CN102520150A - Diagnostic test paper for determination of gamma-hydroxybutanoic acid and preparation method thereof - Google Patents
Diagnostic test paper for determination of gamma-hydroxybutanoic acid and preparation method thereof Download PDFInfo
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- CN102520150A CN102520150A CN2011104058031A CN201110405803A CN102520150A CN 102520150 A CN102520150 A CN 102520150A CN 2011104058031 A CN2011104058031 A CN 2011104058031A CN 201110405803 A CN201110405803 A CN 201110405803A CN 102520150 A CN102520150 A CN 102520150A
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- test paper
- paper
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- mensuration
- hydroxybutyric acid
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Abstract
The invention relates to a diagnostic test paper for determination of gamma-hydroxybutanoic acid and a preparation method thereof. The test paper comprises first phase immersion liquid and second phase immersion liquid. The preparation method comprises the following steps: (1) soaking filter paper in the first phase immersion liquid for 3 to 8 min, drying the filter paper at a temperature of 20 to 50 DEG C, then soaking the filter paper in the second phase immersion liquid for 3 to 8 min and drying the filter paper at a temperature of 20 to 50 DEG C again so as to obtain raw paper of the test paper used for determination; and (2) applying the raw paper of the test paper on a plastic film base and cutting the plastic film base into pieces so as to obtain reagent strips used for determination. The diagnostic test paper provided in the invention can be assembled into a plurality of detection means, has the advantages of convenient operation, rapid detection, good sensitivity, high accuracy and the like, can be used for qualitative and semi-quantitative determination of conventional liquid and can detect the content of residual 4-hydroxybutanoic acid in blood, urine and saliva of a drinker or a victim; therefore, the diagnostic test paper has a good application prospect.
Description
Technical field
The invention belongs to the mensuration field of gamma-hydroxybutyric acid, particularly mensuration diagnose test paper of a kind of gamma-hydroxybutyric acid and preparation method thereof.
Background technology
The pharmacological action of gamma-hydroxybutyric acid: the GHB formal name used at school is gamma hydroxybutyrate.Be a kind of colourless, do not have smell, liquid with no taste, also formulations such as adularescent powder, tablet and capsule.Belong to what depressor of nerve centre tool habituation property, once be used for as anesthetic,general, help for sleep and body-building, GHB does not have legal medical application at present, on the contrary with drug abuse, poisoning, excessive and date rape is relevant.
Its toxicological effect: clinical demonstration produces relexation but the GHB of low dosage can produce the floaty euphoria anxiety reduction; Can produce sedation during high dose, can let the people sleep, at last stupor or dead.Usually oral ten milligrams; Can produce the temporary loss of memory, 30 milligrams can hypnotic; Reach 20 g and will cause poisoning and once use; Similarly symptom comprises: feel sick, vomiting, headache, reflex forfeiture, possibly lose consciousness, stupor and dead very soon, with alcohol also with can more aggravating its danger.
Its abuse property: oral GHB is made into particle or Powdered usually, dissolves in the what liquid for example to eat after the beverage of boiling water, wine or other kinds.GHB takes the back and promptly had an effect in 10 to 20 minutes, can produce similar happiness, the drunk and effect of promoting the sexual maturity after taking, and adds that fast and short time of its drug effect is difficult for clear-headed and can has the effect that produces the temporary loss of memory; After injured party mistake being had a drink encroached on; Also can't remember the injured party, thereby abused, even in order to commit a crime; Among nearest popular what teenager and the adult of youth comparatively, mostly occur within the what club in the place that quilt is abused.Classified as first order controlled drug at U.S. GHB.
Gamma-hydroxybutyric acid is commonly called as " liquid MDMA ", " facial treatment essence ", " G water ", has been the drugs that very seriously spread unchecked in American-European countries, and the abuse object of Hong Kong, Macao, Taiwan is main with the teenager, is control anesthesia similar drug in China.Suck " facial treatment essence " and can make people's song and dance revelry all through the night and untiring, What is more does not sleep two days two nights, and spirit is in extremely excited or illusion state.Some drug trafficker is in order to increase drugs " facial treatment essence " sales volume, and other novel drugs are entrained among " facial treatment essence " to strengthen its toxicity, makes it have more temptation property to the drug addict.Classified as a kind of " date rape ball.”
Therefore, the gesture of the developmenting spread of drugs " facial treatment essence " can not be despised, and very likely develops into the Hou of the methamphetamine that continues, dancing outreach, " K " powder, the novel drug of the serious abuse of another quilt.Violent in view of its oncoming force, harm is big, characteristics such as disguised strong; Prohibition of drug department and public security department attract great attention; Openly investigate and seize and the entertainment management renovation that is prone to be involved in drug traffic in require to strengthen conscientiously inspection, resolution dynamics to all kinds of oral liquids, flexible package punch thing etc.; Therefore fast measuring with detect BHB, it is very necessary that the development detectable just seems.
Summary of the invention
Technical matters to be solved by this invention provides mensuration diagnose test paper of a kind of gamma-hydroxybutyric acid and preparation method thereof, and this test paper has easy to operate, detects fast, and sensitivity is good, and the accuracy advantages of higher can qualitative and half-quantitative detection conventional liq.
The mensuration diagnose test paper of a kind of gamma-hydroxybutyric acid of the present invention (gamma-hydroxybutyric acid, 4 hydroxybutyric acid), said test paper comprises following reagent:
(1) first phase immersion liquid
Stabilizing agent and/or protective agent 1-10g;
Bovine serum albumin(BSA) 20-2000mg; Oxalic acid 200-2000mg;
NAD
+(DPN) 0.1-2.0g; NADP
+(DPN I) 0.01-1.0g;
Diaphorase 200-20000iu; 4 hydroxybutyric acid dehydrogenase 2 00-10000u;
Adding damping fluid to cumulative volume is 100～150ml;
(2) second phase immersion liquid
Chlorination nitro blue tetrazolium NBT 0.1-0.5g;
Azophenlyene dimethyl ester sulfate PMS 0.02-0.2g;
Adding organic solvent to cumulative volume is 100～150ml.
Said stabilizing agent and/or protective agent are one or more in gelatin, Arabic gum, polyglycol, the polyacrylic acid-acrylic ester.
Said damping fluid is 40-80mmol/L Tris-malonic acid damping fluid or the phosphate buffer of pH=5.8-8.8.
Said organic solvent is ethanol or methyl alcohol.
The preparation method of the mensuration diagnose test paper of a kind of gamma-hydroxybutyric acid of the present invention comprises:
(1) filter paper is soaked 3-8 minute in the first phase immersion liquid after, in 20-50 ℃ of drying, in the second phase immersion liquid, soak again after 3-8 minute, in 20-50 ℃ dry once more, obtain measuring the body paper of test paper;
(2) body paper with the said determination test paper sticks on the plastic base, cuts into piece, makes the mensuration reagent strip.
The specification of the reagent strip in the said step (2) is 0.3 * 0.3～0.8 * 0.8cm
2
Reaction principle of the present invention is following:
NAD(P)H+PMS——————→NAD(P)
++PMS·H
2
PMS·H
2+NBT——————→PMS+formazan
Wherein, NAD+: DPN;
NADH: dihydrocoenzyme I;
NADP: DPN I;
NADPH: dihydrocoenzyme II;
Diaphorase: diaphorase;
NBT: nitro blue tetrazolium, one type of developer of chlorination nitro blue tetrazolium;
PMS: azophenlyene dimethyl ester sulfate;
Formazan: Jia Za (being the bluish violet compound);
The colour developing degree of the formazan that generates and the 4 hydroxybutyric acid concentration positive correlation of body fluid to be measured; Can print into standard color card and be attached on the wrappage (sealing bag or instructions), but thereby the degree of colour developing and standard colors comparison semiquantitative determination 4 hydroxybutyric acid concentration.Because the concentration of substrate that is detected is often higher, the frequent highly significant of the chromogenic reaction that detects.
Beneficial effect
Diagnose test paper of the present invention can be assembled into multiple pick-up unit, and has easy to operately, detects fast, and sensitivity is good, and the accuracy advantages of higher can be qualitative and half-quantitative detection conventional liq (milk, drinks, soda or the like).The content of residual 4 hydroxybutyric acid also can be detected in blood (like serum/whole blood), urine and the saliva for the drinking person or the injured party, can be applied to the strike to drug abuse and criminal offence, therefore has a good application prospect.
Description of drawings
Fig. 1 is the pick-up unit that reagent piece and PVC sheet are formed;
Fig. 2 is the color development face of the pick-up unit be made up of reagent piece, hemofiltration film, two PVC sheets;
Fig. 3 is the application of sample face of the pick-up unit be made up of reagent piece, hemofiltration film, two PVC sheets.
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 the restriction scope of the present invention.Should be understood that in addition those skilled in the art can do various changes or modification to 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
Phasel enzyme solution liquid cumulative volume is that the process for preparation of 100mL is following:
1) dissolves in Arabic gum 5g;
2) dissolve in bovine serum albumin(BSA) 500mg;
3) dissolve in oxalic acid 300mg;
4) dissolve in NAD
+0.5g, NADP
+0.2g;
5) dissolve in diaphorase, 10000iu;
6) dissolve in 4 hydroxybutyric acid dehydrogenase 10 000u;
7) the 50mmol/L Tris-malonic acid damping fluid that adds pH8.0 is to 100ml;
The first phase solution impregnation:
After formulations prepared from solutions is good, add impregnator, a volume whatman 3MM filter paper is installed, bake out temperature is set 30 ℃, and chart drive speed is set 3.0mm/ second, begins to flood paper feed, and solution has soaked back filter paper and stopped 1 hour at drying baker, so that oven dry fully;
The second phase formulations prepared from solutions:
Dissolve in chlorination nitro blue tetrazolium 250mg; (being that concentration is 0.25g/100ml)
Azophenlyene dimethyl ester sulfate 100mg; (being that concentration is 0.10g/100ml)
With absolute ethyl alcohol 100ml is solvent.
The second phase solution impregnation:
The second phase solution is added impregnator, and the filter paper that will soak first phase installs, and bake out temperature is set 45 ℃; Chart drive speed is set 3.0mm/ second, begins to flood paper feed, and solution has soaked back filter paper and stopped 1 hour at drying baker; So that oven dry fully, so far body paper completes.
Test paper is made: get one section body paper, single face posts double faced adhesive tape, is cut into the 5mm slice with the 5mm hobboing cutter, and slice is attached on the wide pvc thin slice of 8cm*25cm, is being cut into the wide reagent strip of 5mm with hobboing cutter, is put in the airtight bottle of black, and it is subsequent use to put drying agent well.
The mensuration of soda 4 hydroxybutyric acid concentration:
4 hydroxybutyric acid sodium (sigma)
Cola drink takes by weighing 12.60mg 4 hydroxybutyric acid sodium, adds in the cola drink to be dissolved as 100ml, is diluted to 0 μ mol/L with cola drink respectively, 50 μ mol/L, 100 μ mol/L, 200 μ mol/L, 500 μ mol/L, six kinds of concentration of 1000 μ mol/L L.The reagent strip of getting six roots of sensation embodiment 1 preparation immerses in above-mentioned six kinds of solution simultaneously, and take out about ten seconds, inhales with thieving paper and removes excessive moisture, sees color then in two minutes, and the result divides obviously to each gradient zones of darkviolet of 1000 μ mol/L from the yellow of feminine gender.
Embodiment 2
Phasel enzyme solution liquid cumulative volume is that the process for preparation of 100mL is following:
1) dissolves in Arabic gum 10g;
2) dissolve in bovine serum albumin(BSA) 20mg;
3) dissolve in oxalic acid 200mg;
4) dissolve in NAD
+0.1g, NADP
+0.01g;
5) dissolve in diaphorase, 200iu;
6) dissolve in 4 hydroxybutyric acid dehydrogenase 2 00u;
7) the 40mmol/L Tris-malonic acid damping fluid that adds pH6.0 is to 100ml;
The first phase solution impregnation:
After formulations prepared from solutions is good, add impregnator, a volume whatman 3MM filter paper is installed, bake out temperature is set 30 ℃, and chart drive speed is set 3.0mm/ second, begins to flood paper feed, and solution has soaked back filter paper and stopped 1 hour at drying baker, so that oven dry fully;
The second phase formulations prepared from solutions:
Dissolve in chlorination nitro blue tetrazolium 100mg;
Azophenlyene dimethyl ester sulfate 20mg;
With absolute ethyl alcohol 100ml is solvent.
The second phase solution impregnation:
The second phase solution is added impregnator, and the filter paper that will soak first phase installs, and bake out temperature is set 45 ℃; Chart drive speed is set 3.0mm/ second, begins to flood paper feed, and solution has soaked back filter paper and stopped 1 hour at drying baker; So that oven dry fully, so far body paper completes.
Test paper is made: get one section body paper, single face posts double faced adhesive tape, is cut into the 5mm slice with the 5mm hobboing cutter, and slice is attached on the wide pvc thin slice of 8cm*25cm, is being cut into the wide reagent strip of 5mm with hobboing cutter, is put in the airtight bottle of black, and it is subsequent use to put drying agent well.(Fig. 1)
The mensuration of whole blood sample 4 hydroxybutyric acid concentration:
Get one section body paper and be cut into the 8mm slice with the 8mm hobboing cutter; And then it is subsequent use to be cut into square with scissors, at the one-sided double faced adhesive tape that posts of the wide pvc thin slice of 8cm*25cm, is cut into the wide slice of 8mm with the 8mm hobboing cutter then; In the hole of beating diameter 5mm apart from an end 10mm place; The reagent piece is attached on the position of punching, covers the hemofiltration film of a 10mm*5mm above the reagent piece, subsequent use.(Fig. 2)
Get 10 parts of blood samples and measure with above-mentioned strip with the c8000 of Abbott Laboratories biochemical instruments mensuration 4 hydroxybutyric acid, the concentration of yin and yang attribute, and positive sample as a result, the result of fundamental sum biochemical instruments conforms to.
Embodiment 3
Phasel enzyme solution liquid cumulative volume is that the process for preparation of 100mL is following:
1) dissolves in gelatin 1g;
2) dissolve in bovine serum albumin(BSA) 2000mg;
3) dissolve in oxalic acid 2000mg;
4) dissolve in NAD
+2g, NADP
+1g;
5) dissolve in diaphorase, 20000iu;
6) dissolve in 4 hydroxybutyric acid dehydrogenase 10 000u;
7) the 80mmol/L Tris-malonic acid damping fluid that adds pH8.7 is to 150ml;
The first phase solution impregnation:
After formulations prepared from solutions is good, add impregnator, a volume whatman 3MM filter paper is installed, bake out temperature is set 30 ℃, and chart drive speed is set 3.0mm/ second, begins to flood paper feed, and solution has soaked back filter paper and stopped 1 hour at drying baker, so that oven dry fully;
The second phase formulations prepared from solutions:
Dissolve in chlorination nitro blue tetrazolium 500mg;
Azophenlyene dimethyl ester sulfate 200mg;
With absolute ethyl alcohol 150ml is solvent.
The second phase solution impregnation:
The second phase solution is added impregnator, and the filter paper that will soak first phase installs, and bake out temperature is set 45 ℃; Chart drive speed is set 3.0mm/, begins to flood paper feed, and solution has soaked back filter paper and stopped 1 hour at drying baker; So that oven dry fully, so far body paper completes.
Test paper is made: get one section body paper, single face posts double faced adhesive tape, is cut into the 5mm slice with the 5mm hobboing cutter, and slice is attached on the wide pvc thin slice of 8cm*25cm, is being cut into the wide reagent strip of 5mm with hobboing cutter, is put in the airtight bottle of black, and it is subsequent use to put drying agent well.(Fig. 1)
The mensuration of urine 4 hydroxybutyric acid concentration:
4 hydroxybutyric acid sodium (sigma)
Negative urine 3-hydroxybutyric acid takes by weighing the 12.60mg3-Sodium, adds in the negative urine of 100m to dissolve, and is diluted to 0 μ mol/L with negative urine respectively, 100 μ mol/L, 200 μ mol/L, 500 μ mol/L, five kinds of concentration of 1000 μ mol/L.The reagent strip of getting 3 preparations of 5 instances immerses in above-mentioned 5 kinds of solution simultaneously, and take out about ten seconds, inhales with thieving paper and removes excessive moisture.
Saw color then in two minutes, the result divides obviously to each gradient zones of darkviolet of 1000 μ mol/L from 0 yellow.
Claims (6)
1. the mensuration diagnose test paper of a gamma-hydroxybutyric acid, it is characterized in that: said test paper comprises following reagent:
(1) first phase immersion liquid
(2) second phase immersion liquid
Chlorination nitro blue tetrazolium NBT 0.1-0.5g;
Azophenlyene dimethyl ester sulfate PMS 0.02-0.2g;
Adding organic solvent to cumulative volume is 100～150ml.
2. the mensuration diagnose test paper of a kind of gamma-hydroxybutyric acid according to claim 1, it is characterized in that: said stabilizing agent and/or protective agent are one or more in gelatin, Arabic gum, polyglycol, the polyacrylic acid-acrylic ester.
3. the mensuration diagnose test paper of a kind of gamma-hydroxybutyric acid according to claim 1, it is characterized in that: said damping fluid is 40-80mmol/L Tris-malonic acid damping fluid or the phosphate buffer of pH=5.8-8.8.
4. the mensuration diagnose test paper of a kind of gamma-hydroxybutyric acid according to claim 1, it is characterized in that: said organic solvent is ethanol or methyl alcohol.
5. the preparation method of the mensuration diagnose test paper of a gamma-hydroxybutyric acid comprises:
(1) filter paper is soaked 3-8 minute in the first phase immersion liquid after, in 20-50 ℃ of drying, in the second phase immersion liquid, soak again after 3-8 minute, in 20-50 ℃ dry once more, obtain measuring the body paper of test paper;
(2) body paper with the said determination test paper sticks on the plastic base, cuts into piece, makes the mensuration reagent strip.
6. the preparation method of the mensuration diagnose test paper of a kind of gamma-hydroxybutyric acid according to claim 5, it is characterized in that: the specification of the reagent strip in the said step (2) is 0.3 * 0.3～0.8 * 0.8cm
2
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|---|---|---|---|
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104111253A (en) * | 2014-06-20 | 2014-10-22 | 武汉高丰生物科技有限公司 | Preparation method of 4-hydroxybutyric acid test paper resisting interference of ascorbic acid and pigments |
| CN104198407A (en) * | 2014-08-14 | 2014-12-10 | 上海睿康生物科技有限公司 | Detection kit for detecting content of beta-hydroxybutyrate in serum by adopting stable enzymatic method |
| CN104897667A (en) * | 2015-06-19 | 2015-09-09 | 公安部物证鉴定中心 | Application of Smith reagent in detecting GHB (Gamma-hydroxybutyrate) in liquid sample |
Citations (3)
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| CN1249431A (en) * | 1998-09-28 | 2000-04-05 | 生命扫描有限公司 | Diagnostic reagent based on tetrazole compound |
| CN1419604A (en) * | 2000-03-28 | 2003-05-21 | 生命扫描有限公司 | Reagents and methods for detecting a reduced cofactor |
| CN101900734A (en) * | 2010-02-03 | 2010-12-01 | 上海高丰医疗电器有限公司 | Diagnosis test paper for diabetic ketosis and other symptoms of relatively high ketone body |
-
2011
- 2011-12-08 CN CN2011104058031A patent/CN102520150A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1249431A (en) * | 1998-09-28 | 2000-04-05 | 生命扫描有限公司 | Diagnostic reagent based on tetrazole compound |
| CN1249349A (en) * | 1998-09-28 | 2000-04-05 | 生命扫描有限公司 | Diagnostic reagent based on tetrazole compound |
| CN1419604A (en) * | 2000-03-28 | 2003-05-21 | 生命扫描有限公司 | Reagents and methods for detecting a reduced cofactor |
| CN101900734A (en) * | 2010-02-03 | 2010-12-01 | 上海高丰医疗电器有限公司 | Diagnosis test paper for diabetic ketosis and other symptoms of relatively high ketone body |
Non-Patent Citations (1)
| Title |
|---|
| DAWN T. BRAVO ET AL: "Reliable, Sensitive, Rapid and Quantitative Enzyme-Based Assay for Gamma-Hydroxybutyric Acid (GHB)", 《J FORENSIC SCI》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104111253A (en) * | 2014-06-20 | 2014-10-22 | 武汉高丰生物科技有限公司 | Preparation method of 4-hydroxybutyric acid test paper resisting interference of ascorbic acid and pigments |
| CN104198407A (en) * | 2014-08-14 | 2014-12-10 | 上海睿康生物科技有限公司 | Detection kit for detecting content of beta-hydroxybutyrate in serum by adopting stable enzymatic method |
| CN104897667A (en) * | 2015-06-19 | 2015-09-09 | 公安部物证鉴定中心 | Application of Smith reagent in detecting GHB (Gamma-hydroxybutyrate) in liquid sample |
| CN104897667B (en) * | 2015-06-19 | 2017-10-03 | 公安部物证鉴定中心 | Application of the Smith reagents in detection liquid sample in GHB |
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Application publication date: 20120627 |