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Preparation method of low-abundance low-molecular-weight protein-rich material

A low-molecular-weight, low-abundance technology, used in the preparation of test samples, material inspection products, biological testing, etc., can solve problems such as degradation, interference effects, and easy loss of target proteins, and achieve the effect of avoiding loss and preventing degradation.

Inactive Publication Date: 2012-12-05
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the commonly used protein enrichment material is magnetic mesoporous material, its main disadvantages are: 1) the target protein is easily lost and degraded by other high molecular weight proteases during the separation process; Molecular Interfering Effects

Method used

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  • Preparation method of low-abundance low-molecular-weight protein-rich material

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1 Preparation of porous silicon

[0019] Take the P-type boron-doped silicon wafer (100) crystal form, fix it in the electrolytic cell, add 0.5ml ethanol, 2-3ml hydrofluoric acid with a mass concentration of 40% as the electrolyte, use the silicon wafer as the anode and the platinum electrode as the cathode , Carry out DC electrolytic etching and stripping, set the current intensity to 10~80mA, the etching time is 5 minutes, the porous silicon layer after etching and stripping is washed with ethanol and ultrasonically crushed for 15 minutes, and then dried with nitrogen , The pore size of the obtained porous silicon particles is 5-20nm;

[0020] The above porous silicon particles are subjected to ozone oxidation treatment for 20 minutes to form surface silicon-oxygen bonds;

[0021] The oxidized porous silicon particles were immersed in a mixed solution of 1% aminosilylation reagent and ethanol solvent for 1 hour, and then dried in an oven at 110°C for 15 minutes. The...

Embodiment 2

[0023] Example 2 Processing and testing of insulin samples

[0024] In this example, the porous silicon particles modified with undecylenic acid with a porosity of 26% were used as the enrichment material to directly detect low-concentration insulin samples. The specific steps are as follows:

[0025] 1. Sample adsorption

[0026] The insulin sample in this example is a medical human injection, and the insulin injection is diluted with physiological buffer PBS to 1.0 mg·mL -1 Then as the solution to be tested.

[0027] Adsorption: Take four centrifuge tubes, labeled 1, 2, 3, 4, and add 50 uL of the test solution to each, then add 3 mg of unetched silicon powder to centrifuge tube 2, and 3 mg to centrifuge tube 3. For porous silicon particles with a porosity of 26%, 3 mg of porous silicon particles with a porosity of 26% modified by undecylenic acid were added to the centrifuge tube 4, and the four centrifuge tubes were shaken and incubated for 1 h at room temperature.

[0028] Separati...

Embodiment 3

[0034] Example 3 Processing and testing of patient serum samples

[0035] In this embodiment, the porous silicon particles modified with undecylenic acid with a porosity of 26% are used as the enrichment material to directly detect and analyze the patient's serum sample. The specific steps are as follows:

[0036] 1. Sample adsorption

[0037] The serum sample in this example was obtained from a hospital. The preparation of the serum sample was performed by those skilled in the medical field. Among them, 5-9 are the serum of rectal cancer patients, and 10-14 are the serum of non-cancer patients. The prepared serum samples are stored in- 80 ℃ ultra-low temperature refrigerator.

[0038] Adsorption: Dilute the serum sample 10 times with physiological buffer PBS and add 50 uL to a centrifuge tube, then add 3 mg of undecylenic acid modified porous silicon particles with a porosity of 26% to obtain a suspension Incubate with shaking for 1 h at room temperature.

[0039] Separation: The abo...

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Abstract

The invention discloses a preparation method of a low-abundance low-molecular-weight protein-rich material. The preparation method comprises the following steps of: fixing a P-type boron-doped silicon wafer in an electrolytic cell; adding ethanol and hydrofluoric acid as electrolyte; performing direct current electrolytic etching and demolding by taking the silicon wafer as an anode and a platinum electrode as a cathode; cleaning an etched and demolded porous silicon layer with ethanol, then ultrasonically crushing and blowing with nitrogen; performing ozone oxidation treatment on porous silicon particles; soaking the oxidized porous silicon particles in mixed solution of silylation reagent and organic solvent to react, then drying in a drying box, and soaking and cleaning the porous silicon particles, which are dried by evaporation, with ethanol; and selecting porous silicon with required aperture and performing surface modification by using amino silylation reagent, sulfydryl silylation reagent or epoxy silylation reagent. According to the preparation method, the effect of separating, enriching and detecting low-molecular-weight protein in a blood serum sample in one step is achieved by preparing the porous silicon particles.

Description

Technical field [0001] The invention belongs to the technical field of biochemical detection, and particularly relates to a preparation method of a low-abundance low-molecular-weight protein enrichment material for protein detection. Background technique [0002] In recent years, with the concept of population proteomics and its application in practical research, the distance between experimental research and clinical application will be further shortened. As the content and type changes of proteins in human body fluids caused by diseases are important indicators for the early diagnosis and evaluation of various diseases in clinic, the application of modern biotechnology to find specific protein changes related to diseases is a hot research topic. . However, there are still very few biomarkers that can be used in routine clinical testing. Theoretically, because the molecular weight of protein is relatively large, it is more difficult to enter the body fluid circulation, and som...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N1/28G01N33/68
Inventor 邬建敏谈洁赵伟洁
Owner ZHEJIANG UNIV
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