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Fluorescence detection method, curve establishment method and kit for free zinc ion concentration

A fluorescence detection, zinc ion technology, applied in the field of cell biology and medicine, can solve the problems of complex operation steps, low measurement efficiency, difficult to equip, etc., and achieves the effect of simple operation, cost saving and efficiency improvement of the equipment

Active Publication Date: 2022-04-01
HUAZHONG UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

However, this method needs to obtain the fluorescence intensities of mNeonGreen and mRuby2 in different channels through a dual-channel imaging device, calculate the FRET ratio, and then obtain the concentration of free zinc ions through a very complicated derivation and calculation formula. The above method has the advantages of Two obvious defects: (1) need to use dual-channel fluorescence imaging equipment, such equipment is expensive to purchase, it is not easy to equip general experimental units, and the operation steps are also very complicated; (2) the calculation process is complicated, and the calculation process is more than one step, Moreover, there are many parameters in the formula, and the concentration of free zinc ions in the vesicles cannot be obtained in one step, resulting in low measurement efficiency

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  • Fluorescence detection method, curve establishment method and kit for free zinc ion concentration
  • Fluorescence detection method, curve establishment method and kit for free zinc ion concentration
  • Fluorescence detection method, curve establishment method and kit for free zinc ion concentration

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Embodiment 1

[0066] Example 1 The method of the present invention can realize single-channel imaging to detect the concentration of free zinc ions in vesicles

[0067] Specifically include the following steps:

[0068] 1. Establishment of free zinc ion concentration-mRuby2 fluorescence intensity working curve

[0069] Experimental animals: C57BL / 6J mice, 12 weeks old, purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. (Charles River Laboratories). Type 2 diabetes mellitus (T2DM) model Leprdb / Leprdb (db / db) mice, 12 weeks old, were purchased from Jiangsu GemPharmatech Co., Ltd. The mice used in all experiments in this example were male. All experimental related operations were in compliance with the requirements of the "Guidelines for the Management and Use of Laboratory Animals".

[0070] Experimental equipment: The imaging system includes a high-speed confocal scanning unit (model: Yokogawa CSU-X1), a 4-color laser light source, a multiplying charge-coupled d...

Embodiment 2

[0097] Embodiment 2 Kit of the present invention carries out the method for free zinc ion concentration detection

[0098] The kit of the present invention includes the following components: an adenovirus carrying a target gene and a phosphate buffered saline solution containing 10% glycerol; the target gene is a nucleotide sequence of a metal zinc sensor connected to the NPY gene, and the metal zinc The sensor is based on the circular motif of the human TRIM72 protein, the N-terminal of the circular motif is connected to the mNeonGreen fluorescent protein molecule, and the C-terminal of the circular motif is connected to the mRuby2 fluorescent protein molecule; wherein the adenovirus carrying the target gene is Dry powder, phosphate-buffered saline containing 10% glycerol is used to solubilize adenovirus carrying the gene of interest.

[0099] The specific use process includes the following steps:

[0100] (1) Isolate the pancreatic islet β cells to be tested;

[0101] (2) ...

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Abstract

The invention relates to a fluorescence detection method, a curve establishment method and a kit for the concentration of free zinc ions. The detection method of the invention uses an adenovirus carrying a target gene to infect the pancreatic islet β cells to be tested, and the target gene is a NPY gene linked The nucleotide sequence encoding the metal zinc sensor; then use a single-channel fluorescence microscope to perform in vivo imaging of pancreatic beta cells to obtain the emitted red mRuby2 fluorescence intensity; then calculate the islet to be tested according to the free zinc ion concentration-mRuby2 fluorescence intensity working curve Concentration of free zinc ions in β-cell vesicles. The detection method has low requirements on imaging equipment. After the working curve is established, it only needs to use a single-channel fluorescence imaging equipment, which is easier to operate and does not need to go through complicated calculations. It can directly measure the single-channel mRuby2 fluorescence intensity in one step. Obtain the average free zinc ion concentration in the vesicles, significantly improving the efficiency of the measurement.

Description

technical field [0001] The invention relates to the fields of cell biology and medicine, in particular to a fluorescence detection method, a curve establishment method and a kit for free zinc ion concentration in vesicles of pancreatic beta cells. Background technique [0002] Fluorescence resonance energy transfer (FRET) refers to the energy transfer phenomenon between two fluorescent molecules that are very close (generally within the range of 10nm). When the emission spectrum of the donor fluorescent molecule overlaps with the absorption spectrum of the acceptor fluorescent molecule, and the distance between the two molecules is close enough, a non-radioactive energy transfer, that is, the FRET phenomenon, occurs, which makes the fluorescence of the donor quenched (intensity lower than when it exists alone), while the fluorescence emitted by the acceptor is greatly enhanced. [0003] In patients with type 2 diabetes or other diseases caused by zinc deficiency in β cells,...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/64
CPCG01N21/6402G01N21/6428G01N21/6458G01N2021/6432
Inventor 王意周梦璇咸逸
Owner HUAZHONG UNIV OF SCI & TECH