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A technology for detecting reagents and ethanol, which is applied in the field of clinical detection, can solve the problems of poor specificity of alcohol oxidase, difficulty in maintaining long-term activity, uneven product activity, etc. Effect
Inactive Publication Date: 2009-04-29
SHANGHAI JINLONG BIOLOGICAL TECH
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Problems solved by technology
[0009] These patents all focus on how to accurately measure ethanol and content in samples by dry chemical test paper method, and how to immobilize alcohol oxidase, but ignore several key technical issues: 1) the specificity of alcohol oxidase Poor, it also has a strong oxidation effect on most alcohols such as methanol, isopropanol and other non-ethanol substances, and these substances have a great interference effect in the detection process
2) Alcohol oxidase is extremely unstable, and its activity is difficult to maintain for a long time, resulting in a large difference in the stability of the product at present. 3) The color reaction of hydrogen peroxide and TMB in the reaction system is easily affected by some reducing substances. Such as the interference of ascorbic acid (vitamin C), 4) the chromogenic substrate of the present ethanol detection reagent strip is TMB (N, N, N', N'-tetramethylbenzidine), which is the enzyme of horseradish peroxidase The most commonly used substrate, but it is difficult to dissolve in water. In the current process of preparing ethanol detection reagents, the substrate TMB is dissolved in xylene, and then the organic solvent is removed through volatilization. If the volatilization is not complete, it is undoubtedly harmful to health. of
Secondary drying has a great impact on the activity of temperature-sensitive alcohol oxidase. At the same time, the organic solvent xylene also has different degrees of inhibition on the activity of alcohol oxidase and horseradish peroxidase, resulting in uneven activity of the product
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Embodiment 1
[0025] Example 1. Preparation of saliva ethanol content detection reagent
[0026] 1) Prepare alcohol dehydrogenase and ascorbate oxidase solutions
[0027] Take 10 mg of alginate, 200 mg of gelatin, 500 units of alcohol dehydrogenase, 3.0 grams of coenzyme NAD, and 1000 units of ascorbate oxidase, and dissolve them in 20 ml of 0.1 M phosphate with pH 7.5.
[0028] 2) Preparation of blue tetrazolium solution
[0029] 2.0 g of tetrazolium salt was dissolved in 20 ml of double distilled water.
[0030] 3) Preparation of reaction pad by immobilization of enzyme and substrate:
[0031] Immerse a 0.5CM wide 3M filter paper in the above enzyme / tetrazolium salt (1:1) mixture, take out the filter paper and dry it for 10 minutes under 50OC hot air and away from light.
[0032] 4) Preparation of reagent strips
[0033] Paste the dried reaction pad on the white polyester board with glue on one side at a distance of 1.0 cm from the front end, then paste absorbent paper about 2.5 cm lo...
Embodiment 2
[0034] Embodiment 2. of saliva ethanol content detection reagent
[0035] Prepare ethanol solutions with the saliva of normal people, the concentrations are: 0, 20, 80 and 300 mg / dL respectively, and prepare methanol and isopropanol solutions with the same concentrations at the same time. In addition, ethanol solutions of different concentrations containing 10 mg / ml vitamin C were prepared.
[0036] Insert the water-absorbing end of the reagent strip prepared in Example 1 into the sample for 10 seconds, take it out, and leave it for 5 minutes to observe the color change of the reaction pad.
[0037] result:
[0038] Ethanol (mg / dL) 0 20 80 300 Test results yellowish light blue blue Navy blue Methanol (mg / dL) 0 20 80 300 Test results yellowish yellowish yellowish yellowish Isopropanol (mg / dL) 0 20 80 300 Test results yellowish yellowish yellowish yellowish Ethanol (mg / dL) + Vitamin C (10mg / ml) 0 20 8...
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Abstract
The invention relates to the technical field of clinical detection, in particular to a detection reagent for the content of alcohol in saliva. The invention discloses the detection reagent for the content of the alcohol in the saliva, which is characterized in that 1.25 to 0.5U of alcohol dehydrogenase, 0.05 to 0.2mg of coenzyme NAD and 0.025 to 0.2mg of tetrazolium salt are loaded and contained in a solid phase or semi-solid phase carrier with the area of 5.0*5.0mm<2>. The detection reagent for the content of the alcohol in the saliva has high specificity, high sensitivity, and quick reaction, does not contain poisonous substances, can detect orally, is safe and reliable, shows a result without a composite sensor host, can directly contrast readings with a colorimetric card, has better thermal stability, and can be preserved for 2 years at room temperature. The reagent has the advantages of good repetitiveness of determination results, convenient carrying, simple preparation technology, low cost, easy industry promotion and convenient field detection, and can be used for the detection of drunk drivers and taken as a simple tool for postmortem examination of judicial doctors.
Description
technical field [0001] The invention relates to the technical field of clinical detection, in particular to a detection reagent for ethanol content in saliva. Background technique [0002] In recent years, criminal cases caused by drinking and traffic accident cases caused by drunk driving have been on the rise. At present, many countries have formulated the relationship between blood ethanol concentration and traffic accident responsibility determination, and established a variety of methods that can detect blood ethanol content. For example, GM / MS and other instruments detect ethanol components. Although these methods can accurately quantify, they have the disadvantages of expensive equipment, high detection costs, special training, and inability to quickly detect at the scene of the crime. Breath ethanol analyzers can be used on-site, but they are also very expensive, unhygienic, and prone to cross-infection diseases. [0003] A large number of studies have shown that t...
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