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Method for detecting prostate cancer exosome based on Fe3O4@ SiO2@ TiO2 nanoparticle enrichment and PSMA sensor

A nanoparticle, prostate cancer technology, applied in instruments, measuring devices, scientific instruments, etc., can solve problems such as loss of exocytosis, and achieve the effects of strong specificity, simple and rapid separation method, and high sensitivity

Pending Publication Date: 2020-12-25
NINGBO UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It has been reported in the literature that TiO 2 The enrichment of exosomes can be successfully achieved, but the subsequent solid-liquid separation by centrifugation may lose a large amount of exosomes.

Method used

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  • Method for detecting prostate cancer exosome based on Fe3O4@ SiO2@ TiO2 nanoparticle enrichment and PSMA sensor
  • Method for detecting prostate cancer exosome based on Fe3O4@ SiO2@ TiO2 nanoparticle enrichment and PSMA sensor
  • Method for detecting prostate cancer exosome based on Fe3O4@ SiO2@ TiO2 nanoparticle enrichment and PSMA sensor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Synthetic Fe 3 o 4 @SiO 2 @TiO 2 Nanoparticles

[0048] 1. Add 0.15g Fe 3 o 4 Dissolve in a mixture of ethanol (280mL), deionized water (70mL) and ammonia water (5mL, 28wt%), and sonicate for 15min. Then 4 mL TEOS was added, and the reaction was continuously stirred at room temperature for 10 h to obtain Fe 3 o 4 @SiO 2 . figure 1 a is Fe 3 o 4 TEM image of figure 1 b is Fe 3 o 4 @SiO 2 TEM image. These two figures illustrate that SiO 2 Successfully wrapped in Fe 3 o 4 Outside.

[0049] 2. Add 0.05g Fe 3 o 4 @SiO 2 Dissolve in a mixture of ethanol (100 mL) and ammonia water (0.3 mL, 28 wt%), and after ultrasonication for 15 min, add 0.75 mL of TBOT dissolved in ethanol dropwise with continuous stirring. The reaction was continuously stirred at 45° C. for 24 h, the prepared product was washed three times with ultrapure water and ethanol, and dried at 60° C. overnight. figure 1 c is Fe 3 o 4 @SiO 2 @TiO 2 TEM image of figure 1 d is Fe 3 o 4 ...

Embodiment 2

[0051] Exosome isolation.

[0052] 1. Prepare two parallel samples: LNCaP exosome solution (10 7 individual / μL). Exosomes from one sample were stained with PKH26 dye. Exosomes from another sample were stained with 1.2 mg Fe 3 o 4 @SiO 2 @TiO 2 After incubating at room temperature for 8 minutes, use a magnetic stand for rapid magnetic separation, and wash with PBS repeatedly for 3 times. figure 2 a is the bright-field and fluorescence images after exosome staining, figure 2 b is Fe 3 o 4 @SiO 2 @TiO 2 / bright-field and fluorescence maps of exosomes, figure 2 c is the bright-field and fluorescence images of the washings after repeated washing. These three figures illustrate that Fe 3 o 4 @SiO 2 @TiO 2 Exosomes can be successfully isolated and the number of enriched exosomes is large.

[0053] 2. Prepare exosomes at a concentration of 10 7 cells / μL, stained with PKH26, and the fluorescence intensity was recorded as F 0 . Prepare the same concentration of ex...

Embodiment 3

[0056] Example 3 Exploration of the best conditions for exosome detection, including the following steps:

[0057] 1. Optimization of PSMA sensor. First design and build three PSMA sensors, the PSMA1 sequence is shown in the table below:

[0058]

[0059] After synthesis, the chains were untied at a high temperature of 100 degrees, then annealed, and then the three sensors were diluted to a concentration of 200nM and their fluorescence intensity was measured (excitation: 557nm, emission 580nm). image 3 a is the fluorescence intensity graph of three sensors with the same concentration, among which sensor 3 has the smallest fluorescence intensity and the smallest background, so sensor 3 was selected for exosome detection in the experiment.

[0060] 2. Prepare exosomes with a concentration of 106 / μL, incubate with sensor 3 at different concentrations (0, 20, 50, 100, 200, 300, 400, 500, 600nM) at room temperature for 30 minutes, and measure the fluorescence intensity (excit...

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Abstract

The invention discloses a method for detecting prostate cancer exosome based on Fe3O4@ SiO2@ TiO2 nanoparticle enrichment and a PSMA sensor. The method comprises the following steps of: enriching exosome by adopting Fe3O4@ SiO2@ TiO2 nanoparticles, and then constructing a hairpin-shaped PSMA aptamer sensor to quantify the exosome by measuring the change of fluorescence intensity. By utilizing thedetection method, the exosome of prostate cancer cells can be successfully distinguished from the exosome of normal prostate cells, and the detection limit is 9 * 10 <3> exosomes / microliter. The method is used for clinical prostate cancer patient serum samples and normal human serum samples, can quickly and accurately detect prostate cancer patients, and is verified by a traditional ELISA method. The exosome enrichment method is simple and quick, the enrichment efficiency is high, exosome enrichment can be completed within 8 minutes, and the enrichment efficiency is 91.5%. The sensitivity ishigh, the specificity is high, the detection limit is 9 * 10 <3> exosomes / microliter, and clinical prostate cancer patients and healthy people can be remarkably distinguished.

Description

technical field [0001] The invention is applied to the technical field of detection and analysis of exosome markers, and relates to a TiO-based 2 Rapid and accurate assay of prostate cancer exosomes with and PSMA aptamers. Background technique [0002] Prostate cancer (PCa) is the most common solid malignancy in men worldwide. In the early stage, it has a high cure rate, therefore, the treatment of prostate cancer depends on early diagnosis. Prostate-specific antigen (PSA) is a serum biomarker for prostate cancer diagnosis. However, although PSA has high sensitivity, its specificity is poor, especially in men with serum PSA levels of 2-10 ng / mL, and therefore, other specific PCa biomarkers are needed in urology. Exosomes are biological vesicles encapsulated in lipid bilayer membranes secreted by normal cells and tumor cells, ranging in size from 30 to 200 nm. Exosomes play different roles in intercellular communication and are widely found in body fluids, including blood...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/574G01N33/543G01N21/64
CPCG01N33/57484G01N33/57434G01N33/56966G01N33/54346G01N21/6428G01N2021/6432
Inventor 李巧玉施海梅余绍宁
Owner NINGBO UNIV
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