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A glass nanopore sensor for detecting oh and its preparation and application

A nanopore sensor and nanopore technology, applied in instruments, measuring devices, scientific instruments, etc., can solve the problems of difficult probe synthesis, low steady-state concentration, high reactivity, and achieve selectivity and sensitivity, rapid response, and economical detection. The effect of simple cost and process flow

Active Publication Date: 2021-07-27
EAST CHINA NORMAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, OH has high reactivity and poor stability (the average lifespan is only 10 -6 s), the steady-state concentration in the cell is extremely low, and there are also interferences from various oxides and antioxidants in the cell
[0004] Traditional methods for detecting OH mainly include electron spin resonance, chromatography, and fluorescence methods, but few of them can achieve non-destructive detection in a single living cell, even though the detection limit of some fluorescent probes has reached the single-cell level. However, fluorescent probes for OH generally have limitations such as difficulty in probe synthesis, low detection selectivity, high background signal, strong cytotoxicity, and poor photostability.
These challenges make it difficult to achieve substantial breakthroughs in the detection technology of OH in vivo

Method used

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  • A glass nanopore sensor for detecting oh and its preparation and application
  • A glass nanopore sensor for detecting oh and its preparation and application
  • A glass nanopore sensor for detecting oh and its preparation and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Embodiment 1 Utilizes the Fenton reaction to detect the OH produced in the aqueous solution

[0071] (1) Preparation of glass nanoporous sensors

[0072] Use drawing instrument to draw out aperture 70nm, the glass conical nanopore of conical nanopore angle 5 °, inject the ethanol solution (V of 8mmol / L chloroauric acid 氯金酸 :V 乙醇 =3:2) 10 μL, irradiated for 2 hours under a 254 nm ultraviolet lamp, washed the nanopore with ethanol, dried at room temperature, and baked at 100° C. for 1 hour, so that the gold film was firmly covered on the inner wall of the nanopore. Then configure 10 -2 mol / L ethanol solution of cysteamine hydrochloride, inject 10 μL into the nanopore, and let it stand at room temperature for 8 hours, so that the sulfhydryl group in the cysteamine molecule can bind to the gold film through the Au-S bond, and then wash with ethanol to remove excess or weakly bound cysteamine. Finally, take the protoporphyrin solution (10 -5 mol / L), (get commercially av...

Embodiment 2

[0078] Example 2 uses drugs to stimulate and inhibit cells, and detects OH in cells

[0079] (1) Preparation of glass nanoporous sensors

[0080] The preparation method is the same as in Example (1).

[0081] (2) Detection of OH in cells

[0082] Before detection, inject 10 into the glass nanopore sensor -4 mol / L Zn 2+ Solution, at room temperature (25°C±3°C), let it stand for 8 hours to remove unbound or weakly bound Zn 2+ solution and washed with distilled water. Then inject pH7.0 electrolyte solution (containing 0.1mol / L KCl, pH7.0, 10mmol / L HEPES buffer solution) into the hole.

[0083] RAW264.7 mouse mononuclear macrophages in cell culture medium (composition includes 10% bovine serum albumin, 100unit / mL penicillin, 100ug / mL streptomycin), 37 ℃, 5% CO 2 On the day before the test, the cells were transferred to a petri dish with a diameter of 35 mm and a height of 14 mm, allowing the cells to adhere and grow freely for 24 hours. Before the test, under a confocal mic...

Embodiment 3

[0087] Example 3 Non-destructive cell insertion experiment

[0088] (1) Preparation of glass nanoporous sensors

[0089] The preparation method is the same as in Example (1).

[0090] (2) Non-destructive cell insertion experiment

[0091] Before detection, inject 10 into the glass nanopore sensor -4 mol / L Zn 2+ Solution, at room temperature, let it stand for 8 hours, remove unbound or weakly bound Zn 2+ solution and washed with distilled water. Then inject pH7.0 electrolyte solution (containing 0.1mol / L KCl, pH7.0, 10mmol / L HEPES buffer solution) into the hole.

[0092] In this example, Hela cells were used, and the culture method was the same as that of the mouse mononuclear macrophage RAW264.7 in Example 2. Before the experiment, the cells were stained with fluorescent dyes: Calcein-AM was used to stain live cells and PI was used to stain dead cells. Insert the glass nanopore sensor into the cell for 15 minutes and then take it out, and compare the changes in the numb...

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Abstract

The invention belongs to the technical field of sensing and detection of glass nanopores, and in particular relates to a glass nanopore sensor for detecting OH, its preparation and an electrochemical analysis method based on the principle of ion rectification. The method for the nanopore sensor of the present invention comprises: firstly using a drawing instrument to draw out a glass tapered nanopore, then evenly coating a layer of gold film on the inner wall of the nanopore, and finally through the strong interaction of the Au-S bond and the amide reaction, followed by modification of cysteamine molecules and protoporphyrin molecules on the gold film, and finally a functionalized glass nanopore sensor. The present invention also provides a method for detecting ·OH by the glass nanopore sensor. The technological process of the present invention is simple, does not need expensive reagents and large-scale instruments, and can realize high selectivity to OH in a complex system, high-sensitivity and fast response, and is an ideal solution for OH in the fields of food safety, medical hygiene, and environmental monitoring. Detection provides a portable and efficient detection platform and method design.

Description

technical field [0001] The invention belongs to the technical field of sensing and detection of glass nanopores, and in particular relates to a glass nanopore sensor for detecting hydroxyl radicals (OH) and its preparation and an electrochemical analysis method for OH based on the principle of ion rectification. Background technique [0002] Glass nanopore is a nanoscale tip formed by drawing a glass capillary with a drawing device. The opening shape and pore size at the tip of the nanopore can be adjusted by setting different heating temperatures, speeds, delay times, and pulling force values ​​for the drawing device. parameters to adjust. Place the prepared nanopore in an electrochemical workstation to build a three-electrode system, apply a symmetrical voltage to the nanopore, and an asymmetric current will be obtained. This I-V curve is called ionic current rectification (ICR). It is found that the degree and direction of ion rectification depend on the surface charge d...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N27/36G01N27/327
CPCG01N27/3278G01N27/36
Inventor 陈文婷朱安伟施国跃
Owner EAST CHINA NORMAL UNIV
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