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Method for detecting endotoxin content of liquid

An endotoxin and liquid technology, applied in the direction of electrochemical variables of materials, etc., can solve the problems of detection specificity, optical measurement interference, ecological damage, etc., which is beneficial to industrial production, avoids the interference of G factor bypass, alleviates The effect of ecological pressure

Active Publication Date: 2014-03-26
SUZHOU INST OF BIOMEDICAL ENG & TECH CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0009] The detection of the limulus test is easily interfered by the chromogenic substances in the sample, and the coagulation process of the limulus reagent also interferes with the optical measurement. In addition, the G factor bypass in the limulus reagent reaction will also affect the specificity of the detection
The cost of preparing Limulus reagents from rare animals such as Limulus is high, and it will cause a certain degree of ecological damage. Based on the above factors, the wide application of Limulus test has certain limitations, and the detection of endotoxin requires the development of new sensing strategies.

Method used

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  • Method for detecting endotoxin content of liquid

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Experimental program
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Effect test

Embodiment 1

[0039] 1) RNA was extracted from Limulus blood, primers were artificially synthesized, and cDNA of Limulus factor C was obtained using a reverse transcription kit.

[0040] 2) By means of genetic engineering, the cDNA is loaded into the yeast expression vector, recombinantly expressed Limulus factor C, and the expression conditions are further optimized.

[0041] 3) The recombinantly expressed Limulus factor C was purified by affinity chromatography, and identified by Western blotting.

[0042] 4) Make a saturated aqueous solution of Limulus factor C, and then mix it with the liquid to be tested to obtain a mixed solution. The endotoxin in the liquid to be tested can activate Limulus factor C to produce active Limulus factor C.

[0043]5) Artificially synthesize a polypeptide sequence containing limulus tripeptide and modify it on the surface of the electrode. The C-terminus of the polypeptide sequence is designed as cysteine, and the number of amino acids contained in the pol...

Embodiment 2

[0048] 1) RNA was extracted from Limulus blood, primers were artificially synthesized, and cDNA of Limulus factor C was obtained using a reverse transcription kit.

[0049] 2) By means of genetic engineering, the cDNA is loaded into the insect expression vector, recombinantly expresses Limulus factor C, and the expression conditions are further optimized.

[0050] 3) The recombinantly expressed Limulus factor C was purified by ion exchange chromatography, and identified by Western blotting.

[0051] 4) Make a saturated aqueous solution of Limulus factor C, and then mix it with the liquid to be tested to obtain a mixed solution. The endotoxin in the liquid to be tested can activate Limulus factor C to produce active Limulus factor C.

[0052] 5) Artificially synthesize a polypeptide sequence containing limulus tripeptide and modify it on the surface of the electrode. The C-terminus of the polypeptide sequence is designed as cysteine, and the number of amino acids contained in t...

Embodiment 3

[0057] 1) RNA was extracted from Limulus blood, primers were artificially synthesized, and cDNA of Limulus factor C was obtained using a reverse transcription kit.

[0058] 2) By means of genetic engineering, the cDNA is loaded into the expression vector of Escherichia coli, recombinantly expressed Limulus factor C, and the expression conditions are further optimized.

[0059] 3) The recombinantly expressed Limulus factor C was purified by ultrafiltration and identified by Western blotting.

[0060] 4) Make a saturated aqueous solution of Limulus factor C, and then mix it with the liquid to be tested to obtain a mixed solution. The endotoxin in the liquid to be tested can activate Limulus factor C to produce active Limulus factor C.

[0061] 5) Artificially synthesize a polypeptide sequence containing limulus tripeptide and modify it on the surface of the electrode. The C-terminus of the polypeptide sequence is designed as cysteine, and the number of amino acids contained in t...

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Abstract

The invention discloses a method for detecting the endotoxin content of a liquid. According to the method, the concentration of endotoxin in the liquid to be detected is learned by detecting the electrochemical response of an electrode in electrolyte after the liquid to be detected and a restructuring limulus factor C are mixed and a contact reaction is performed on the mixture and a peptide modified electrode; when the restructuring limulus factor C is prepared, the restructuring limulus factor C is recombined and is expressed by adopting a means of genetic engineering. Through the scheme, a result is obtained directly by an electrochemical technology; the interference of color samples in the conventional methods, such as a colorimetric method, is avoided; the using of tachypleus amebocyte lysate is avoided, so that the detection cost can be reduced, and moreover, the ecological pressure is lightened to a certain degree; the method specifically reacts to the endotoxin, the interference of factor G bypass in the conventional tachypleus amebocyte lysate reaction is avoided; meanwhile, the genetic engineering method is used, so that the uniformity among reagent batches in the detection method is guaranteed, and the industrial production is facilitated.

Description

technical field [0001] The invention relates to the technical field of endotoxin detection, in particular to an electrochemical analysis method for detecting endotoxin content in liquid. Background technique [0002] Endotoxin is a complex composed of polysaccharides, lipids and proteins, and is one of the main components of the cell wall of Gram-negative bacteria. When bacteria rupture, endotoxins are released, and endotoxins are ubiquitous in the environment. Endotoxin can cause febrile reaction, leukocyte reaction, endotoxemia, endotoxic shock and disseminated intravascular coagulation. Therefore, endotoxin detection is of great significance in the fields of environmental monitoring, quality control of pharmaceutical factories, and pyrogen monitoring of medical devices. [0003] Endotoxin has the following properties: strong stability and heat resistance; weak antigenicity, less antibodies formed after immunization of animals. At present, there are two main methods for...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N27/26
Inventor 缪鹏王弼陡罗刚银钱庆王钟周程文播
Owner SUZHOU INST OF BIOMEDICAL ENG & TECH CHINESE ACADEMY OF SCI
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