Preparation method of immobilized enzyme nanofiber membrane for detecting alcohol content in spit

A technology of nanofiber membranes and immobilized enzymes, which is applied in the direction of immobilized enzymes, biochemical equipment and methods, fiber treatment, etc., can solve the problems of large detection errors, inaccurate detection results, and increased detection errors, and achieve improved detection Sensitivity and precision, the effect of improving sensitivity and accuracy, and increasing the number of cycles

Inactive Publication Date: 2013-08-07
SOUTHWEST JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This preparation method is simple to operate, but due to the dense filter paper, the enzyme is only physically adsorbed on the surface of the filter paper, resulting in poor thermal stability and acid-base stability, resulting in large detection errors and inaccurate detection results.
At the same time, because the surface of the filter paper is dense, the specific surface area is small, the amount of adsorbed enzyme is small and it is easy to fall off, which makes the detection sensitivity low and the detection error is large.
Moreover, alcohol oxidase is composed of eight subunits, and any subunit of the enzyme falls off during immobilization and use, which will cause the loss of enzyme activity to varying degrees and increase the detection error; while all eight subunits are immobilized On the base carrier, the spatial structure of the enzyme will be destroyed, and the steric hindrance of the enzyme when it acts on the substrate will be increased, thereby affecting the activity of the enzyme and reducing its detection sensitivity and accuracy.
Therefore, the current immobilized alcohol oxidase detection system fails to meet the requirements of accuracy, sensitivity, and repeated use.

Method used

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  • Preparation method of immobilized enzyme nanofiber membrane for detecting alcohol content in spit
  • Preparation method of immobilized enzyme nanofiber membrane for detecting alcohol content in spit

Examples

Experimental program
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Effect test

Embodiment 1

[0034] A preparation method of an immobilized enzyme nanofiber membrane for detecting alcohol content in saliva, the steps of which are:

[0035] A. A polystyrene nanofiber membrane with amino groups on the surface was prepared by an electrospinning device, and the amino density was 2.0 nmol / cm 2 (That is, the amount of amino groups contained in the nanofiber membrane per square centimeter is 2.0 nanomoles).

[0036] B. The nanofiber membrane of step A is immersed in the carboxyl-terminated polyethylene glycol solution for 1 hour and then taken out to dry to obtain a nanofiber membrane grafted with a carboxyl-terminated polyethylene glycol polymer, and the graft density is 0.4 nmol / cm 2 (that is, 0.4 nanomoles of carboxyl-terminated polyethylene glycol polymers are grafted on the nanofiber membrane per square centimeter);

[0037] C, the nanofiber membrane of the grafted polymer in step B is immersed in the alcohol oxidase solution for 6 hours and taken out to obtain the nano...

Embodiment 2

[0047] A preparation method of an immobilized enzyme nanofiber membrane for detecting alcohol content in saliva, the steps of which are:

[0048] A. A polystyrene nanofiber membrane with amino groups on the surface was prepared by an electrospinning device, and the amino density was 3.0 nmol / cm 2 ;

[0049] B. The nanofiber membrane of step A is immersed in the carboxyl-terminated polyethylene glycol solution for 3 hours and then taken out to dry to obtain a nanofiber membrane grafted with a carboxyl-terminated polyethylene glycol polymer, and the graft density is 0.2 nmol / cm 2 ;

[0050] C, the nanofiber membrane of the grafted polymer in step B is immersed in the alcohol oxidase solution for 7 hours, and taken out to obtain the nanofibrous membrane of multi-site covalent grafting of alcohol oxidase, the nanofiber membrane of alcohol oxidase on the nanofiber membrane The grafting amount is 100mg / g;

[0051] D, immerse the nanofiber membrane of grafted alcohol oxidase in po...

Embodiment 3

[0054] A preparation method of an immobilized enzyme nanofiber membrane for detecting alcohol content in saliva, the steps of which are:

[0055] A. A polystyrene nanofiber membrane with hydroxyl groups on the surface was prepared by an electrospinning device, and the hydroxyl density was 3.5 nmol / cm 2 ;

[0056] B. The nanofiber membrane of step A is immersed in the carboxyl-terminated polyethylene glycol solution for 3 hours and then taken out to dry to obtain a nanofiber membrane grafted with a carboxyl-terminated polyethylene glycol polymer, and the graft density is 0.7 nmol / cm 2 ;

[0057] C, the nanofiber membrane grafted with polymer in step B is immersed in the alcohol oxidase solution for 8 hours and taken out to obtain the nanofiber membrane of multi-site covalent grafting alcohol oxidase; The grafting amount is 180mg / g;

[0058] D, immerse the nanofiber membrane of grafted alcohol oxidase in polyhistidine solution for 1 hour, fix multiple subunits of alcohol oxid...

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Abstract

The invention discloses a preparation method of an immobilized enzyme nanofiber membrane for detecting the alcohol content in spit. The preparation method comprises the steps of: covalently binding alcohol oxidase through a polymer such as polyethylene glycol or polyethyleneimine as an arm molecular multi-site; further fixing a plurality of subunits of the alcohol oxidase by the polyethyleneimine, polylysine, poly-histidine or chitosan and the like through physical action among charges; then absorbing indigo blue as a chromogenic substrate; generating hydrogen peroxide after ethanol in a sample is oxidized by the alcohol oxidase in detection; leading the indigo blue to fade by the hydrogen peroxide, and conveniently calculating the ethanol concentration in the sample by comparing the fading degree of the indigo blue with a standard color atla. The preparation method is high in sensitivity, simple and convenient to operate, and low in price, can be repeatedly used for a plurality of times, and can be conveniently used for investigating and treating drunk driving by a law-enforcing department; and the reliable evidence is provided for the law enforcement process.

Description

technical field [0001] The invention belongs to the technical field of alcohol concentration detection, in particular to a preparation method of an immobilized enzyme nanofiber membrane for detecting alcohol content in saliva. Background technique [0002] Many industries require qualitative or quantitative detection and analysis of ethanol content. For example, various countries have strict requirements on the blood alcohol content of motor vehicle drivers. In my country, a driver's blood alcohol concentration of 0.2 mg / ml is considered to be driving under the influence of alcohol, and when the alcohol concentration reaches 0.8 mg / ml, it is regarded as drunk driving. In recent years, tragedies caused by drink-driving accidents are not uncommon. Although the traffic law enforcement authorities have stepped up enforcement and punishment, there are always drivers who take the risk; in addition, some drivers drive illegally because they cannot accurately determine their own al...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/00C12Q1/26D06M15/53D06M15/61
Inventor 李孝红刘清杰赵龙严仕力
Owner SOUTHWEST JIAOTONG UNIV
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