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

A nanofiber membrane, immobilized enzyme technology, applied in immobilized enzymes, biochemical equipment and methods, enzymes and other directions, can solve the problems of large detection errors, inaccurate detection results, increase detection errors, etc., to improve detection sensitivity and precision, improve sensitivity and accuracy, and increase the effect of cycle times

CN103232987BInactive Publication Date: 2015-02-11SOUTHWEST JIAOTONG UNIV
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Patent Information

Authority / Receiving Office
CN · China
Patent Type
Patents(China)
Current Assignee / Owner
Publication Date
2015-02-11
Estimated Expiration
Not applicable · inactive patent

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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.
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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 whose blood alcohol concentration reaches 0.2mg / ml is considered to be drunk driving, and when the alcohol concentration reaches 0.8mg / ml, it is considered to be drunk driving. In recent years, tragedies of drunk driving causing tragic accidents are not uncommon. Although the traffic law enforcement department has stepped up law enforcement and punishment, there are always drivers who take risks; in addition, some drivers drive illegally because they cannot accurately judge their own alcohol cont...

Examples

Embodiment 1

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

[0035] A. Using an electrospinning device to prepare a polystyrene nanofiber membrane with amino groups on the surface, the amino group density is 2.0nmol / cm 2 (That is, the amount of amino groups contained in each square centimeter of the nanofibrous membrane is 2.0 nanomoles).

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

[0037] C. Immerse the nanofibrous membrane grafted with polymer in step B in the alcohol oxidase solution for 6 hours, take it out, and obtain the nanofibrous ...

Embodiment 2

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

[0048] A. Using an electrospinning device to prepare a polystyrene nanofiber membrane with amino groups on the surface, the amino group density is 3.0nmol / cm 2 ;

[0049] B. Immerse the nanofibrous membrane of step A in the carboxyl-terminated polyethylene glycol solution for 3 hours and take it out to dry to obtain a nanofiber membrane grafted with carboxyl-terminated polyethylene glycol polymer, and the grafting density is 0.2nmol / cm 2 ;

[0050] C. Immerse the nanofiber membrane grafted with polymer in step B in the alcohol oxidase solution for 7 hours, take it out, and obtain the nanofiber membrane of multi-site covalently grafted alcohol oxidase, and the alcohol oxidase on the nanofiber membrane The grafting amount is 100mg / g;

[0051] D. Immerse the nanofibrous membrane grafted with alcohol oxidase in a polylysine solution for 3 hours, immob...

Embodiment 3

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

[0055] A. Using an electrospinning device to prepare a polystyrene nanofiber membrane with hydroxyl groups on the surface, the density of hydroxyl groups is 3.5nmol / cm 2 ;

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

[0057] C, immerse the nanofibrous membrane grafted with polymer in step B in the alcohol oxidase solution for 8 hours, take it out, and obtain the nanofibrous membrane of multi-site covalently grafted alcohol oxidase; the alcohol oxidase on the nanofibrous membrane Grafting amount is 180mg / g;

[0058] D. Immerse the nanofibrous membrane grafted with alcohol oxidase in a polyhistidine solution for ...