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FSAP detection probe and preparation method and application thereof

A technology for detection probes and detection kits, which is applied in the interdisciplinary field of medicine, can solve the problems of undetectable, mixed free PD-L1, cumbersome steps, etc., and achieve the effects of maintaining detection ability, good photostability, and strong fluorescence interference

Pending Publication Date: 2022-07-29
WUHAN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, ultracentrifugation not only requires an expensive ultracentrifuge, but also is cumbersome and time-consuming. More importantly, the recovery rate of extracellular vesicles in this method is low (accounting for 5% of the total extracellular vesicles), and free PD- L1 will also be mixed with
Therefore, ultracentrifugation can lead to the loss of large amounts of PD-L1-positive EVs, which cannot be detected by ELISA methods

Method used

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  • FSAP detection probe and preparation method and application thereof
  • FSAP detection probe and preparation method and application thereof
  • FSAP detection probe and preparation method and application thereof

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[0045] According to a typical embodiment of the present invention, a preparation method of a FSAP detection probe is provided, the method comprising:

[0046] Step S1, pass a protective gas in the octadecene solvent, then add silver acetate and octanethiol and heat to 160-170 ° C to obtain a mixed solution; inject ODE-Se precursor into the mixed solution and rapidly cool down to 110 ~130℃ for reaction to obtain near-infrared hydrophobic Ag 2 Se quantum dots;

[0047] In the step S1,

[0048] The preparation method of the ODE-Se precursor includes: under a protective gas atmosphere, dissolving selenium powder in an octadecene solvent, heating to 200-220 DEG C until it becomes a transparent solution, and then maintaining for a period of time to obtain ODE -Se precursor.

[0049] The molar ratio of the silver acetate, the ODE-Se precursor and the octanethiol is 1:1:(18-22). This molar ratio range is favorable for preparing quantum dots with high crystallinity and complete rea...

Embodiment 1

[0069] Embodiment 1, FSAP detection probe and its preparation method and application

[0070] 1. FSAP detection probe and preparation method thereof

[0071] (1) Pass protective gas in octadecene solvent, then add 0.2 mmol silver acetate and 700 μL octanethiol and heat to 165 ° C to obtain a mixed solution; inject 0.2 mmol ODE-Se precursor into the mixed solution and rapidly cool down Reaction at 120 °C to obtain near-infrared hydrophobic Ag 2 Se quantum dots;

[0072] (2) the hydrophobic Ag 2 20 mg of Se quantum dots were coated and modified with 50 mg of octylamine-grafted polyacrylic acid OPA in chloroform solution, and then the excess OPA was removed by centrifugation, and dispersed in PBS to obtain a hydrophilic concentration of 0.4 μM with carboxyl groups on the surface. Ag 2 Se-OPA quantum dots;

[0073] (3) the Ag 2 500 μL of Se-OPA quantum dots were subjected to amidation reaction with 50 μL (100 μM) of PD-L1 aptamer with amino group at one end at 25°C to obtain...

Embodiment 2

[0077] Embodiment 2, FSAP detection probe and its preparation method and application

[0078] 1. FSAP detection probe and preparation method thereof

[0079](1) Pass protective gas in octadecene solvent, then add 0.2 mmol silver acetate and 700 μL octanethiol and heat to 160° C. to obtain a mixed solution; inject 0.2 mmol ODE-Se precursor into the mixed solution and rapidly cool down Reaction at 130 °C to obtain near-infrared hydrophobic Ag 2 Se quantum dots;

[0080] (2) the hydrophobic Ag 2 20 mg of Se quantum dots were coated and modified with 50 mg of octylamine-grafted polyacrylic acid OPA in chloroform solution, and then the excess OPA was removed by centrifugation, and dispersed in PBS to obtain a hydrophilic concentration of 0.4 μM with carboxyl groups on the surface. Ag 2 Se-OPA quantum dots;

[0081] (3) the Ag 2 500 μL of Se-OPA quantum dots were subjected to amidation reaction with 50 μL (100 μM) of PD-L1 aptamer with amino group at one end at 25°C to obtain ...

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Abstract

The invention discloses an FSAP detection probe and a preparation method and application thereof.The method comprises the steps that shielding gas is introduced into an octadecene solvent, then silver acetate and octyl mercaptan are added, the mixture is heated to 160-170 DEG C, and a mixed solution is obtained; an ODE-Se precursor is injected, the temperature is rapidly reduced to 110-130 DEG C for a reaction, and the near-infrared hydrophobic Ag2Se quantum dots are obtained; the preparation method comprises the following steps: carrying out coating modification on a hydrophobic Ag2Se quantum dot in a chloroform solution by using octylamine grafted polyacrylic acid OPA to obtain a hydrophilic Ag2Se-OPA quantum dot with carboxyl on the surface; and carrying out amidation reaction on the FSAP and a PD-L1 aptamer with amino at one end to obtain the FSAP. The FSAP detection probe can detect the PD-L1 level of the extracellular vesicles, the PD-L1 positive extracellular vesicles do not need to be separated in advance, interference of free PD-L1 is avoided, and the detection efficiency is greatly improved.

Description

technical field [0001] The invention relates to the field of medical cross-technology related to fluorescence anisotropy detection technology and extracellular vesicle PD-L1 level, in particular to a FSAP detection probe and a preparation method and application thereof. Background technique [0002] Malignant tumors have high mortality and high recurrence rate, and are still one of the important diseases that threaten human health and life. In recent years, tumor immunotherapy has become another method of tumor treatment after traditional surgery, radiotherapy and chemotherapy, bringing hope to patients with advanced malignant tumors. American immunologist James Allison and Japanese biologist Tasuku Honjo won the 2018 Nobel Prize in Physiology or Medicine for their "discovery of negative immunomodulatory treatments for tumors." Among them, immunotherapy targeting programmed cell death 1 ligand 1 (PD-L1) and its corresponding receptor PD-1 is a star molecule in the current i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/532G01N33/569G01N33/574G01N33/58C09K11/88C09K11/02
CPCG01N33/532G01N33/588G01N33/56966G01N33/57488C09K11/881C09K11/025G01N2333/70532
Inventor 田智全陈刚余自力刘婧许瑞
Owner WUHAN UNIV