratio (S / N) and sensitivity. Through comparative experiments, it is fully proved that the signal amplification...

Embodiment 3

[0083] Embodiment 3: detection condition optimization

[0084] (1) Optimization of reaction time

[0085] By detecting the fluorescence intensity of magnetic beads with different ATRP reaction time, the relationship between the reaction time of ATRP and the fluorescence intensity was studied. The result is as Figure 4 As shown, the fluorescence intensity gradually increased with the prolongation of the reaction time and reached a stable level at 120min. Therefore, the ATRP reaction time was chosen as 120min.

[0086] (2) Optimization of fluorescent monomer concentration

[0087] Fluorescent monomer concentration can affect the length of the polymer chain and thus the detection performance. When the content of fluorescent monomers in the system is insufficient, the fluorescence intensity cannot reach the maximum. The fluorescence intensity of the magnetic beads with the fluorescent monomer concentration of 0.025mM, 0.05mM, 0.1mM, 0.2mM, 0.3mM and the bisphenol A concentra...

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Abstract

The invention discloses a bisphenol A fluorescence detection method based on atom transfer radical polymerization and truncated aptamers. The DNA is unfolded, and the unfolded hairpin DNA can be connected to the ATRP reaction initiator PBIB through a click reaction, and a large number of fluorescent monomers are connected and polymerized on the unfolded hairpin DNA through the ATRP reaction. The concentration is positively correlated, and the sensitive and accurate determination of the concentration of bisphenol A has been successfully realized. The results show that the method of the invention has high sensitivity, selectivity, anti-interference and accuracy for the detection of bisphenol A, and has great practical application value in food safety and environmental monitoring.

Description

technical field [0001] The invention relates to a bisphenol A fluorescence detection method based on an atom transfer radical polymerization reaction and a truncated aptamer, and belongs to the technical field of biological analysis. Background technique [0002] Bisphenol A (BPA), full name 2,2-bis(4-hydroxyphenyl)propane, is an important synthetic raw material for polycarbonate and epoxy resin, and is also a component of many common daily necessities such as food packaging materials, tableware, baby bottles, etc. Manufacturing raw materials. BPA can seep into food or beverages through food packaging containers or plastic films. Repeated use of these items or exposure to high heat environments can lead to the leaching of BPA, which can then be ingested by the human body. In addition, there is also a large amount of bisphenol A in the environment due to the landfilling and discarding of domestic waste in daily life and the discharge of bisphenol A containing wastewater in m...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/64
CPCG01N21/6486
Inventor 杨怀霞刘艳菊郭壮壮李曼曼王笑阳王柯
Owner HENAN UNIV OF CHINESE MEDICINE
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