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Intracellular detection method based on double-excitation ratio type up-conversion fluorescence probe

A detection method and excitation light technology, which can be used in fluorescence/phosphorescence, material excitation analysis, measurement devices, etc., and can solve problems such as the inability to achieve accurate intracellular quantitative detection.

Active Publication Date: 2021-03-05
FUJIAN INST OF RES ON THE STRUCTURE OF MATTER CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In order to solve the problem that dye-sensitized up-conversion nanoprobes cannot achieve accurate intracellular quantitative detection in the prior art, the present invention provides an intracellular detection method based on dual-excitation ratio up-conversion fluorescent probes

Method used

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  • Intracellular detection method based on double-excitation ratio type up-conversion fluorescence probe
  • Intracellular detection method based on double-excitation ratio type up-conversion fluorescence probe
  • Intracellular detection method based on double-excitation ratio type up-conversion fluorescence probe

Examples

Experimental program
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Effect test

Embodiment 1

[0078] This example uses dye-sensitized Yb-doped up-conversion nanoparticles IR808-UCNP@Yb-F127 to detect hypochlorous acid:

[0079] (1) 1mmol oil-soluble nanoparticles NaYF 4 :Yb,Er@NaYF 4 : The dye IR808 of Yb and 1 μ mol is dispersed in 20mL solvent methylene chloride, mixes one hour, obtains solution one;

[0080] (2) Dissolve 400mg of F127 in 2mL of dichloromethane, ultrasonically mix for 2min, and add dropwise to solution 1 under constant stirring to obtain solution 2;

[0081] (3) The solution is rotated and evaporated to remove the solvent dichloromethane, and after the volatilization of the dichloromethane is completed, it is redispersed in the aqueous solution;

[0082] (4) After the step (3) is completed, centrifuge and wash twice with deionized water to obtain the water-soluble dye-sensitized up-conversion nanoprobe.

[0083] (5) reacting the nanoprobe in step (4) with the target detection substance (hypochlorous acid) of 0-100 μ M for 30 minutes;

[0084] (6)...

Embodiment 2

[0089] This example uses dye-sensitized Nd-doped up-conversion nanoparticles IR808-UCNP@Nd-F127 to detect hypochlorous acid:

[0090] (1) 1mmol oil-soluble nanoparticles NaYF 4 :Yb,Er@NaYF 4 : Nd and 1 μmol of the dye IR808 were dispersed in 20mL of dichloromethane, mixed for one hour to obtain solution 1;

[0091] (2) Dissolve 400mg of F127 in 2mL of dichloromethane, ultrasonically mix for 2min, and add dropwise to solution 1 under constant stirring to obtain solution 2;

[0092] (3) The solution is rotated and evaporated to remove the solvent dichloromethane, and after the volatilization of the dichloromethane is completed, it is redispersed in the aqueous solution;

[0093] (4) After step (3) is completed, centrifuge and wash twice with deionized water to obtain a water-soluble dye-sensitized up-conversion nanoprobe;

[0094] (5) reacting the nanoprobe in step (4) with the target detection substance (hypochlorous acid) of 0-100 μ M for 30 minutes;

[0095] (6) Measure t...

Embodiment 3

[0100] refer to image 3 , near-infrared dual-excitation confocal microscopy system, including 2 independent lasers (980nm laser 1 and 808nm laser 2 2), laser scanning confocal microscope and fluorescence signal acquisition device. Among them, the laser scanning confocal microscope includes basic components such as an oil mirror 5, a dichroic mirror 1 6, a dichroic mirror 2 7, and a mirror 8; the fluorescence signal acquisition device includes a spectrometer 3 and a CCD camera 4 (as an image controller) .

[0101] The excitation light emitted by the excitation light source is focused by the oil lens to the cell to be observed, and the up-conversion light emitted by the nano-probe in the cell to be tested passes through the oil lens 5, dichroic mirror 1 6, and dichroic mirror 2 7 in sequence. , mirror 8, lens one 11, pinhole 9, lens two 12, optical filter 10 and lens three 13, captured by spectrometer 3. The up-converted light emitted by the nano-probe in the cell to be teste...

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Abstract

The invention belongs to the technical field of nano biological materials, and discloses an intracellular detection method based on a double-excitation ratio type up-conversion fluorescence probe. Themethod comprises the steps of carrying out contact reaction on a nanoprobe and a to-be-detected target object in cells, and detecting the concentration of the to-be-detected target object by monitoring the luminous intensity of the nanoprobe by adopting double exciting light, wherein the nanoprobe is dye-sensitized rare earth doped up-conversion nanoparticles, and the target object to be detectedis selected from intracellular active oxygen and active nitrogen related to many physiology and pathology, or intracellular metal ions. According to the method, the energy transfer process of the up-conversion nanoparticles is efficiently sensitized by using the dye, so that the detection sensitivity is improved; a double-excitation ratio probe model is provided as a setting reference, so that the detection deviation caused by an intracellular complex environment, probe distribution and instruments and equipment is reduced; a design scheme of a near-infrared double-excitation confocal microscope system is provided, and a new thought is provided for detecting substances in cells.

Description

technical field [0001] The invention belongs to the technical field of nanobiological materials, and in particular relates to an intracellular detection method based on a double-excitation ratio type up-conversion fluorescent probe. Background technique [0002] The development of non-invasive fluorescent probes to monitor biomolecules or physiological processes in living cells is crucial to understanding cell biology, pathology, and other biomedical-related sciences. However, the current intracellular analysis is mainly through targeted imaging or non-quantitative fluorescent labeling, which cannot meet the actual needs. One reason for this is the low sensitivity of the probe. For example, the concentration of hypochlorous acid in cells is very low. If there is no external stimulus, it is difficult for general probes to detect endogenous hypochlorous acid. Although the in vitro detection limit of some dye probes can also reach very low, their poor water solubility and sho...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/64
CPCG01N21/6486
Inventor 卢珊陈学元柯建熙商晓颖刘李幸俊宋晓荣
Owner FUJIAN INST OF RES ON THE STRUCTURE OF MATTER CHINESE ACAD OF SCI
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