Detection method for protein with nucleic acid aptamer target gathering and laser enzymolysis

A nucleic acid aptamer and protein technology, applied in the field of chemical biology, can solve the problems of difficult detection and low concentration, and achieve the effect of simple method, accurate qualitative and quantitative, and large specific surface area

Inactive Publication Date: 2013-06-05
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, at present, many protein molecular markers with clinical application are often at very low concentration levels in body fluids, making detection very difficult

Method used

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  • Detection method for protein with nucleic acid aptamer target gathering and laser enzymolysis
  • Detection method for protein with nucleic acid aptamer target gathering and laser enzymolysis
  • Detection method for protein with nucleic acid aptamer target gathering and laser enzymolysis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1:

[0041] The preparation of the target plate with surface modified gold film, the preparation process is as follows figure 1 Shown:

[0042] (1) Using the classic Frens method to synthesize gold colloid under the protection of sodium citrate, the specific steps are:

[0043] (a) Add 1mmol / L chloroauric acid (HAuCl 4 ) solution and boil;

[0044] (b) Add 38.8mmol / L trisodium citrate solution, and reflux for 15min;

[0045] (c) cooling. The obtained red solution was filtered through a 0.22 μm filter membrane to obtain gold nanocolloids with a size of 13 nm.

[0046] (2) Take 10 mL of gold gel, centrifuge at 20,000 rcf for 20 min, and discard the supernatant. Adjust the gold colloid concentration to 92 nmol / L according to the absorbance at 260 nm;

[0047] (3) Spot 2 μL 92 nmol / L gold glue on a clean target plate and let it dry naturally overnight;

[0048] (4) Adjust the oven to 200 °C, put in the target plate, and calcinate for 2 h;

[0049] (5) Cool...

Embodiment 2

[0051] Example 2: Using lysozyme as a model protein, nucleic acid aptamers were used for on-target enrichment, laser enzymatic hydrolysis and MALDI-TOF MS qualitative and quantitative analysis:

[0052] (1) Immobilization of the thiol-modified lysozyme aptamer: The thiol-modified lysozyme aptamer was treated with 2.5 mM tris(2-carboxyethyl)phosphine (TCEP) for 1 h, and 2 μL was spotted on the target plate. Put the target plate in a wet box overnight in the dark;

[0053] (2) Configuration of standard protein solution: Dilute lysozyme protein to 5000 ng / mL, 1000 ng / mL, 100 ng / mL, 14 ng / mL;

[0054] (3) Enrichment: pipette 2 μL of lysozyme protein solution onto the MALDI target plate, and react in a wet box at room temperature for 1 hour. Rinse with 0.1% Tween 20 and deionized water for 1 min. dry;

[0055] (4) Laser enzymolysis: Prepare 25 ng / μL trypsin solution with 25 mmol / L ammonium bicarbonate buffer. Take 2 μL and spot on the target plate enriched with lysozyme. Irradi...

Embodiment 3

[0058] Example 3: Adding lysozyme to human urine, using nucleic acid aptamers for on-target enrichment, laser enzymolysis and MALDI-TOF MS qualitative and quantitative analysis:

[0059] (1) Preparation of urine samples: Collect morning urine from healthy people, centrifuge to remove precipitate, filter with 0.22 μm filter membrane, and store at -20°C. Add lysozyme directly to the urine sample to concentrations of 5000 ng / mL, 500 ng / mL, and 100 ng / mL;

[0060] (2) According to steps (3), (4) and (5) of Example 2, enrichment, laser enzymatic hydrolysis and mass spectrometry were performed on the sample. Qualitative and quantitative analysis of lysozyme;

[0061] (3) Detection of endogenous lysozyme in urine: No protein was added to the urine sample, and the blank urine sample was directly enriched as described in Steps (3), (4) and (5) of Example 2, and laser Enzymatic digestion and mass spectrometry analysis. MS / MS analysis was performed on the peptide m / z=1400.6, and compa...

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Abstract

The invention belongs to the field of chemicobiology and relates to a detection method for protein with nucleic acid aptamer target gathering and laser enzymolysis. Firstly, nanogold with the diameter of 13nm and a target board are in epoxy dripping mode, the anogold is dryed and calcined, and even and thick gold films are formed on the target board. With the method, the gold films which are with a loose hole-type structure are formed on the surface finish of the target board, large specific surface area is provided, excellent stability and biocompatibility are achieved, and the method for protein with nucleic acid aptamer target gathering and laser enzymolysis can be applied to various functionalized modifications. Secondly, nucleic acid aptamers of mercapto modification is fixed on the target board. Thirdly, target protein is gathered and target quick laser enzymolysis is supplemented, and samples are sent to matrix-assisted laser desorption ionization (MALDI)-time-off-flight mass spectrometer (TOFMS) to accept qualitative and quantitative analysis. The whole experiment process is simple, quick, accurate and can be widely applied into high-throughput qualitative and quantitative analysis of important protein.

Description

technical field [0001] The invention belongs to the field of chemical biology, and specifically relates to a simple, rapid and accurate qualitative and quantitative analysis of target protein molecules by using aptamer targets to enrich target proteins, supplemented by laser enzymolysis, and MALDI-TOF MS analysis. Methods of Quantitative Analysis. Background technique [0002] Protein molecular markers are an important class of biological macromolecules that can provide information on the occurrence and development of diseases. The detection of protein molecular markers can promote the early diagnosis of diseases, monitor the progress of diseases, promote subsequent treatment, etc., so it is very important. However, many protein molecular markers with clinical applications are often at very low concentrations in body fluids, making detection very difficult. The traditional method is to use antibody-based immunization methods, such as ELISA, because antibodies have high aff...

Claims

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Application Information

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IPC IPC(8): G01N27/64
Inventor 邓春晖张学洋熊娅张祥民
Owner FUDAN UNIV
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