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Relaxation time immunosensing analysis method based on magnetic separation

An immunosensing, relaxation time technology, applied in analytical materials, measurement devices, instruments, etc., can solve problems such as poor stability and operability, and limited applications

Active Publication Date: 2015-05-13
THE NAT CENT FOR NANOSCI & TECH NCNST OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method is based on the change of the state of the immunomagnetic beads, and the change of the state of the immunomagnetic beads is affected by many factors, such as the size and concentration of the nano-magnetic beads, the number of binding sites on the surface of the analyte, the immune reaction time and other conditions. Both will affect the change of relaxation time, resulting in poor stability and operability of the method, which limits the further application of the method

Method used

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  • Relaxation time immunosensing analysis method based on magnetic separation
  • Relaxation time immunosensing analysis method based on magnetic separation
  • Relaxation time immunosensing analysis method based on magnetic separation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Embodiment 1 large magnetic bead-antibody conjugate (SMB 大 - Preparation of Ab1)

[0079] (1) SMB 大 Activation: Take 200 μL of superparamagnetic nanospheres with a particle diameter of 350 nm and put them into a centrifuge tube, magnetically separate them, then add 1000 μL of deionized water, place them in a vortex shaker for 2 minutes; then add 15 μL with a mass concentration of 50 mg / mL of NHS and 15 μL of EDC with a mass concentration of 25 mg / mL were shaken for 20 minutes to activate; excess EDC, NHS and by-products were separated by magnetic field, and then dissolved in 200 μL of PBS solution with a molar concentration of 0.01M and pH=7.4 to obtain Activation of superparamagnetic nano-microsphere liquid.

[0080] (2) Coupling of coated antibody (Ab1) and large-sized superparamagnetic nano-magnetic beads: Take 80 μL of the above-mentioned activated microsphere solution, add it to a centrifuge tube, and then add 0.2 mg of anti-biological macromolecule antibody (Ab...

Embodiment 2

[0081] Embodiment 2 small magnetic bead-antibody conjugate (SMB 小 - Preparation of Ab1)

[0082] (1) SMB 小 Activation: Take 400 μL of superparamagnetic nano-magnetic beads with a particle diameter of 30 nm and place them in a vortex shaker for 2 minutes; then add 10 μL of NHS with a mass concentration of 50 mg / mL and 10 μL of EDC with a mass concentration of 25 mg / mL, and shake for 20 minutes. Activation; adding 1000 μL of PBS solution with a molar concentration of 0.01M and pH=7.4 to obtain an activated superparamagnetic nano-magnetic bead solution.

[0083] (2) Coupling of the capture antibody (Ab2) that recognizes Salmonella and the supercisive nano-magnetic beads of small particle size: add 0.1 mg anti-biological macromolecule antibody (Ab2) to the above-mentioned activated supercisive nano-magnetic beads to avoid Shake with light for 1 hour, separate and remove the unreacted antibody by gradient magnetic field separation column, pour off the waste liquid; redissolve the...

Embodiment 3

[0084] Example 3 Comparison of different separation speeds of magnetic beads with different particle sizes in the same magnetic field

[0085] Superparamagnetic nano-magnetic beads with particle sizes of 250nm and 30nm were selected as comparison objects, and placed on a magnetic separation rack with a magnetic field of 0.01T. Observe the aggregation of magnetic beads.

[0086] Such as figure 2 As shown, under the same magnetic field conditions (0.01T), SMB with a particle size of 250nm 大 -Ab1 quickly aggregated to the bottom due to the action of the magnetic field within 30s, and the SMB with a particle size of 30nm 小 -Ab2 is not separated by the magnetic field after 12 hours due to its low saturation magnetization, and is still in a suspended state in the glass bottle.

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Abstract

The invention relates to a relaxation time immunosensing analysis method based on magnetic separation. The method comprises the following steps: selecting two magnetic beads (one can be quickly separated and the other cannot be separated) different in saturated magnetization intensity and remarkably different in separation speed in the same magnetic field, so that the magnetic beads can be separated in the magnetic field; coupling the magnetic beads to an antibody used for identifying different sites of the same target object respectively to prepare immunomagnetic beads; producing an immunoreaction of the immunomagnetic beads and a to-be-detected sample; performing magnetic separation on a mixed system, measuring transverse relaxation time for supernatant liquor subjected to magnetic separation, and determining the concentration of biomacromolecules in the to-be-detected sample according to change of the transverse relaxation time. The immunomagnetic beads different in saturated magnetization intensity are different in separation speed in the same magnetic field, the magnetic separation is combined with magnetic relaxation time analysis, the reaction time only needs 30 minutes, and the method can be used for quickly detecting bacteria, viruses and proteins and has a very good application prospect in an aspect of biomarker detection.

Description

technical field [0001] The invention relates to a magnetic relaxation time immunosensing analysis method, in particular to a relaxation time immunosensing analysis method based on magnetic separation. Background technique [0002] Infectious plant pathogenic bacteria and animal viruses are the key monitoring objects in entry-exit inspection and quarantine work and public health safety. Timely, accurate and convenient detection of these infectious harmful substances is crucial to the protection of human health and life safety. , It is of great significance to ensure the security of the country and maintain social stability. 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 method is simple and easy to implement, but due to its low detection sensitivity and long time consumption, it needs to occupy a large amoun...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/531
CPCG01N33/54333G01N33/569G01N33/6803
Inventor 蔣兴宇陈翊平曹丰晶张晓青吴景王卓牛亚静
Owner THE NAT CENT FOR NANOSCI & TECH NCNST OF CHINA
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