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High-sensitivity multi-target quantitative analysis method based on gas pressure detection

A technology of air pressure detection and quantitative detection method, which is applied in the field of high-sensitivity quantitative analysis, can solve the problems of high cost of experimental methods and expensive instruments and equipment, and achieves the effects of reliable detection results, rapid detection and good selectivity.

Inactive Publication Date: 2014-08-20
XIAMEN UNIV
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  • Description
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Problems solved by technology

[0006] The present invention aims at the disadvantages of the existing high-sensitivity quantitative detection and analysis method and its instruments and equipment are expensive, and the cost of the experimental method is high, and develops a quantitative detection and analysis method based on air pressure detection with high sensitivity, high selectivity, high accuracy, and cheap and portable , which can be used in the laboratory for high-sensitivity and high-selectivity inorganic ions, small molecules, and biomacromolecules such as proteins, DNA, and even viruses, bacteria, cells, etc. POC quantitative detection analysis

Method used

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  • High-sensitivity multi-target quantitative analysis method based on gas pressure detection
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  • High-sensitivity multi-target quantitative analysis method based on gas pressure detection

Examples

Experimental program
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Effect test

Embodiment 1

[0035] Embodiment 1 Synthesis of acrylic acid phosphoramidite monomer

[0036] The synthesis of acrylic acid phosphoramidite monomer is divided into three steps: 1) 6-amino-1-hexanol (1g, 8.53mmol), triethylamine (2.36mL, 17mmol) are added to ice bath in 100mL dichloromethane, Methacryloyl chloride (2.67 g, 2.55 mmol) was added dropwise under anhydrous and oxygen-free conditions, and then the whole system was stirred and reacted at room temperature for 2 hours. 2) The solvent was spin-dried, and 10 mL of ethanol and 15% NaOH (4 mL) were added to the product to selectively hydrolyze the product into N-(6-hydroxyhexyl)methacrylamide. N-(6-hydroxyhexyl)methacrylamide was separated with ethyl acetate on a silica gel column for the next reaction. 3) Dissolve the purified N-(6-hydroxyhexyl)methacrylamide (0.50g, 2.70mmol) in 10mL of anhydrous dichloromethane, and add N,N-diisopropylethylamine dropwise at 0°C ( DIPEA, 0.98g, 7.5mmol), and 2-cyanoethyl N,N-diisopropylphosphoramidite...

Embodiment 2

[0037] Example 2 Synthesis and purification of nucleic acid molecules modified by methacryl groups, biotin groups and mercapto groups

[0038] Using ordinary CPG as a solid phase carrier, using DNA monomer bases as raw materials, synthesize strand A, strand B, linker nucleic acid aptamer, detection DNA, ELISA DNA, and final strand from 3' to 5' on a DNA synthesizer The 5' ends of A and B were modified (modified on a synthesizer) with the phosphoramidite acrylic acid monomer synthesized in Example 1, and the modified sulfhydryl groups on the DNA were detected. The specific synthesized sequences are shown in Table 1, Table 2, and Table 3. The biotin-modified CPG was used as the solid phase carrier, and the DNA monomer base was used as the raw material, and the capture DNA was synthesized from the 3' end to the 5' end on a DNA synthesizer. The specific synthesized sequence is shown in Table 2. After the synthesis, the above CPG was transferred to a 2 mL clean and sterilized Eppen...

Embodiment 3

[0046] Example 3 Preparation of Hydrogel

[0047]Dissolve chain A and chain B in ultrapure water to prepare high-concentration DNA storage solution. Prepare 10% ammonium persulfate and 5% TEMED (N,N,N',N'-tetramethylethylenediamine) respectively, that is, dissolve 0.05g ammonium persulfate in 0.5mL ultrapure water and 25μl TEMED in 0.5mL ultrapure water. DNA and acrylamide with a final concentration of 4% were prepared as a mixture, and put into a vacuum desiccator for 10 minutes to vacuum and degas. Add freshly prepared initiator (ammonium persulfate) and accelerator (TEMED) with a final concentration of 1.4%, mix the reaction system in a vacuum desiccator, and react under vacuum at 30°C for 15 minutes to obtain chain A and The linear polymer products PS-A and PS-B of chain B. After mixing PS-A and PS-B according to 1:1, add 1 μL of 10× reaction buffer, (cocaine uses PB buffer: 77mM Na 2 HPO 4 ,23mMNaH 2 PO 4 , 50mM NaCl, 5mM MgCl 2 ,pH7.3; Adenosine uses Tris-HCl buf...

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Abstract

The invention discloses a high-sensitivity quantitative analysis method based on gas pressure detection, which can be used for high-sensitivity quantitative detection on many targets including inorganic ions, micro-molecules, and bio-macromolecules, such as proteins, DNA, viruses, bacteria and cells. The high-sensitivity quantitative analysis method utilizes an enzyme or nano particles and the like to catalyze hydrogen peroxide to generate a lot of gas; when a detection signal of a target molecule is converted into a gas pressure intensity signal, signal amplification is realized; finally, pressure intensity change is converted into an electric signal by a barometer, and then the electric signal is read so as to finish high-sensitivity quantitative detection. The barometer and three different detection systems including a DNA hydrogel system, a functionalized DNA sensing system and an ELISA (Enzyme-Linked Immuno Sorbent Assay) system are utilized to prove the feasibility, wide applicability and reliability of the high-sensitivity quantitative analysis method.

Description

technical field [0001] The invention discloses a high-sensitivity quantitative analysis method based on air pressure detection, which can be used for high-sensitivity quantification of various targets such as inorganic ions, small molecules, and biomacromolecules such as proteins, DNA, and even viruses, bacteria, and cells. It belongs to the fields of medical equipment, biological reagents, analytical instruments and the like. Background technique [0002] The development of novel, sensitive, highly selective, cheap and convenient quantitative detection and analysis methods has always been the most popular topic in scientific research. Existing quantitative detection and analysis methods usually rely on various large precision instruments, such as spectrometers, mass spectrometers, chromatographs, electrophoresis, etc. After a long period of development of these quantitative analysis methods, mature detection methods and technologies have been established, so they are widel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N7/18
CPCG01N7/18C12Q1/30G01N33/54373G01N33/581G01N33/587G01N33/543G01N33/56983G01N2333/11
Inventor 杨朝勇官志超郏莎莎朱志刘丹张明霞林水潮李久兴庄峙厦
Owner XIAMEN UNIV
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