Magnetic resonance immune sensing method for detecting biomacromolecule

A biomacromolecule and immunosensing technology, applied in the field of biomacromolecule detection, can solve the problems of complex operation and low sensitivity of immunoassay methods

Inactive Publication Date: 2013-09-04
CHINESE ACAD OF INSPECTION & QUARANTINE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Using the method of the present invention, PS microspheres are used as immunoaffinity carriers to perform rapid extraction and enrichment of biological macromolecule Ag in sample pretreatment, and at the same time, the Ag captured by PS microspheres is coupled to the MRS in the detection system The specific immunoreaction of Ab, using low-field magnetic resonance analyzer, realizes the sensitive, simple and rapid detection of biological macromolecules, and overcomes the shortcomings of current molecular biology detection methods such as complicated operation and low sensitivity of immunological detection methods

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  • Magnetic resonance immune sensing method for detecting biomacromolecule

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Embodiment 1: the preparation of simulated sample

[0035] Take the detection of the content of Fusarium wilt in the sample as an example.

[0036] Take 20 corn seed samples in a 50mL centrifuge tube, soak and disinfect with 30mL 3-5% NaClO for 5min-15min, and rinse with 10mL sterile water for 3 times. Soak with 40mL PBS buffer solution (0.01mol / L, pH 7.4), overnight at 4°C. Transfer the soaking liquid into a centrifuge tube, centrifuge at 1000r / min for 10min, take the supernatant and transfer it to another centrifuge tube, centrifuge at 10000r / min for 15min, discard the supernatant, suspend and precipitate with 100mL of the above PBS buffer solution, and prepare corn seed matrix liquid. Use this matrix solution as diluent to prepare different content corn blight bacteria (10 7 , 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 10and0cfu / mL) series of positive samples. Negative samples were prepared according to the above steps without adding corn wilt bacteria.

Embodiment 2

[0037] Embodiment 2: Preparation of composite reagent PS-Ab conjugate

[0038] Take the detection of the content of Fusarium wilt in the sample as an example.

[0039] 1. Preparation of phosphate buffer solution: disodium hydrogen phosphate (0.2M) and sodium dihydrogen phosphate (0.2M) according to (Na 2 HPO 4 / NaH 2 PO 4 =4:1 ratio) and mixed to prepare 0.01M phosphate buffer solution, pH=7.4.

[0040] 2. Preparation of blocking solution: Bovine serum albumin (BSA) was dissolved in PBS with a molar concentration of 0.01M and a pH of 7.4 to make the concentration by weight 0.5%.

[0041] 3. Activation of PS microspheres: Take 5mg of PS microspheres with a particle diameter of 1000nm carboxyl groups in a 1.5mL centrifuge tube, place them in an ultrasonic cleaner for 30s, put them in a vortex oscillator for 10s, and centrifuge at a centrifugal force of 6000rpm After 8 minutes, pour out the waste liquid, and then wash with 200 μL phosphate-sodium hydroxide buffer solution ea...

Embodiment 3

[0043] Embodiment 3: Preparation of magnetic resonance magnetic relaxation time sensing probe (MRS)

[0044] Take the detection of the content of Fusarium wilt in the sample as an example.

[0045] 1. SMP microsphere activation: Take 300μL of SMP microspheres with a particle diameter of 50nm in a 1.5mL centrifuge tube, place them in an ultrasonic cleaner for 30s, put them in a vortex oscillator and vibrate for 10s, then add 10μL with a mass concentration of 50mg / ml of NHS and 10 μL of EDC with a mass concentration of 25 mg / ml were activated by shaking for 15 min, and then purified by a magnetic separation column.

[0046] 2. Magnetic separation column purification: put a magnetic separation column with an inner diameter of 1 mm on a magnetic separation rack with a gradient magnetic field, take 500 μL of the above-mentioned activated SMP microsphere suspension into the magnetic separation column, pass through the column to make it adsorb on the magnetic separation column Then...

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Abstract

The invention relates to a magnetic resonance immune sensing method for detecting a biomacromolecule. Polystyrene microspheres coupled with specific antibody (1) are used to capture biomacromolecule antigens (2) in a sample and can specifically bonded with relaxation time sensing probes (4) based on superparamagnetic nanoparticles, thereby allowing the relaxation time sensing probes (4) to change from a dispersed state to an aggregate cluster state, and resulting in the change of relaxation time of surrounding water molecule protons. The detection is carried out according to a function relationship between the change amount and the biomacromolecule (2) by using a low field nuclear magnetic resonance spectrometer. The conjugated polystyrene microspheres-antibody conjugate (1) is employed to overcome steric hindrance of the magnetic resonance immunoreactions and increase specific binding sites. Compared with a conventional magnetic resonance immune sensing method, the method provided by the invention can significantly increased detection sensitivity of the biomacromolecule (2), is simple in sample pretreatment step and fast in analytic speed, and has very wide application prospects and development value.

Description

technical field [0001] The present invention relates to a method for detecting biomacromolecules, in particular to a sensing detection method and related reagents which use polystyrene microspheres as immunoaffinity carriers to enrich biomacromolecules to be tested and directly participate in magnetic resonance immune reactions Methods of preparation and use. Background technique [0002] Infectious plant pathogenic bacteria and animal viruses are the key monitoring objects in the entry-exit inspection and quarantine work. Timely, accurate and convenient detection of these infectious harmful substances is essential for protecting human health and life safety and ensuring national security. , Maintaining social stability is of great significance. [0003] The current detection methods for detecting the pathogenic bacteria and viruses mainly include methods such as isolation and culture detection, immunological detection, and molecular biology. The isolation and culture meth...

Claims

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Application Information

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IPC IPC(8): G01N24/08G01N33/531G01N33/569
Inventor 邹明强陈翊平
Owner CHINESE ACAD OF INSPECTION & QUARANTINE
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