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A kind of pH fluorescent dye, pH fluorescent probe, preparation method and application

A technology of fluorescent dyes and fluorescent probes, which is applied in the field of biomedical materials, can solve problems such as poor sensitivity, selectivity and photostability, uneven distribution of reference reagents, and influence on the accuracy of test results, and achieve good ratio measurement effect and convenient Measuring ratiometric fluorescence and the effect of improving detection accuracy

Active Publication Date: 2021-01-19
SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing fluorescent dyes generally have some shortcomings in practical applications, such as uneven distribution of sensitive reagents sensitive to pH changes and reference reagents insensitive to pH changes, which is not conducive to ratio measurement and affects the accuracy of detection results. It is greatly affected by environmental factors, has a narrow pH response range to pH changes, and has poor sensitivity, selectivity and photostability, etc.
Most of the existing pH detection probes are also obtained based on the above-mentioned dyes, which have the disadvantages of these dyes; in addition, the existing pH fluorescent probes also have the following disadvantages: nanoparticle probes are easily wrapped by secretory vesicles or attached to On the endosome, which limits its further application in biological systems; the fluorescence of the small molecule probe system is not strong and unstable to light, and is easy to be quenched; it is chelated with other molecules in the cell, which makes the fluorescence spectrum shift

Method used

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  • A kind of pH fluorescent dye, pH fluorescent probe, preparation method and application
  • A kind of pH fluorescent dye, pH fluorescent probe, preparation method and application
  • A kind of pH fluorescent dye, pH fluorescent probe, preparation method and application

Examples

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preparation example Construction

[0134] In the present invention, there is no special limitation on the preparation method of polyurethane microspheres and other carriers, and the preparation methods commonly used in this field can be used for preparation.

[0135] Preferably, the preparation method of the carrier includes but is not limited to any one of self-emulsification-solidification method, electrostatic spray method, microfluidic method and ion coacervation method, preferably self-emulsification-solidification method;

[0136] Preferably, the carrier is polyurethane microspheres, and the preparation of polyurethane microspheres by self-emulsification-curing method includes: adding the polyurethane solution as an oil phase to the water phase dropwise, stirring, and the toluene solvent volatilizes during the stirring process, so that the polyurethane self-cures into balls , wherein the concentration of the polyurethane solution is preferably 5% to 10%, and the stirring speed is preferably 200 to 800 rpm....

Embodiment 1

[0140] A pH fluorescent probe comprises a polyurethane carrier and a pH fluorescent dye encapsulated in the polyurethane carrier, and the pH fluorescent dye includes a pH-sensitive protein solution and a reference pH-sensitive Nile Red solution.

[0141] The preparation method of the pH fluorescent probe comprises the following steps:

[0142] (1) Preparation of pH-sensitive protein solution: Weigh 500mg of protein (BSA) into a 15mL centrifuge tube, dissolve in 10mL double-distilled water, invert upside down to fully dissolve; let stand, remove air bubbles; pipette the prepared Put 8 mL of BSA solution in a beaker, stir at a medium speed at room temperature, add 16 mL of 75% ethanol dropwise into it; add 200 μL of 8% glutaraldehyde (w:w%) dropwise to the above solution, avoid light, Stir and cross-link for 18 hours; centrifuge the obtained BSA solution at 3500 rpm for 15 minutes to remove precipitated impurities and obtain a pH-sensitive protein solution;

[0143] (2) Prepara...

Embodiment 2

[0149] A pH fluorescent probe comprises a polyurethane carrier and a pH fluorescent dye wrapped in the polyurethane carrier, the fluorescent dye includes a pH-sensitive protein solution and a reference pH-sensitive Nile Red solution.

[0150] The difference between the preparation method of the pH fluorescent probe and Example 1 lies in step (1) preparation of the pH-sensitive protein solution.

[0151] In this example, (1) Preparation of pH-sensitive protein solution: Weigh 500 mg of protein (BSA) and place it in a 15 mL centrifuge tube, dissolve it in 10 mL of double-distilled water, invert it upside down to fully dissolve it; let stand, remove air bubbles ; Take 8 mL of the prepared BSA solution and place it in a beaker, stir at a medium speed at room temperature, and add 16 mL of acetone dropwise into it; add 200 μL of 8% glutaraldehyde (w:w%) dropwise to the above solution, and keep away from light , medium-speed stirring and cross-linking for 18 hours; the obtained BSA s...

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Abstract

The invention belongs to the technical field of biomedical materials, and relates to pH fluorochrome, a pH fluorescent probe, preparation methods and application. The pH fluorochrome comprises a pH sensitive protein solution and a reference pH-sensitivity fluorochrome solution. The pH fluorescent probe comprises a carrier and the fluorochrome wrapped with the carrier. The pH fluorescent probe is amicron-level pH fluorescent probe. According to the pH fluorochrome and the pH fluorescent probe, pH values can be analyzed quickly and accurately in real time, and the good ratio measurement effectis achieved; and fluorescence can exist for a long time during co-culture of the pH fluorescent probe and cells, the microenvironment of the cells is detected in real time, and the change of the microenvironment in vivo is effectively reflected.

Description

technical field [0001] The invention belongs to the technical field of biomedical materials, and relates to a fluorescent probe, in particular to a pH fluorescent dye, a pH fluorescent probe, a preparation method and an application. Background technique [0002] Intracellular pH plays an important role in living organisms, and it is closely related to the maintenance of normal cell functions. The progress or completion of chemical reactions, many important physiological processes of cells and organelles, etc. are closely related to the pH value. For example, intracellular pH plays a crucial role in a series of tissue activities such as enzyme activity, cell proliferation and apoptosis, drug resistance, ion transport, endocytosis and muscle contraction. Abnormal cell function, growth, and division are often directly or indirectly related to abnormal pH values. Excessive acidity and alkalinity can lead to heart, lung or neurological diseases, and may also cause diseases such ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N21/64
CPCG01N21/6486
Inventor 赵晓丽刘缘
Owner SHENZHEN INST OF ADVANCED TECH CHINESE ACAD OF SCI
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