Method for detecting human epididymis protein 4 based on resonance energy transfer of up-conversion nano material and BHQ-1

A resonance energy transfer, BHQ-1 technology, applied in the field of nanomaterials and analytical chemistry, can solve the problem of low detection sensitivity, achieve high sensitivity, improve accuracy and stability, and strong specificity.

Active Publication Date: 2021-02-23
SOUTH CENTRAL UNIVERSITY FOR NATIONALITIES
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] The purpose of the present invention is mainly to solve the problem of low detection sensitivity and provide a method for det

Method used

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  • Method for detecting human epididymis protein 4 based on resonance energy transfer of up-conversion nano material and BHQ-1
  • Method for detecting human epididymis protein 4 based on resonance energy transfer of up-conversion nano material and BHQ-1
  • Method for detecting human epididymis protein 4 based on resonance energy transfer of up-conversion nano material and BHQ-1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0051] A method for preparing a water-soluble up-conversion fluorescent nanomaterial, comprising the steps of:

[0052] 1) Using rare earth chloride as a raw material, oleic acid as a ligand, and 1-octadecene as a solvent, high-temperature solvothermal method is used to synthesize rare earth up-conversion nanoparticles wrapped in oleic acid. The specific method is as follows:

[0053] a) Rare earth chloride raw material LnCl 3 Synthesized by air bath method:

[0054] 6mmol of Y with a molar ratio of 79.8:20:0.2 2 o 3 , Yb 2 o 3 and Er 2 o 3 The formed mixture was added into a one-necked bottle, 25mL of concentrated hydrochloric acid (36wt%-38wt%, the same below) was added thereto and heated until the solution was clear; then the temperature was raised and the solvent was evaporated to dryness and cooled to room temperature; then ultrapure water was added thereto simultaneously , absolute ethanol, and n-hexane, heated at 70°C for 5h, and after cooling to room temperature...

Embodiment 2

[0066] Preparation of upconversion fluorescent probes: Amidation reaction was used to modify the surface of the target cancer antigen HE4 aptamer on the surface of water-soluble upconversion fluorescent nanomaterials. The specific method is as follows:

[0067] Add 100 μL of 10 mg / mL water-soluble up-conversion fluorescent nanomaterial dispersion prepared in Example 1 (the up-conversion fluorescent nanoparticles wrapped with polyacrylic acid prepared in Example 1 are dispersed with ultrapure water) into 700 μL MES buffer solution ( 10mM, pH=5.5); then 100uL 5mg / mL EDC solution (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride EDC·HCl was dissolved in MES buffer solution (10mM, pH=5.5)) and 100uL 10mg / mL N-hydroxysulfosuccinimide solution Sulfo-NHS (prepared in MES buffer solution (10mM, pH=5.5)) were added to the above solution, and then the mixture was heated at 35 Incubate at ℃ for 1 h to activate the carboxyl groups on the water-soluble up-conversion fluorescent na...

Embodiment 3

[0071] In order to investigate the effect of different fluorescent acceptor concentrations on the fluorescence quenching efficiency, this example, on the basis of Example 2, added different amounts of HE4 aptamer-BHQ-1 dispersions to the above prepared in Example 1. Convert the nanoparticles into the buffer solution and incubate at 37°C for 1 h to obtain the detection probe, then measure the upconversion fluorescence intensity of each mixture under a 980nm laser, and obtain the corresponding upconversion fluorescence intensity in the mixture solution with the largest fluorescence quenching efficiency. Convert fluorescent probe concentration and fluorescent acceptor concentration.

[0072] Figure 5 , 6 It is the result figure of the efficiency of the acceptor to the fluorescence quenching of the water-soluble up-conversion nanoparticles (the fluorescence intensity of the blank sample is F 0 , the fluorescence intensity of each group of mixed solutions after adding the HE4 ap...

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Abstract

The invention discloses a method for detecting HE4 based on fluorescence resonance energy transfer of an up-conversion nano material and a BHQ1 quenching group. The preparation method comprises the following steps of synthesizing polyacrylic acid modified up-conversion nanoparticles by a solvothermal method, and coupling and immobilizing an aptamer of which the 5 '-end is modified with amino and the 3'-end is modified with BHQ1 onto the surfaces of the particles by amidation reaction to construct an up-conversion nanoparticle probe. As fluorescence resonance energy transfer can occur between the BHQ1 and the up-conversion nanoparticles, fluorescence of the up-conversion nanoparticles can be quenched by the BHQ1; after the target object is added, affinity between the target object and the aptamer is stronger, the distance between an energy donor and an energy receptor is increased, fluorescence resonance energy transfer is blocked, and then fluorescence of the up-conversion nanoparticles is recovered; quantitative determination of the tumor marker HE4 can be realized according to the fluorescence intensity recovery degree of the upconversion nanoparticles under different concentrations of a target object. The method disclosed by the invention is simple and convenient to operate, high in sensitivity and strong in specificity, and is expected to be used for early diagnosis of ovarian malignant tumors.

Description

technical field [0001] The invention relates to the technical fields of nanomaterials and analytical chemistry, in particular to a method for detecting HE4 based on upconversion nanomaterials and BHQ-1 quenching group fluorescence resonance energy transfer. Background technique [0002] Malignant ovarian tumor is one of the most common gynecological cancers at home and abroad. Its incidence rate ranks third after cervical cancer and uterine body cancer, with an incidence rate of 1.4%. With the continuous development of science and technology, society and medical facilities, scientists have made considerable efforts in tumor treatment such as new drug development, improvement of medical facilities and optimization of treatment plans, etc., but its mortality rate still ranks first among gynecological malignancies. Five-year survival rate is only 3 to 19%. Among them, one of the important reasons causing its high mortality rate is the lack of accurate analysis methods for earl...

Claims

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Application Information

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IPC IPC(8): G01N21/64G01N33/574G01N33/533C09K11/02C09K11/85B82Y20/00B82Y30/00B82Y40/00
CPCG01N21/6428G01N33/57449G01N33/57488G01N33/533C09K11/02C09K11/7773B82Y20/00B82Y30/00B82Y40/00G01N2021/6432G01N2021/6439G01N2333/81
Inventor 李春涯马秀清叶绵泰王炎英
Owner SOUTH CENTRAL UNIVERSITY FOR NATIONALITIES
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