CN101271120A - Production method of reagent for detecting alcoholicity in saliva - Google Patents
Production method of reagent for detecting alcoholicity in saliva Download PDFInfo
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- CN101271120A CN101271120A CNA200810036780XA CN200810036780A CN101271120A CN 101271120 A CN101271120 A CN 101271120A CN A200810036780X A CNA200810036780X A CN A200810036780XA CN 200810036780 A CN200810036780 A CN 200810036780A CN 101271120 A CN101271120 A CN 101271120A
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Abstract
The invention relates to a preparation method of a reagent for detecting the alcohol content in saliva, comprising: 1) the excessive immersion liquid is absorbed after a filter paper is soaked in the first-phase immersion liquid, the rapid warm-hot drying is carried out at the temperature of 20 to 30 DEG C, then the soaking in the second-phase immersion liquid is carried out, the re-drying is carried out at the temperature of 20 to 30 DEG C and a piece of original paper of a piece of measurement test paper is obtained; 2) the original paper of the measurement test paper is adhered on a plastic sheet, and the small measurement test strips are formed by cutting. The method can accelerate the reaction process; the reaction products are removed, so as to be conductive to the more complete reaction; when the sample to be detected contains higher concentration of ethanol, the interferences of the reactants and the reaction products can be reduced; the method is conductive to remove the inhibitory reaction in the measurement of the people with high alcohol content; the method can also be provided with two test blocks simultaneously on the same test strip, thus having more accurate judgment of the result.
Description
Technical field
The invention belongs to and detect the alcohol field in the saliva, particularly relate to a kind of preparation method who detects the reagent of alcohol content in the saliva.
Background technology
The society of alcohol detection uses very extensive, and in recent years, the criminal case that causes after drinking and the traffic hazard case that causes of driving when intoxicated constantly rise.Many countries have all formulated the relation of blood alcohol concentration and traffic accident responsibility identification, have set up the multiple method that can detect alcohol content in the blood.Blood sampling detects alcohol content, safe and sanitary hidden danger is always arranged, thereby more favor in getting saliva and carry out alcohol detection but in practice.
At present, a lot of portable detectable bars having occurred, is CN1837821A and CN1904619A as publication number, below they have all utilized, and principle:
ALO: alcohol oxidase enzyme
HRP: horseradish peroxidase
TMB:3,3 ' 5,5 '-tetramethyl benzidine
But for high capacity for liquor crowd, after excessive drinking, the phenomenon that testing result can occur reducing on the contrary, therefore when carrying out the detection of traffic law, foundation for a punishment is difficult to carry out on the contrary.
Summary of the invention
The purpose of this invention is to provide a kind of preparation method who detects the reagent of alcohol content in the saliva, this method can be improved the problems referred to above, removes the inhibitory reaction of measuring among the high capacity for liquor crowd, makes reaction result more accurate.
A kind of preparation method who detects the reagent of alcohol content in the saliva of the present invention comprises the following steps:
(1) after filter paper soaks in the first phase immersion liquid, inhale and go too much immersion liquid, 20-30 ℃ of rapid warm drying soaked in the second phase immersion liquid again, and 20-30 ℃ dry once more, obtains measuring the body paper of test paper;
(2) stick on the plastic sheet with the body paper of double faced adhesive tape, cut into the mensuration test strips of fritter the said determination test paper.
The process for preparation that the described first phase immersion liquid cumulative volume is 10mL is as follows:
1) joins the 0.1mol/L～2mol/L Tris-malonic acid damping fluid of pH7.2 or the 0.5mol/L PB (phosphate buffer) of pH7.8;
2) dissolve in alcohol oxidase, contain 10～200iu in the 10mL damping fluid;
3) dissolve in horseradish peroxidase, contain 200～5000iu in the 10mL damping fluid;
4) dissolve in acetaldehyde dehydrogenase, contain 2～100iu in the 10mL damping fluid, NAD
+0.01-0.1g;
5) dissolve in stabilizing agent and protective agent, concentration 0.1%～3% (g/100mL).
Described stabilizing agent and protective agent are natural or artificial polymerization macromolecule colloids, are one or more potpourris in gelatin, Arabic gum, bovine serum albumin(BSA), polyglycol, the polyacrylic acid-acrylate.
The described second phase immersion liquid is to adopt 3,3 ' 5, and 5 '-tetramethyl benzidine developer is dissolved in organic solvent, and concentration is 0.2%～1.5% (g/100mL).
Adopt toluene, dimethylbenzene, chloroform, methylene chloride, ethylene dichloride or dimethyl formamide as solvent.
The mensuration test strips that cuts into fritter in the described step (2) is the mensuration test strips that cuts into the fritter that contains 0.4 * 0.4～0.6 * 0.6cm.
Principle of the present invention:
ALO: alcohol oxidase enzyme
HRP: horseradish peroxidase
TMB:3,3 ' 5,5 '-tetramethyl benzidine
Aldehyde-DH: acetaldehyde dehydrogenase
NAD
+: coenzyme I
NADH: dihydrocoenzyme I
Beneficial effect of the present invention:
1) can the accelerated reaction process;
2) cleaning reaction product, it is more complete to help reaction;
3) when containing higher concentration ethanol in the sample, can reduce the interference of reactant and reaction product;
4) help removing the inhibitory reaction of measuring among the high capacity for liquor crowd, make reaction result more accurate.
5) acetaldehyde of drinking and afterwards producing for high capacity for liquor crowd head strong is measured by the enzymatic chromogenic reaction.
Description of drawings
Fig. 1 is preparation flow figure of the present invention.Fig. 2 is the alcoholometry reagent strip.Fig. 3 be alcohol and alcohol in vivo metabolic product acetaldehyde binomial measure reagent strip.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Embodiment 1
1. prepare the first phase immersion liquid
In the 0.2mol/L of pH7.2 Tris-malonic acid damping fluid 10mL, the alcohol oxidase of dissolving 100iu adds horseradish peroxidase 2500iu again, stirs, and fully after the dissolving, adds acetaldehyde dehydrogenase 100iu, NAD again
+20mg, 20mg gelatin (molten in advance).
2. soak evenly with No. 3 whatman quantitative test test paper, take out, inhale and remove unnecessary liquid, in 28 ℃ of following incubators, dry up.
3. prepare the second phase immersion liquid
Take by weighing TMB 100mg, with the dissolving of 10mL dimethyl formamide.
The filter paper of the dry leaching first phase immersion liquid is soaked in the second phase immersion liquid once more, take out rapidly, in 20 ℃ of following incubators, dry up.
4. the dry filter paper after will leaching sticks on the plastic cards with double faced adhesive tape, is cut into strip, and every length is 8cm, and the top has the mensuration indicator paper block of 0.5 * 0.5cm.(Fig. 2)
Embodiment 2
1. in the 0.5mol/L of pH7.8 PB damping fluid 10mL, the alcohol oxidase of dissolving 200iu adds horseradish peroxidase 1000iu again, stirs, and fully after the dissolving, adds aldehyde dehydrogenase 2 0iu, NAD again
+10mg, final concentration are the BSA (bovine serum albumin(BSA)) of 3% (w/v).
2. soak evenly with No. 3 whatman quantitative test test paper, take out, inhale and remove unnecessary liquid, in 28 ℃ of following incubators, dry up.
3. prepare the second phase immersion liquid
Take by weighing TMB 100mg, with the dissolving of 10mL dimethyl formamide.
The filter paper of the dry leaching first phase immersion liquid is soaked in the second phase immersion liquid once more, take out rapidly, in 28 ℃ of following incubators, dry up.Dry filter paper after the leaching is sticked on the plastic cards with double faced adhesive tape, be cut into strip, every length is 8cm, and the top has the mensuration indicator paper block of 0.5 * 0.5cm.(Fig. 2)
Embodiment 3
1. preparation acetaldehyde is measured the immersion liquid of reagent piece
In the 0.2mol/L of pH7.2 Tris-malonic acid damping fluid 10mL, dissolving 120iu acetaldehyde dehydrogenase, NAD
+50mg, diaphorase 60iu and chlorination nitro tetrazole blue 200mg, bovine serum albumin(BSA) 100mg.
2. soak evenly with No. 3 Whatman quantitative test test paper, take out, inhale and remove unnecessary liquid, in 28 ℃ of following incubators, dry up.
3. the dry body paper after will leaching sticks on the plastic cards with double faced adhesive tape, as second determination block, is cut into strip.(Fig. 3)
Claims (8)
1. one kind is detected in the saliva alcohol content and the preparation method of the reagent of metabolic product in vivo thereof, comprises the following steps:
(1) after filter paper soaks in the first phase immersion liquid, inhale and go too much immersion liquid, 20-30 ℃ of rapid warm drying soaked in the second phase immersion liquid again, and 20-30 ℃ dry once more, obtains measuring the body paper of test paper;
(2) body paper with the said determination test paper sticks on the plastic sheet, cuts into the mensuration test strips of fritter.
2. the preparation method of the reagent of alcohol content in the detection saliva according to claim 1 is characterized in that the process for preparation that the described first phase immersion liquid cumulative volume is 10mL is as follows:
1) joins the 0.1mol/L～2mol/L Tris-malonic acid damping fluid of pH6.5-8.5 or the 0.5mol/L phosphate buffer of pH7.2-8.0;
2) dissolve in alcohol oxidase, contain 10～500iu in the 10mL damping fluid;
3) dissolve in horseradish peroxidase, contain 500～10000iu in the 10mL damping fluid;
4) dissolve in acetaldehyde dehydrogenase, contain 2～100iu in the 10mL damping fluid, NAD
+0.01-0.1g;
5) dissolve in stabilizing agent and protective agent, concentration 0.1%～3% (g/100ml).
3. the preparation method of the reagent of alcohol content in the detection saliva according to claim 2; it is characterized in that; described stabilizing agent and protective agent are natural or artificial polymerization macromolecule colloids, are one or more potpourris in gelatin, Arabic gum, bovine serum albumin(BSA), polyglycol, the polyacrylic acid-acrylate.
4. the preparation method of the reagent of alcohol content in the detection saliva according to claim 1, it is characterized in that the described second phase immersion liquid is 3,3 ' 5,5 '-tetramethyl benzidine (TMB) developer solution is in organic solvent, and concentration is 0.2%～1.5% (g/100ml).
5. the preparation method of the reagent of alcohol content is characterized in that TMB is dissolved in toluene, dimethylbenzene, chloroform, methylene chloride, ethylene dichloride, the dimethyl formamide in the detection saliva according to claim 4.
6. the preparation method of the reagent of alcohol content is characterized in that the mensuration test strips that cuts into fritter in the described step (2) is the mensuration test strips that cuts into the fritter that contains 0.4 * 0.4～0.6 * 0.6cm in the detection saliva according to claim 1.
7. the alcohol content and the preparation method of reagent thereof of metabolic product in vivo thereof in the detection saliva according to claim 1 is characterized in that the process for preparation that the described mensuration alcohol metabolism product acetaldehyde first phase immersion liquid cumulative volume is 10mL is as follows:
1) joins the 0.1mol/L～2mol/L Tris-malonic acid damping fluid of pH6.5-8.5 or the 0.5mol/L phosphate buffer of pH7.2-8.0;
2) dissolve in acetaldehyde dehydrogenase, contain 20～200iu in the 10mL damping fluid, NAD
+0.01-0.1g;
3) dissolve in NAD diaphorase 10-100iu;
4) dissolve in the blue 0.01-0.5g of chlorination nitro tetrazole;
5) dissolve in stabilizing agent and protective agent, concentration 0.1%～3% (g/100ml).
8. the alcohol metabolism product acetaldehyde that contains in the detection saliva according to claim 7 is measured the reagent piece, is cut into the fritter that contains 0.4 * 0.4～0.6 * 0.6cm, and is listed on the mensuration reagent strip.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200810036780 CN101271120B (en) | 2008-04-29 | 2008-04-29 | Production method of reagent for detecting alcoholicity in saliva |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200810036780 CN101271120B (en) | 2008-04-29 | 2008-04-29 | Production method of reagent for detecting alcoholicity in saliva |
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| Publication Number | Publication Date |
|---|---|
| CN101271120A true CN101271120A (en) | 2008-09-24 |
| CN101271120B CN101271120B (en) | 2013-04-17 |
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ID=40005215
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|---|---|---|---|
| CN 200810036780 Expired - Fee Related CN101271120B (en) | 2008-04-29 | 2008-04-29 | Production method of reagent for detecting alcoholicity in saliva |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102564979A (en) * | 2011-11-21 | 2012-07-11 | 宁波美康生物科技有限公司 | Method for determining alcohol concentration by using enzyme cycling method and alcohol determination kit |
| CN102901727A (en) * | 2012-10-22 | 2013-01-30 | 公安海警学院 | Ethanol concentration detection kit by using circulatory enzyme method and preparation method thereof |
| CN104977296A (en) * | 2015-06-30 | 2015-10-14 | 江苏大学 | Novel alcohol degree detection method and apparatus |
| CN107607730A (en) * | 2017-10-26 | 2018-01-19 | 南通伊仕生物技术股份有限公司 | For detecting the reagent strip of alcohol content, preparation method and kit in saliva |
| CN108593633A (en) * | 2018-04-19 | 2018-09-28 | 中山大学 | A kind of Test paper for quickly detecting saliva uric acid |
| CN111735811A (en) * | 2020-08-12 | 2020-10-02 | 民康医疗科技（天津）有限公司 | Triglyceride detection reagent, triglyceride detection test paper and preparation method of triglyceride detection test paper |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1749751A (en) * | 2004-09-15 | 2006-03-22 | 王志洪 | Detecting test paper strip for measuring alcohol content in human saliva |
| CN1837821A (en) * | 2005-03-24 | 2006-09-27 | 丁国兴 | Preparation method of test paper for detecting alcohol content in saliva and test paper prepared thereby |
| CN2812002Y (en) * | 2005-03-24 | 2006-08-30 | 丁国兴 | Test paper and test paper package for detecting alcohol content in saliva |
-
2008
- 2008-04-29 CN CN 200810036780 patent/CN101271120B/en not_active Expired - Fee Related
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102564979A (en) * | 2011-11-21 | 2012-07-11 | 宁波美康生物科技有限公司 | Method for determining alcohol concentration by using enzyme cycling method and alcohol determination kit |
| CN102901727A (en) * | 2012-10-22 | 2013-01-30 | 公安海警学院 | Ethanol concentration detection kit by using circulatory enzyme method and preparation method thereof |
| CN104977296A (en) * | 2015-06-30 | 2015-10-14 | 江苏大学 | Novel alcohol degree detection method and apparatus |
| CN107607730A (en) * | 2017-10-26 | 2018-01-19 | 南通伊仕生物技术股份有限公司 | For detecting the reagent strip of alcohol content, preparation method and kit in saliva |
| CN108593633A (en) * | 2018-04-19 | 2018-09-28 | 中山大学 | A kind of Test paper for quickly detecting saliva uric acid |
| CN111735811A (en) * | 2020-08-12 | 2020-10-02 | 民康医疗科技（天津）有限公司 | Triglyceride detection reagent, triglyceride detection test paper and preparation method of triglyceride detection test paper |
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|---|---|
| CN101271120B (en) | 2013-04-17 |
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Granted publication date: 20130417 |