CN1904619A - Reagent stripe for detecting alcohol content in saliva and box kit - Google Patents
Reagent stripe for detecting alcohol content in saliva and box kit Download PDFInfo
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- CN1904619A CN1904619A CN 200610029610 CN200610029610A CN1904619A CN 1904619 A CN1904619 A CN 1904619A CN 200610029610 CN200610029610 CN 200610029610 CN 200610029610 A CN200610029610 A CN 200610029610A CN 1904619 A CN1904619 A CN 1904619A
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Abstract
The invention discloses an agent tape and kit for testing alcohol content in spit. The content of alcohol in spit would be semi-quantification tested according to the color of agent tape after ethanol oxidase and peroxidase oxygenating N, N, N', N'-tetramethyl benzidine.
Description
Technical field
The present invention relates to a kind of reagent strip and kit that detects ethanol, relate in particular to a kind of reagent strip and kit that detects ethanol content in the saliva.
Background technology
In recent years, the criminal case that causes after drinking and the traffic hazard case that causes of driving when intoxicated constantly rise.At present, many countries have all formulated the relation of ethanol in blood concentration and traffic accident responsibility identification, have set up the multiple method that can detect ethanol in blood content.As instrument detecting alcohol components such as GM/MS, though these methods can be accurately quantitative, exist the purchase instrument and cost an arm and a leg, detect the cost height, need special training, can't be in the shortcoming of spot fast detecting.Expiration formula alcohol determining instrument can use at the scene, but price is also very expensive, and unhygienic, and cross infection disease takes place easily.
Therefore, occurred a lot of portable detectable bars in recent years on the market, disclosed wherein a kind of detectable bar as patent publication No. CN1749751, active unstable but these reagent strips all exist under the high temperature, holding time is short, is unfavorable for the defective of on-the-spot outdoor detection.
Summary of the invention
Technical matters to be solved by this invention is to provide a kind of reagent strip and kit that detects ethanol content in the saliva, to solve defective of the prior art.
The present invention is according to saliva and the conforming experiment basis of ethanol in blood content, on the basis of zymetology reaction, set up chromogenic reaction, at first, alcohol oxidase (ALO) becomes acetaldehyde to discharge hydrogen peroxide simultaneously oxidation of ethanol, under the situation that peroxidase (HRP) exists, and TMB (N, N, N ', N '-tetramethyl benzidine) oxidation takes place and develop the color, concrete reaction principle is as follows:
In above reaction, the content of ethanol becomes linear dependence in the depth of TMB blueness and the speed of color speed and the sample, and finally as a kind of index of semiquantitative detection.
The Tris-malonic acid that adopts 1M during concrete preparation is as buffer system, and as stabilizing agent, under this condition, alcohol oxidase can keep activity to reach one month under 37 ℃ situation with algin and gelatin.And under the condition that does not have stabilizing agent to exist, it is active in 24 hours that alcohol oxidase only can keep under 37 ℃ situation.
The present invention at first provides a kind of reagent strip that detects ethanol content in the saliva, and this reagent strip prepares by the following method:
1. prepare alcohol oxidase and horseradish peroxidase solution
Active unit's ratio is 1: the alcohol oxidase of 2-10 and horseradish peroxidase, be dissolved in the 1M Tris-malonic acid damping fluid of pH 7.2, and making the gross activity unit of enzyme in every milliliter the damping fluid is 150-550, adds and the isopyknic water of damping fluid again;
2. prepare the TMB/ xylene solution
The TMB hydrochloride is soluble in water, be mixed with the aqueous solution of 10-30mg/ml, with the processing of decolouring of the chroman hydrochloride propylhomoserin of 10mg/ml, add and the isopyknic dimethylbenzene of TMB hydrochloride aqueous solution then, with NaOH neutralization hydrochloric acid wherein; Layering behind the solution left standstill discards aqueous solution, obtains the TMB/ xylene solution;
3. the immobilization of enzyme and substrate
In filter paper immersion enzyme mixed liquor 1., take out the filter paper drying; The TMB/ xylene solution is dropped on the dry filter paper drying;
4. prepare reagent strip
Dried filter paper is attached to the front end that single face contains the white polyester plate of glue, is cut into the rectangular of wide 0.5-1cm, promptly make the detectable bar with paper cutter.
Wherein step in 1. alcohol oxidase and horseradish peroxidase solution in also contain algin and gelatin, wherein the content of algin is 0.25-0.75mg/ml in the enzyme solutions, gelatine content is 5-15mg/ml.The content of algin and gelatin is respectively 10mg and 200mg in the enzyme solutions of preferred every 20ml.
Ethanol detectable bar finally is a content of judging ethanol by the colour developing of TMB, the consumption of TMB directly causes the difference of reacted color in this reaction system, if use heavy dose of TMB, then the result shows from the green to the blueness, be the graded of ratio with concentration of alcohol, if use low dose of TMB, high concentration ethanol is shown as brown or black, and low concentration then is shown as blueness.The present invention has adjusted the amount of TMB according to the regulation of the relevant ethanol content of China, makes colour developing result obviously differentiation 0,0.2,0.8,1.5 and 3.0mg/ml, is used for the standard comparison card that ethanol detects thereby prepared.
Other purpose of the present invention is to provide a kind of kit that is used for detecting the saliva ethanol content, this kit comprises the mentioned reagent bar, standard comparison card and and reaction terminating liquid, the described reaction terminating liquid concentration that is weight percentage is 0.2% SDS (sodium dodecylsulphonate) solution.
TMB (N, N, N ', N '-tetramethyl benzidine) is a kind of non-carcinogenic developer, and after the effect of superoxide enzyme, colour developing in about 40 minutes reaches the peak, weakens gradually immediately, can disappear to colourless to 2 hours.SDS is the inhibitor of enzyme, can make reaction terminating, and color is kept do not take off in 12-24 hour, is the good terminator that naked eyes are judged.After this product was equipped with the SDS terminator, testing result did not change after 24 hours, very helped the preservation of evidence at the scene.
This product has following characteristics:
1. Gao Du thermal stability is convenient to use under the normal temperature and preserve.Product of the present invention can be preserved 1 year at normal temperatures, can preserve under 50 ℃ more than 24 hours, and the sensitivity of its detection still can reach 0.1mg/ml.Compare with other external like product, external product all can only be preserved 1-3 hour under 50 ℃ of high temperature.
2. highly sensitive, the sensitivity of this test strips can reach 0.1mg/ml.And do not produce false positive.
3. reaction velocity is fast.Just can finish the detection of a person-portion in 20 seconds.
4. aberration is good.Not only feminine gender exists obvious color different with the sample of positive 0.2mg/ml, and also can be easy to judge the aberration of 0.2mg/ml and 0.8mg/ml with naked eyes.
5. sample variation.According to user's needs, this product can be used for detecting the concentration of alcohol in urine or other body fluid or the exudate, though colour developing is slightly different with saliva, can obviously distinguish the aberration of feminine gender (0.0O) and positive (0.4mg/ml) and 0.8mg/ml equally.(concentration of alcohol in the urine is higher than the concentration in the saliva)
Characteristics by visible ethanol test strip of the present invention in the following table:
Table 1 saliva ethanol content test-strips detects the comparison of discussing with expiration
| Products characteristics | Saliva ethanol content test-strips | Ethanol expiration detector |
| With the haemoconcentration relation | Direct relation | Letter connects conversion relation |
| The on-the-spot detection | Easy to carry, the particularly suitable field quick detection | Because price is more expensive, being not easy to each traffic police portably uses, and after each detection use all arranged convalescence, can not use continuously. |
| Wholesomeness | Single packaged, disposable use, be not subjected to time, place, climate effect, avoided infection and cross-infection. | Because same instrument uses repeatedly, often need check and correction and maintenance safe and sanitary to have hidden danger |
| Detection sensitivity | 0.1mg/ml | Have only expensive expiration instrument just can reach 0.1mg/ml |
| Quantitative or qualitative | Qualitative or half-quantitative detection | Sxemiquantitative or detection by quantitative |
Embodiment
Embodiment 1: preparation detectable bar
1. prepare alcohol oxidase and horseradish peroxidase solution
Get algin 10mg, alcohol oxidase 500 units, horseradish peroxidase 2500 units, gelatin 200mg is dissolved in the 1M Tris-malonic acid damping fluid of 10ml pH7.2, adds the distilled water of 10ml again.
2. prepare TMB solution
Take by weighing the 400mgTMB hydrochloride and be dissolved in the distilled water of 20ml,, add the dimethylbenzene of 20ml again, with NaOH neutralization hydrochloric acid wherein with the processing of decolouring of the 10mg/ml chroman hydrochloride propylhomoserin of 0.1ml.Mix back TMB and be dissolved in dimethylbenzene, leave standstill back solution layering, discard aqueous solution.Obtain the TMB/ xylene solution.
3. the immobilization of enzyme and substrate
Filter paper that 0.5CM is wide immerses in the above enzyme mixed liquor, takes out filter paper under 30 ℃ of hot blasts, lucifuge dry 20 minutes.The TMB/ xylene solution is dropped on the dry filter paper equably under 30 ℃ of hot blasts, lucifuge dry 20 minutes again.
4. prepare reagent strip
Dried filter paper is attached to the front end that single face contains the white polyester plate of glue, is cut into the rectangular of wide 0.5cm, promptly make the detectable bar with paper cutter.
Then this reagent strip, standard color comparison card and reaction terminating liquid are made into kit.
Embodiment 2: the on-the-spot testing result of reagent strip
1. the comparison of saliva ethanol detectable bar and gas chromatography head space method blood testing data:
4 Traffic Warden Subteams apply in Shanghai City to distribute reagent strip, carry out on-the-spot test, and the result of all tests and gas chromatography head space method (GC/MS) detect relatively.The result shows, the detection of gas chromatography head space method conforms to substantially in saliva ethanol detectable bar testing result and the blood.Accuracy rate is 98.0%.
2. sample collection mode:
1) test-strips is directly put into mouth, take out after waiting for 10 seconds, observe change color; Or
2) saliva is spat in the detection vessel.
Claims (9)
1. reagent strip that detects ethanol content in the saliva is characterized in that this reagent strip is prepared into by following method:
1. prepare alcohol oxidase and horseradish peroxidase solution
Active unit's ratio is 1: the alcohol oxidase of 2-10 and horseradish peroxidase, be dissolved in the 1MTris-malonic acid damping fluid of pH 7.2, and the gross activity unit that makes enzyme in every milliliter of damping fluid is 150-550, adds and the isopyknic water of damping fluid again;
2. prepare the TMB/ xylene solution
The TMB hydrochloride is soluble in water, be mixed with the aqueous solution of 10-30mg/ml, with the processing of decolouring of the chroman hydrochloride propylhomoserin of 10mg/ml, add and the isopyknic dimethylbenzene of TMB hydrochloride aqueous solution then, with NaOH neutralization hydrochloric acid wherein; Layering behind the solution left standstill discards aqueous solution, obtains the TMB/ xylene solution;
3. the immobilization of enzyme and substrate
In filter paper immersion enzyme mixed liquor 1., take out the filter paper drying; The TMB/ xylene solution is dropped on the dry filter paper drying;
4. prepare reagent strip
Dried filter paper is attached to the front end that single face contains the white polyester plate of glue, slitting with paper cutter, promptly make the detectable bar.
2. reagent strip according to claim 1, it is characterized in that also containing algin and gelatin in alcohol oxidase during step 1. and the horseradish peroxidase solution, wherein the content of algin is 0.25-0.75mg/ml in the enzyme solutions, and gelatine content is 5-15mg/ml.
3. reagent strip according to claim 1 and 2 is characterized in that step 2., and TMB hydrochloride content soluble in water is 20mg/ml.
4. reagent strip according to claim 1 and 2 is characterized in that immersing during step 3. filter paper after the enzyme mixed liquor under 30 ℃ of hot blasts, lucifuge dry 20 minutes; The dry filter paper that dripped the TMB/ xylene solution under 30 ℃ of hot blasts, lucifuge dry 20 minutes again.
5. a method for preparing the described reagent strip of claim 1 is characterized in that this method comprises the steps:
1. prepare alcohol oxidase and horseradish peroxidase solution
Active unit's ratio is 1: the alcohol oxidase of 2-10 and horseradish peroxidase, be dissolved in the 1MTris-malonic acid damping fluid of pH 7.2, and the gross activity unit that makes enzyme in every milliliter of damping fluid is 150-550, adds and the isopyknic water of damping fluid again;
2. prepare the TMB/ xylene solution
The TMB hydrochloride is soluble in water, be mixed with the aqueous solution of 10-30mg/ml, with the processing of decolouring of the chroman hydrochloride propylhomoserin of 10mg/ml, add and the isopyknic dimethylbenzene of TMB hydrochloride aqueous solution then, with NaOH neutralization hydrochloric acid wherein; Layering behind the solution left standstill discards aqueous solution, obtains the TMB/ xylene solution;
3. the immobilization of enzyme and substrate
In filter paper immersion enzyme mixed liquor 1., take out the filter paper drying; The TMB/ xylene solution is dropped on the dry filter paper drying;
4. prepare reagent strip
Dried filter paper is attached to the front end that single face contains the white polyester plate of glue, slitting with paper cutter, promptly make the detectable bar.
6. method according to claim 5 is characterized in that also containing algin and gelatin in alcohol oxidase during step 1. and the horseradish peroxidase solution, and wherein the content of algin is 0.25-0.75mg/ml in the enzyme solutions, and gelatine content is 5-15mg/ml.
7. according to claim 5 or 6 described methods, it is characterized in that step is 2., TMB hydrochloride content soluble in water is 20mg/ml.
8. according to claim 5 or 6 described methods, it is characterized in that immersing during step 3. filter paper after the enzyme mixed liquor under 30 ℃ of hot blasts, lucifuge dry 20 minutes; The dry filter paper that dripped the TMB/ xylene solution under 30 ℃ of hot blasts, lucifuge dry 20 minutes again.
9. kit that detects ethanol content in the saliva is characterized in that this kit comprises the described reagent strip of claim 1, standard comparison card and reaction terminating liquid, and the described reaction terminating liquid concentration that is weight percentage is 0.2% SDS solution.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200610029610 CN1904619A (en) | 2006-08-01 | 2006-08-01 | Reagent stripe for detecting alcohol content in saliva and box kit |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200610029610 CN1904619A (en) | 2006-08-01 | 2006-08-01 | Reagent stripe for detecting alcohol content in saliva and box kit |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1904619A true CN1904619A (en) | 2007-01-31 |
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Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200610029610 Pending CN1904619A (en) | 2006-08-01 | 2006-08-01 | Reagent stripe for detecting alcohol content in saliva and box kit |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102384971A (en) * | 2011-10-18 | 2012-03-21 | 郑州炜盛电子科技有限公司 | Spittle alcoholicity test strip and production method thereof |
| CN103267758A (en) * | 2013-05-17 | 2013-08-28 | 浙江东方基因生物制品有限公司 | Dry chemical method rapid diagnostic reagent strip for testing content of alcohol in saliva and preparation method thereof |
| 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 |
| CN109765366A (en) * | 2019-01-31 | 2019-05-17 | 中国农业科学院兰州兽医研究所 | A kind of kit and its detection method detecting foot and mouth disease virus 3AB antibody |
| CN110095457A (en) * | 2019-04-17 | 2019-08-06 | 广东环凯微生物科技有限公司 | A kind of the measurement test paper and rapid assay methods of chlorine residue |
-
2006
- 2006-08-01 CN CN 200610029610 patent/CN1904619A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102384971A (en) * | 2011-10-18 | 2012-03-21 | 郑州炜盛电子科技有限公司 | Spittle alcoholicity test strip and production method thereof |
| CN103267758A (en) * | 2013-05-17 | 2013-08-28 | 浙江东方基因生物制品有限公司 | Dry chemical method rapid diagnostic reagent strip for testing content of alcohol in saliva and preparation method thereof |
| CN103267758B (en) * | 2013-05-17 | 2015-05-13 | 浙江东方基因生物制品有限公司 | Dry chemical method rapid diagnostic reagent strip for testing content of alcohol in saliva and preparation method thereof |
| 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 |
| CN109765366A (en) * | 2019-01-31 | 2019-05-17 | 中国农业科学院兰州兽医研究所 | A kind of kit and its detection method detecting foot and mouth disease virus 3AB antibody |
| CN110095457A (en) * | 2019-04-17 | 2019-08-06 | 广东环凯微生物科技有限公司 | A kind of the measurement test paper and rapid assay methods of chlorine residue |
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Effective date of registration: 20070105 Address after: Shanghai City Caoxi road 258 Lane 23 No. 3 No. 2 floor Applicant after: Shanghai hehe Mdt InfoTech Ltd Address before: Room 2, building 2715, 311-315 Wu Long Road, Shanghai, Xuhui District Applicant before: Shanghai Weizhuo Biological Sci. & Tech. Co., Ltd. |
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| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |