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Phthalic acid ester plastifier single-stranded DNA nucleic acid aptamer and screening and representing method and electrochemical sensor thereof

A technology of monobutyl phthalate and nucleic acid aptamer, which is applied in the biological field, can solve the problems of no specific recognition of phthalate plasticizer nucleic acid aptamers reported, and achieve high affinity And selectivity, high affinity, good specificity effect

Active Publication Date: 2019-04-12
CAPITAL NORMAL UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there are many nucleic acid aptamers obtained by in vitro screening technology, and many of them have achieved online rapid and high-sensitivity detection, but there is no report on the specific recognition of phthalate plasticizer nucleic acid aptamers

Method used

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  • Phthalic acid ester plastifier single-stranded DNA nucleic acid aptamer and screening and representing method and electrochemical sensor thereof
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  • Phthalic acid ester plastifier single-stranded DNA nucleic acid aptamer and screening and representing method and electrochemical sensor thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: Technical route for screening single-stranded DNA aptamers of phthalate plasticizers using SELEX and high-throughput sequencing technology

[0036] Such as figure 1 As shown, the technical route of the present invention for screening single-stranded DNA nucleic acid aptamers of phthalate plasticizers using SELEX and high-throughput sequencing technology includes the following steps: (1) DBP-NH 2 Chemical synthesis and characterization; (2) DBP-NH 2 Epoxy-activated Sepharose microspheres (Epoxy-activated Sepharose TM 6B) coupling and characterization; (3) 6×10 14 (1nmol) of commercially synthesized starting single-stranded DNA library (pool 0 , Table 1) and DBP-NH 2 The coupled agarose microspheres are mixed and incubated; (4) the agarose microspheres bound with the nucleic acid aptamer are separated and washed from the DNA sequence not bound to the agarose microspheres; (5) the washed agarose The nucleic acid aptamers on the microspheres were eluted by heat; (6)...

Embodiment 2

[0037] Example 2: The amino derivative of DBP (DBP-NH 2 ) Synthesis and characterization

[0038] DBP-NH 2 The synthetic route is as figure 2 Shown. The reagents used in this example are all analytically pure, each step of the reaction process is monitored in real time by thin layer chromatography, and the product is separated and purified by a silica gel column. The specific synthesis method, conditions and characterization are as follows (1-3):

[0039] (1) Preparation of monobutyl phthalate (compound 1)

[0040] Add n-butanol (5ml) to a 50ml dry round bottom flask containing phthalic anhydride (8.5g, 57mmol), anhydrous tetrahydrofuran (7.5ml) and clean and dry magnets. Stir magnetically Heat to reflux at 60°C for 9 hours. An insoluble white solid is formed. After standing at room temperature, filter with suction, wash the bottle with tetrahydrofuran and filter with suction again. A white paste-like substance was obtained after rotary steaming. Join CH 2 Cl 2 Afterwards, the...

Embodiment 3

[0045] Example 3: DBP-NH 2 Coupling on epoxy activated agarose microspheres

[0046] 0.1 g of epoxy-activated agarose microspheres were swollen with deionized water, and washed repeatedly with 20 mL of deionized water to obtain 350 μL wet spheres. Then use 0.2M Na 2 CO 3 (pH≈12) Wash the agarose balls. Add 46.7mg (0.15mmol) DBP-NH to the 500μL reaction system 2 , Placed at room temperature to shake for 48 hours. After the reaction, use pH 4.5 sodium acetate buffer solution (0.1M sodium acetate, 0.5M NaCl) and pH 12 sodium carbonate buffer solution (0.2M NaHCO 3 / Na 2 CO 3 , 0.5M NaCl) alternately wash the agarose microspheres three times, and finally wash with water, and dilute to 500μL, and store in a refrigerator at 4°C for later use. Elemental analysis (Elementar Vario MICROCUBE, Germany) showed that the proportions of the constituent elements C, H, N, and S of the agarose spheres were 33.61%, 9.26%, 0.92% and 1.09%, respectively. Coupling DBP-NH 2 After that, the proportion...

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Abstract

The invention relates to a phthalic acid ester plastifier single-stranded DNA nucleic acid aptamer and a screening and representing method and an electrochemical sensor thereof. The method comprises the following steps of step I, preparing a phthalic acid ester derivative with an amino group at the tail end of one liner fat side chains through chemical synthesis; step II, performing condensation reaction with the amino and ethylene oxide activating groups on agarose microspheres to perform covalent linkage on DBP-NH2 to the agarose microspheres; step III, screening the nucleic acid aptamer based on solid phase separation; step IV, after screening, performing high-flux sequencing on the obtained enrichment library; step V, testing the affinity and the selectivity of two sequences of DBP-1 and DBP-2 being the highest in occurrence frequency during the high-flux sequencing; and step VI, constructing an electrochemical sensor based on DBP-1. The invention can be used for developing variousdetection techniques and biosensors, and the phthalic acid ester plastifier single-stranded DNA nucleic acid aptamer has important application value on convenient detection of a phthalic acid ester plastifier in foods or environment.

Description

[0001] This application is that the application date is October 26, 2016, the application number is 201610949635.5 and the name is "a phthalate plasticizer single-stranded DNA nucleic acid aptamer, and its screening and characterization method and its application Divisional application of the invention patent application for chemical sensors. Technical field [0002] The invention relates to a phthalate plasticizer single-stranded DNA nucleic acid aptamer, a screening and characterization method thereof, and an electrochemical sensor using the same, and belongs to the field of biotechnology. Background technique [0003] Phthalate Esters (Phthalate Esters or Phthalic Acid Esters, PAEs) is a general term for esters formed by phthalic acid, and its chemical structure is composed of a planar aromatic hydrocarbon and two fatty side chains. PAEs are mainly used in polyvinyl chloride materials to make polyvinyl chloride change from hard plastic to elastic plastic and play a plasticizing...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/115C12N15/10G01N33/53
CPCG01N33/5308C12N15/1048C12N15/115C12N2310/16C12Q2535/122C12Q2563/149C12Q2525/205
Inventor 娄新徽何苗韩宇刁冬林郭倩
Owner CAPITAL NORMAL UNIVERSITY
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