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Method for detecting microorganism isoelectric point by adopting immobilized pH gradient capillary isoelectric focusing (CIEF)

An isoelectric focusing and capillary technology, applied in the field of biological analysis, can solve the problems of reducing the stability and sensitivity of microbial detection, unstable UV detection signal, uneven distribution of ampholytes, etc. The effect of faster migration

Inactive Publication Date: 2012-01-11
BEIJING INSTITUTE OF TECHNOLOGYGY
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  • Abstract
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Problems solved by technology

Because the ampholyte used has a strong ultraviolet background absorption, and the ampholyte is unevenly distributed in the capillary, if the electroosmotic flow is used to drive the migration of microorganisms in the tube, etc., the ultraviolet detection signal of the microorganism will be unstable and the background noise signal will be greatly reduced. Robustness and sensitivity of microbial detection
At present, there is no general method and report for determining the isoelectric point of microorganisms in biology.

Method used

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  • Method for detecting microorganism isoelectric point by adopting immobilized pH gradient capillary isoelectric focusing (CIEF)
  • Method for detecting microorganism isoelectric point by adopting immobilized pH gradient capillary isoelectric focusing (CIEF)
  • Method for detecting microorganism isoelectric point by adopting immobilized pH gradient capillary isoelectric focusing (CIEF)

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Embodiment Construction

[0025] Fused silica capillary (100μm i.d. capillary column length 38.5, effective length 30cm, detection window 8.5cm from the outlet end) was washed with 0.1M HCl for 10min, water for 5min, 1M NaOH for 20min, 0.1M NaOH for 20min, water for 20min and methanol for 20 minutes, blown dry with nitrogen and set aside. Add 0.3ml of isocyanate to 5ml of anhydrous pyridine, mix well, inject into the capillary with a manual pump, and react at 70°C for 12 hours to bond the isocyanate to the inner wall of the capillary. After the reaction, wash the capillary with toluene, methanol, triple distilled water, and phosphate buffer saline (PBS, 20 mM pH 7.0) for 10 min each. A carrier ampholyte solution (Ampholine pH 3.5-10.0, dissolved in 20 mM phosphate buffer solution at a volume ratio of 4%) was injected into the inner wall of the capillary with a manual pump. Cathode buffer is 20mM NaOH solution, anode buffer is 20mM H 3 PO 4 solution, focusing at 15kV. After focusing for 10 minutes, ...

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Abstract

The invention relates to a method for detecting microorganism isoelectric point by adopting immobilized pH gradient capillary isoelectric focusing (CIEF), belonging to the field of bioanalysis. The method comprises the steps of: firstly carrying out isoelectric focusing on carrier ampholyte solution by adopting constant voltage in a capillary to lead ampholyte with continuous pH gradient to be formed on the inner wall of the capillary; secondly injecting sample solution to the capillary and carrying out isoelectric focusing by adopting the continuous voltage; then adopting immobilized atmospheric pressure to push the sample from focusing section inside the capillary to pass a detecting window at equal speed so as to determine focus point position of the sample; and finally figuring out pHvalue of the focus point according to linear relation between the pH gradient and the length of the capillary. The method can be applied to the simultaneous online detection of various microorganismsand has universality for microorganism isoelectric point detection.

Description

technical field [0001] The invention relates to a method for measuring the isoelectric point of microorganisms by isoelectric focusing of an immobilized pH gradient capillary, belonging to the field of biological analysis. Background technique [0002] Microorganisms have a surface charge and are amphoteric, possessing an isoelectric point (pI). Microbial surface charge and isoelectric point characteristics are determined by the overall charged properties of molecules such as amino acid residues, glycoproteins, and lipids on the surface of microbial cell membranes or cell walls. Therefore, different microorganisms and microorganisms in different growth and metabolic states can be characterized by their surface charge characteristics and isoelectric point characteristics. Studying the charging characteristics of the surface charge of microorganisms can characterize the physiological and biochemical characteristics of the microorganisms themselves, and has important theoretic...

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N27/447
Inventor 屈锋蔡波太马文韬
Owner BEIJING INSTITUTE OF TECHNOLOGYGY
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