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A method for detecting hydrogen peroxide based on solid-state nanopores

A technology of hydrogen peroxide and nanopores, applied in measuring devices, material analysis through electromagnetic means, instruments, etc., can solve problems such as difficult to reflect the kinetic characteristics of enzymes and substrates, and achieve easy design and processing, and easy physical and chemical modification. sexual effect

Active Publication Date: 2019-12-03
南通大学技术转移中心有限公司
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Problems solved by technology

Traditional hydrogen peroxide sensors are mostly studied from a relatively macroscopic level, and the resulting parameters are often difficult to reflect the kinetic characteristics of the reaction between the enzyme and the substrate at the molecular level. Therefore, to construct an integrated single-molecule horseradish peroxidase sensor for It is particularly important to detect hydrogen peroxide quantitatively in real time.

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  • A method for detecting hydrogen peroxide based on solid-state nanopores
  • A method for detecting hydrogen peroxide based on solid-state nanopores
  • A method for detecting hydrogen peroxide based on solid-state nanopores

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preparation example Construction

[0038] 1. Preparation of Nanopores

[0039] The research on the process of preparing high-insulation all-solid-state silicon nitride film has successfully obtained ultra-thin silicon nitride films with various film thicknesses of 30-100nm. Through the optimization of the process, 10-100nm ultra-thin silicon nitride film that meets the drilling requirements will be obtained; through the optimization of the focused electron beam or ion beam processing conditions in the TEM, FIB, and HIM processing system platforms, various thicknesses, The pore size is the aspect ratio, the shape of the nanopore.

[0040] 2. Modification of nanopores and construction of unimolecular enzyme sensors

[0041] Various silanization treatments (methylsilane, trifluorosilane, carboxysilane, etc.) are used to achieve differential modification of the nanopore surface and inner wall. The peroxidase is coupled to the inner wall of the nanopore through a silane coupling molecule. For example, comb-like g...

Embodiment

[0052] Reagents and materials: APTES, Sigma; potassium chloride (KCl), Sigma; methanol, Sigma; dimethyl sulfoxide, Hefei Xinyuan Biotechnology Co., Ltd.; horseradish peroxidase (HRP), Sigma; ABTS, 98% Sigma; Hydrogen Peroxide (H2O2), 30% SL Labor-Service; Methanol, 98% Sigma; Aminosilane, 98% Sigma;

[0053] Test equipment: field emission scanning electron microscope, Malvern particle size analyzer, analytical balance, vibration isolation table, shielding box, silver wire, pipette gun, ultrapure water, patch clamp,

[0054] Experimental solution preparation

[0055] A. Preparation of 1mol / L KCl solution: Weigh 0.7458g of KCl with an analytical balance, dissolve it, put it in a 10mL volumetric flask, shake well, filter it with an Anotop 0.02μm filter membrane, and then dilute to 0.1mol / L KCl.

[0056] B. Preparation of piranha solution: piranha solution, the ratio is 3:1 (volume ratio) of concentrated sulfuric acid (98%) and hydrogen peroxide solution (30%), prepared in a fum...

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Abstract

The invention relates to a method for detecting hydrogen peroxide based on solid nanopores. The method achieves differential modification of surfaces and inner walls of the nanopores by using varioussilanization treatment and punching different micropores on 10-100 nm ultrathin silicon nitride film, coupling peroxidase to the inner walls of the nanopores through silane coupling molecules to construct a controllable, stable and sensitive nanohole single-molecule horseradish peroxidase sensor, and by analyzing nanopore ion current signals of enzymatic reactions and a model mechanism and law ofthe enzymatic reactions Km and Kcat of single enzyme molecule in the nanopore, quantitatively detecting hydrogen peroxide in real time. The specific small-molecule real-time sensing detection method is established through the differential modification inside and outside the nanopore and the controllable modification of the horseradish peroxidase single molecule in the nanopore, and the precision of the method is less than nanomolar.

Description

technical field [0001] The invention relates to nanopore sensing and detection, in particular to a method for detecting hydrogen peroxide based on solid-state nanopores. Background technique [0002] Nanopore sensing detection technology has been considered as one of the powerful technologies for rapid unlabeled single molecule detection since it was proposed in 1996. Nanopores are mainly divided into solid-state nanopores and biological nanopores. Nanopore sensing technology has become a new, important and potential analysis and detection method in the fields of chemistry and biology. It has broad application prospects and great potential value in the fields of DNA sequencing research, single-molecule detection, single-molecule chemical reaction and protein folding. Nanopores originated from the phenomenon of transmembrane ion channel transport on the surface of cell membranes, using the concept of resistance effect in solution. Similar to the concept, the micropore sens...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N27/327
CPCG01N27/327
Inventor 谭生伟刘全俊
Owner 南通大学技术转移中心有限公司
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