CN103969454B - Melatonin method for quick and detection thereof block and preparation method - Google Patents
Melatonin method for quick and detection thereof block and preparation method Download PDFInfo
- Publication number
- CN103969454B CN103969454B CN201310698818.0A CN201310698818A CN103969454B CN 103969454 B CN103969454 B CN 103969454B CN 201310698818 A CN201310698818 A CN 201310698818A CN 103969454 B CN103969454 B CN 103969454B
- Authority
- CN
- China
- Prior art keywords
- melatonin
- line
- detection
- colloidal gold
- gold
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Endocrinology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
Disclosure one melatonin method for quick and detection thereof block and preparation method, aim to provide a kind of easy to use, fast and convenient, the highly sensitive method for quick detecting the melatonin that adulterates in food, health food or medicine and detection card used thereof, technical points: 1) product that need to detect is added organic solvent dissolution, filter after adding slow releasing agent dilution, collect filtrate; 2) filtrate added drop-wise step 1) collected is on melatonin colloidal gold chromatographic detection card, it is judged that testing sample is positive or negative, if controlling C line and the detection equal displaing amaranth of T line, then negative; If control line C displaing amaranth, detection line T does not develop the color, then be positive; Control line C on colloidal gold test and detection line T does not all develop the color, or detection line T colour developing only occurs, then illustrative experiment is invalid; Belong to technical field of biological.
Description
Technical field
The present invention relates to a kind of detection method, specifically, be method for quick and the test kit of a kind of melatonin, belong to technical field of biological.
Background technology
Melatonin is also known as melatonin, and chemical name is MLT, is a kind of indole amine bormones of pinus secretion in human brain. Its secretion reduces along with the increase of illumination, therefore the dark hormone that is otherwise known as. Melatonin is mainly used in regulating the clear-headed of human body and sleep cycle, and induced physiological is slept, and anti-ageing waits for a long time.
Melatonin is a kind of endogenous hormone, there are some researches show, a large amount of use melatonin may cause breathing problem, produces hangover effect, and melatonin can affect the effect of some calming soporific medicine such as clonazepam simultaneously. In Europe, the cycle of taking of melatonin, applicable crowd and dosage all have strict restriction. In the U.S., melatonin is sold with the form of dietary supplement, but needs clearly to indicate content and using dosage in its description. In China, melatonin only can as health food, and have clear and definite use to specify: day taking dose be 1��3mg, other compositions except vitamin B6 must not be added, should indicating in points for attention to be engaged in during driving, mechanical work or risky operation person do not want before operation or operate and eat, autoimmune syndromes (rheumatoid etc.) and hyperthyroid patient are cautious use of.Therefore doctor's advice is preferably followed when melatonin need to be taken, with clear and definite using dosage, child uses with greater need for caution.
But in recent years, some lawless persons utilize the feature of the calmness of melatonin, the good action of hypnosis and health-oriented products complicated component, sedative hypnotic health-oriented products add melatonin without authorization, user takes this series products in unwitting situation, easily causing adverse consequences, healthy to the people causes serious harm.
At present, the context of detection of melatonin of whether adulterating in tranquillizing and allaying excitement class Chinese patent medicine and health food mainly has following several method:
1, the quick screening method of physical and chemical reaction
Composition to be measured in sample, after ethyl acetate is extracted, adds dimethylaminocinnamaldehyde ethanol solution hydrochloride in extracting solution, by observing the change of solution colour judges whether contain melatonin chemical composition in sample. If solution colour presented obvious blue to green in 30 seconds, then prompting sample is likely to containing melatonin composition.
The advantage of the method is in that analytical tool that need not be expensive, and testing cost is low, can detect by quick, easy carrying out, less demanding to environment, requires also not high to the specialty of operator, can meet the demand of Site Detection. But the method there is also following shortcoming:
Reagent with to dimethylaminocinnamaldehyde ethanol solution hydrochloride for developer, stablizing of this reagent is poor, and long-time placement is easily caused reagent sensitivity deficiency, needs prepared before use, for relatively complicated Site Detection.
The ethanol solution hydrochloride used in reagent, has certain corrosivity and extractant ethyl acetate, has certain abnormal smells from the patient, have certain hazardness for site operation personnel. And the sensitivity of method is relatively low, the color by sample itself is disturbed relatively big, affects the judgement of experimental result, it is easy to produce erroneous judgement.
2, thin layer chromatography
Composition to be measured in sample is through methanol extraction, and gained need testing solution and reference substance solution are launched through developing solvent on silica gel plate, takes out, dries, put and inspect under uviol lamp 254nm. Under the effect of developing solvent, owing to the migration velocity of different material is different, different material diverse location on chromatographic sheet occurs with the form of speckle, according in position corresponding with reference substance, whether testing sample occurs this experimental phenomena of speckle judges whether contain target component in testing sample. If speckle occur in need testing solution thin-layer chromatogram and melatonin reference substance relevant position, then prompting sample is likely to containing melatonin composition.
The advantage of thin layer chromatography is analytical tool that need not be expensive. This method shortcoming is: chromatographic resolution rate is relatively low, Chinese patent medicine complicated component, and interference factor is many, it is easy to produce erroneous judgement.
Development of chromatogram and to dry required time longer, is insufficient for the requirement of quickly mensuration. Chromatographic sheet needs to dry and dry to deposit, and separating effect is relatively big by such environmental effects, is not suitable for Site Detection. Need fixing place, improper make the Site Detection that mobility is big. The professional technique of testing staff is required higher, is not suitable for basic staff execute-in-place.
3, high performance liquid chromatography
High performance liquid chromatography is conventional Modern Methods, and under identical chromatographic condition, different materials has different chromatographic retentions. Under identical chromatographic condition, according to chromatographic retention, melatonin reference substance solution and sample extraction gained need testing solution sample introduction respectively, can determine whether whether testing sample contains melatonin composition.
The advantage of high performance liquid chromatography is that chromatographic isolation efficiency is high, highly sensitive.But currently also having the disadvantage in that instrument price is expensive, time for sample pretreatment is long, chromatographic column vulnerable to pollution, and analysis cost is high; When the composition that coexists chromatographic peak retention time and melatonin chromatographic retention close to time easily cause erroneous judgement; Instrument is high to the requirement of environment, needs fixed placement, is not suitable for Site Detection.
4, tablets by HPLC-MS
Using high performance liquid chromatograph as separator, mass spectrograph is as the sample analysis that tablets by HPLC-MS is suitable for complicated component, ambient interferences is serious of analyzer, and this analysis method can improve the reliability of testing result. But tablets by HPLC-MS is that a kind of analysis cost is higher than high performance liquid chromatography, operates more complicated analysis method. Instrument is higher to the requirement of environment, it is necessary to fixed placement, improper Site Detection. These inspections at present can only carry out in a few experiments room, not easily promotes the use of.
In sum, the context of detection of the current melatonin that whether adulterates in Chinese patent medicine and health food, although the physical and chemical reaction quick screening method having applicable Site Detection is issued, but still a kind of sensitivity, simple to operate, the method carrying out rapid screening melatonin, for pharmacy and basis medicine inspection unit, the quick screening method of the illegal melatonin that adulterates in exploitation Chinese patent medicine and health food, at supervision scene, sample is quickly analyzed, supervise for Chinese patent medicine and health food and evidence is provided in time, hit the imitation behavior of lawless person and ensure that the drug safety of the people is very important.
Summary of the invention
For above-mentioned deficiency, it is an object of the invention to provide a kind of easy to use, fast and convenient, the quick screening method of the melatonin that adulterates in highly sensitive detection food, health food or medicine and detection card used thereof.
For solving above-mentioned technical problem, previous technical scheme provided by the invention is such that the method for quick of this melatonin, comprises the steps: successively
1) product that need to detect is added organic solvent dissolution, filter after adding slow releasing agent dilution, collect filtrate;
2) filtrate added drop-wise step 1) collected is on melatonin colloidal gold chromatographic detection card, starts timing, after 3��5min, according to the detection T line of colloidal gold strip and the colour developing controlling C line, it is judged that testing sample is positive or negative;
3) step 2) described in colloidal gold test on control C line and detection the equal displaing amaranth of T line, then be judged as feminine gender, i.e. undoped p melatonin chemical composition in testing sample; Control line C displaing amaranth on colloidal gold test, detection line T does not develop the color, then be judged as the positive, namely containing melatonin chemical composition in testing sample; Control line C on colloidal gold test and detection line T does not all develop the color, or detection line T colour developing only occurs, then illustrative experiment is invalid, applies new colloidal gold test and again detects.
The organic solvent described in method for quick step 1) of above-mentioned melatonin is alcohol organic solvent or chloroform.
Further, the method for quick of above-mentioned melatonin, described alcohol organic solvent is ethanol.
Further, the method for quick of above-mentioned melatonin, the slow releasing agent described in step 1) is PBS.
Another one technical method of the present invention, it is to provide a kind of detection card detecting melatonin, form including the sample pad being connected successively in PVC board and PVC board, gold-marking binding pad, coated film and adsorptive pads, it is characterized in that: described gold mark joint sheet is the glass fibre of absorption melatonin monoclonal antibody-colloidal gold composite and rabbit igg-colloidal gold composite, the orthoscopic recessiveness detection line T line printed with the solution of melatonin coupling carrier albumen successively on coated film, the orthoscopic recessiveness nature controlling line C line printed with goat anti-rabbit igg solution, two lines is arranged in parallel.
Further, above-mentioned detection card, described coated film is nitrocellulose filter or cellulose acetate membrane, and the consumption of described rabbit igg is 0.2��0.9 �� g/cm2; The consumption of described melatonin monoclonal antibody-colloidal gold composite is 0.09��0.5 �� g/cm2; The consumption of described melatonin monoclonal antibodies is 0.03��0.1 �� g/mm; The concentration of described goat anti-rabbit igg is 4mg/ml, and the consumption of described goat anti-rabbit igg is 0.03��0.05 �� g/mm.
The preparation method that last technical scheme of the present invention provides this detection card, the sample pad being connected successively in PVC board, gold-marking binding pad, coated film and adsorptive pads;
Wherein:
1) preparation method of coated film is:
With pH7.4,0.01MPBS buffer, 10%��20% sucrose by melatonin antigen diluent to 0.3��1.0mg/ml, being sprayed onto on nitrocellulose membrane, the discharge rate of every 300mm is 30ul, for T line; Goat anti-rabbit igg is diluted to 0.3mg/ml��0.5mg/ml, and the discharge rate of every 300mm is 30ul, and for C line, the spacing of C, T line is 4.5mm, places 12��18 hours in 45 DEG C of baking oven;
2) preparation method of gold-marking binding pad is:
With high purity water by 1% gold chloride diluted concentration be 0.01%, put into magnetic stir bar, it is placed in temperature constant magnetic stirring heating to put and boil, acutely every disposable trisodium citrate being rapidly added 0.7ml of 100ml gold chloride diluent after boiling, continue to boil, keep redness no longer to change to color and stop heating, after being cooled to room temperature, supplement the high purity water of loss, obtain bright, the gold colloidal of free from admixture and floating thing;
Take the colloid gold particle that radius is 15nm��40nm, adjust pH value to 7.0��7.4, under agitation add melatonin monoclonal antibody by 10 �� g antibody/ml gold colloidal, last every 1ml adds 100ul10%BSA and stabilizes it, and at the centrifugal 30min of 12000rpm/s, abandons supernatant, the Tris-HCl buffer with the 0.01MpH8.5 of 1/10th initial colloid gold volumes will be precipitated, 1 �롫5 �� PVP-40,5%BSA, 0.1 �롫0.5 �� NaN3Resuspended;
By the Tris-HCl buffer of melatonin monoclonal antibody-colloidal gold composite and rabbit igg-colloidal gold composite 0.01MpH8.0,1 �롫5 �� PVP-40,5%BSA, 1 �롫5 �� TritonX-100,0.1 �롫0.5 �� NaN3It is diluted to 3%��10%, takes glass fibre, every coating 20ml��30ml, puts baking oven, is incubated 18��24 hours at 37 DEG C; Or PBS, 5%BSA of every to melatonin monoclonal antibody-colloidal gold composite and rabbit igg-colloidal gold composite 100ul 0.01MpH8.0,0.1 �롫0.5 �� NaN3It is diluted to 150��300ul, then adds 20% sucrose, dissolve mixing, be sprayed onto with metal-spraying equipment on processed polyester film or glass fibre;
3) sample pad processed
Taking glass fibre, every coating 20ml��30ml coating solution is PBS, 1%BSA, 1%TWEEN-20,0.1 �롫0.5 �� NaN of 0.01MpH7.43, put baking oven and dry 18��24 hours at 37 DEG C.
Compared with prior art, technical scheme provided by the invention, have the advantage that
1, operating procedure of the present invention is simple, and detection speed is fast, and result is easily observed, it may be achieved the rapid screening to batch samples, is substantially reduced testing cost, reduces workload, has practical significance in Food and drug administration;
2, the present invention is directed to testing sample complicated component problem, sample pretreating method is optimized, required sample size is few, it is only necessary to extracts, dilute two steps and can complete sample pre-treatments.
Accompanying drawing explanation
Fig. 1 is detection examination card structure schematic diagram provided by the invention;
Fig. 2 is detection card result of determination provided by the invention is schematic diagram time negative;
Fig. 3 is detection card result of determination provided by the invention is schematic diagram time positive;
Fig. 4 is detection card result of determination provided by the invention schematic diagram when being invalid.
Detailed description of the invention
Below in conjunction with detailed description of the invention; the claim of invention is described in further detail; but not constituting any limitation of the invention, the amendment of anyone limited number of time made within the scope of the claims in the present invention, still in the claims of the present invention.
Embodiment 1
The method for quick of this melatonin, comprises the steps: successively
1) product that need to detect adding alcohol organic solvent dissolve, preferably alcohol organic solvent is ethanol, then adds filtration after PBS slow releasing agent dilutes, and collects filtrate;
Wherein: different dosage form sample dissolving method is as follows:
Hard capsule: capsule shells of outwarding winding, takes about 1 intragranular tolerant.
Soft capsule: cutting off capsule shells with shears, extrusion about 1 intragranular is tolerant.
Tablet: be crushed to powder, takes about 1 amount.
Pill: take about 1/2 each serving consumption, crushes or shreds.
Organic solvent is added, it is preferred to ethanol, the consumption that makes of organic solvent is advisable with submergence testing sample, and the present invention selects 1��2mL's to make consumption, substantially can cover the needs of different dosage form sample to any one sample of above-mentioned sample.
For ensureing to extract completely, the jolting time should no less than 30 seconds, to adapt to the needs of various dosage form.
Being found through experiments, drop to the filtrate on melatonin colloidal gold strip, if the concentration of organic solvent is more than 50%, then melatonin colloidal gold strip can not normally use, thus the PBS that need to add more than 1mL is diluted. Simultaneously, it is contemplated that extracting solution needs the solution that loss is certain in filter process, therefore the present invention adds PBS and is diluted to cumulative volume being 10mL, has both guaranteed the sensitivity of reagent paper, ensures again have enough extracting solution to carry out application of sample.
Liquid dosage form: without pre-treatment, directly detects.
2) filtrate added drop-wise step 1) collected is on melatonin colloidal gold chromatographic detection card, starts timing, after 3��5min, according to the detection T line of colloidal gold strip and the colour developing controlling C line, it is judged that testing sample is positive or negative;
3) step 2) described in colloidal gold test on control C line and detection the equal displaing amaranth of T line, then be judged as feminine gender, i.e. undoped p melatonin chemical composition in testing sample; Control line C displaing amaranth on colloidal gold test, detection line T does not develop the color, then be judged as the positive, namely containing melatonin chemical composition in testing sample; Control line C on colloidal gold test and detection line T does not all develop the color, or detection line T colour developing only occurs, then illustrative experiment is invalid, applies new colloidal gold test and again detects.
The detection card used during detection, form including the sample pad 1 being connected successively in PVC board and PVC board 5, gold-marking binding pad 2, coated film 3 and adsorptive pads 4, consult Fig. 1, sample pad 1 is pressed in about the 1/2��1/3 of gold-marking binding pad 2, gold-marking binding pad 2 is pressed in 0.1��0.2cm, T linear distance coated film 3 lower end 6mm, the C linear distance coated film 3 upper end 6mm of coated film 3, C, T spacing is 4.5mm, and adsorptive pads is pressed in coated film 0.1��0.2cm place.
Wherein: gold-marking binding pad is the glass fibre of absorption melatonin monoclonal antibody-colloidal gold composite and rabbit igg-colloidal gold composite, the orthoscopic recessiveness detection line T line printed with the solution of melatonin coupling carrier albumen on coated film successively, the orthoscopic recessiveness nature controlling line C line printed with goat anti-rabbit igg solution, two lines is arranged in parallel.
Described coated film is nitrocellulose filter or cellulose acetate membrane, and the consumption of described monoclonal-rabbit igg is 0.2��0.9 �� g/cm2, it is preferable that 0.25��0.45 �� g/cm2, more excellent is chosen as 0.3 �� g/cm2, the consumption of described melatonin monoclonal antibody-colloidal gold composite is 0.09��0.5 �� g/cm2, it is preferable that 0.15 �� g/mm, the concentration of described goat anti-rabbit igg is 4mg/ml, the consumption of described goat anti-rabbit igg is 0.03��0.05 �� g/mm, preferably 0.04 �� g/mm, the consumption of described melatonin monoclonal antibodies is 0.03��0.1 �� g/mm, it is preferable that 0.05 �� g/mm.
The preparation method of this detection card is the sample pad, gold-marking binding pad, coated film and the adsorptive pads that are connected successively in PVC board;
Wherein:
1) preparation method of coated film is:
With pH7.4,0.01MPBS buffer, 10%��20% sucrose by melatonin antigen diluent to 0.3��1.0mg/ml, being sprayed onto on nitrocellulose membrane, the discharge rate of every 300mm is 30ul, for T line; Goat-anti rabbit is diluted to 0.3mg/ml��0.5mg/ml, and the discharge rate of every 300mm is 30ul, and running speed is 80 mm/second, pump pressure 100Pa pressure, and movable length 290cm is C line, and the spacing of C, T line is 4.5mm, puts 45 DEG C of oven overnight 12��18 hours;
2) preparation method of gold-marking binding pad is:
With high purity water by 1% gold chloride be diluted to 0.01%, put into magnetic stir bar, it is placed in temperature constant magnetic stirring heating to put and boil, acutely every disposable trisodium citrate being rapidly added 0.7ml of 100ml after boiling, continues to boil, and keeps redness no longer to change to color and stops heating, the moisture content of loss is supplemented after being cooled to room temperature, obtain bright, the gold colloidal of free from admixture and floating thing, can preserve one month and not change.
Take the colloid gold particle that radius is 15nm��40nm, adjust pH value to 7.0��7.4, under agitation add melatonin monoclonal antibody by 10 �� g antibody/ml gold colloidal, the BSA that last every 1ml adds 100ul10% stabilizes it, and at the centrifugal 30min of 12000rpm/s, abandons supernatant, by the precipitation Tris-HCl buffer with the 0.01MpH8.5 of the 100ul of 1/10th initial colloid gold volumes, 1 �롫5 �� PVP-40,5%BSA, 0.1 �롫0.5 �� NaN3Resuspended, put 4 DEG C and can put about 2 months;
The Tris-HCl buffer of melatonin monoclonal antibody-colloidal gold composite and rabbit igg-colloidal gold composite 0.01MpH8.0,1 �롫5 �� PVP-40,5%BSA, 1 �롫5 �� TritonX-100,0.1 �롫0.5 �� NaN3It is diluted to 3%��10%, takes the glass fibre that area is 25*30cm, every coating 20ml��30ml, puts baking oven, places 18��24 hours at 37 DEG C; Or PBS, 5%BSA of every to melatonin monoclonal antibody-colloidal gold composite and rabbit igg-colloidal gold composite 100ul 0.01MpH8.0,0.1 �롫0.5 �� NaN3It is diluted to 150��300ul, then adds 20% sucrose, dissolve mixing, be sprayed onto with metal-spraying equipment on processed polyester film or glass fibre; Equipment pressure 0.015��0.02MPa used, metal spraying parameter is 0.05��0.2ul/mm, running speed is 80 mm/second, it is sprayed onto on processed polyester film or glass fibre, polyester film or glass fibre processing method be: it is 3 �롫5 �� Tris that polyester film or glass fibre are placed on treatment fluid, 5%BSA, 0.1 �롫0.5 �� NaN3Middle immersion 2 hours, liquid should not have polyester film or glass fibre completely, puts 37 DEG C of baking ovens 12��18 hours.
3) sample pad is prepared
Taking 25*30cm glass fibre, every coating 20ml��30ml coating solution is PBS, 1%BSA, 1%TWEEN-20,0.1 ��-0.5 �� NaN of 0.01MpH7.43, put 37 degree of baking oven 18��24 hours.
Detection example 1
Analysis of functional food sold brand board U.S.'s dormancy melatonin capsule (capsule, indicating dose is each 2, identifies " containing melatonin ", and functional component and content show: every 100g is containing melatonin 282mg)
Take this product capsule 1, capsule shells of outwarding winding, take that about 1 intragranular is tolerant to be placed in sample cell, add 1��2mL chloroform and dissolve, after jolting is about 1min energetically, then adds PBS slow releasing agent and dilute as liquid to be measured. Draw 3 liquid to be measured with disposable little suction pipe and vertically drip in the well of melatonin colloidal gold chromatographic detection card, start timing, after 3min, result of determination is, consult Fig. 3, control line C displaing amaranth on colloidal gold test, detection line T does not develop the color, then judge containing melatonin chemical composition in testing sample, points out result consistent with functional component and content.
Adopting efficient liquid phase liquid chromatography technology that match beauty board U.S. dormancy melatonin capsule of the present embodiment is verified detection, testing result this product every is containing melatonin 1.1mg, consistent with the detection method result of the present invention.
Detection example 2
Certain brand health food A(capsule commercially available, indicating dose is each 1��4, does not identify " containing melatonin ")
Take this product capsule 1, capsule shells of outwarding winding, take that about 1 intragranular is tolerant to be placed in sample cell, add 1��2mL ethanol, after jolting is about 1min energetically, then adds PBS slow releasing agent and dilute as liquid to be measured. Draw 3 liquid to be measured with disposable little suction pipe and vertically drip in the well of melatonin colloidal gold chromatographic detection card, start timing, result of determination after 3min, consult Fig. 3: the control line C displaing amaranth on colloidal gold test, detection line T does not develop the color, then judge to add melatonin chemical composition containing illegal in testing sample.
Adopting efficient liquid phase liquid chromatography technology that the analysis of functional food sold A of the present embodiment is verified detection, testing result this product every is containing melatonin 0.9mg, consistent with the detection method result of the present invention.
Detection example 3
Certain brand health food ANSHEN BUNAO YE commercially available (oral liquid, indicating dose is each 1, does not identify " containing melatonin ")
Draw 3 oral liquids with disposable little suction pipe and vertically drip in the well of melatonin colloidal gold chromatographic detection card, start timing, result of determination after 3min, consult Fig. 2: the control line C on colloidal gold test, the detection equal displaing amaranth of line T, then judge testing sample does not have melatonin chemical composition. If there is situation described in Fig. 4, control line C and detection line T on colloidal gold test all do not develop the color, or detection line T colour developing only occur, then illustrate that detection is invalid.
Adopting efficient liquid phase liquid chromatography technology that this brand health food ANSHEN BUNAO YE is verified detection, testing result is not for detect melatonin composition, consistent with the detection method result of the present invention.
Claims (5)
1. the detection card of a melatonin, it is characterized in that, including the sample pad being connected successively in PVC board and PVC board, gold-marking binding pad, coated film and adsorptive pads, it is characterized in that: described gold-marking binding pad is the glass fibre of absorption melatonin monoclonal antibody-colloidal gold composite and rabbit igg-colloidal gold composite, the orthoscopic recessiveness detection line T line printed with the solution of melatonin coupling carrier albumen on coated film successively, the orthoscopic recessiveness nature controlling line C line printed with goat anti-rabbit igg solution, two lines is arranged in parallel;
Wherein:
The consumption of described rabbit igg-colloidal gold composite is 0.2��0.9 �� g/cm2; The consumption of described melatonin monoclonal antibody-colloidal gold composite is 0.09��0.5 �� g/cm2; The consumption of described melatonin coupling carrier albumen is 0.03��0.1 �� g/mm; The concentration of described goat anti-rabbit igg is 0.3-0.5mg/ml, and the consumption of described goat anti-rabbit igg is 0.03��0.05 �� g/mm;
The detection card of described melatonin is prepared successively by following step: sample pad, gold-marking binding pad, coated film and the adsorptive pads being connected successively in PVC board;
1) preparation method of coated film is:
With pH7.4, melatonin coupling carrier albumen is diluted to 0.3��1.0mg/ml by the 0.01MPBS buffer containing 10%��20% sucrose, is sprayed onto on nitrocellulose membrane, and the discharge rate of every 300mm is 30 �� l, for T line; Goat anti-rabbit igg is diluted to 0.3mg/ml��0.5mg/ml, and the discharge rate of every 300mm is 30 �� l, and for C line, the spacing of C, T line is 4.5mm, places 12��18 hours in 45 DEG C of baking oven;
2) preparation method of gold-marking binding pad is:
With high purity water, 1% gold chloride being diluted to concentration is 0.01%, put into magnetic stir bar, it is placed in temperature constant magnetic stirring heating to put and boil, acutely every disposable trisodium citrate being rapidly added 0.7ml of 100ml gold chloride diluent after boiling, continue to boil, keep redness no longer to change to color and stop heating, after being cooled to room temperature, supplement the high purity water of loss, obtain bright, the gold colloidal of free from admixture and floating thing;
Take the colloid gold particle that radius is 15nm��40nm, adjust pH value to 7.0��7.4, melatonin monoclonal antibody is added under agitation by 10 �� g antibody/ml gold colloidal, last every 1ml adds 100 �� l10%BSA and stabilizes it, at the centrifugal 30min of 12000rpm/s, abandon supernatant, by precipitation with 1/10th initial colloid gold volumes containing 1 �롫5 �� PVP-40,5%BSA, 0.1 �롫0.5 �� NaN3The Tris-HCl buffer of 0.01MpH8.5 is resuspended, by melatonin monoclonal antibody-colloidal gold composite and rabbit igg-colloidal gold composite pH8.0, containing 1 �롫5 �� PVP-40,5%BSA, 1 �롫5 �� TritonX-100,0.1 �롫0.5 �� NaN30.01MTris-HCl buffer be diluted to 3%��10%, take glass fibre, every coating 20ml��30ml, put baking oven, 37 DEG C be incubated 18��24 hours; Or every to melatonin monoclonal antibody-colloidal gold composite and rabbit igg-colloidal gold composite 100 �� l 0.01M, pH8.0 containing 5%BSA, 0.1 �롫0.5 �� NaN3PBS be diluted to 150��300 �� l, then add 20% sucrose, dissolve mixing, be sprayed onto on processed glass fibre with metal-spraying equipment;
3) sample pad processed
Taking glass fibre, every coating 20ml��30ml, coating solution is pH7.4, containing 1%BSA, 1%TWEEN-20,0.1 �롫0.5 �� NaN3The PBS of 0.01M, put baking oven and dry 18��24 hours at 37 DEG C.
2. the method for quick of the melatonin detection card described in claim 1, it is characterised in that comprise the steps: successively
1) product that need to detect is added organic solvent dissolution, filter after adding slow releasing agent dilution, collect filtrate;
2) by step 1) filtrate added drop-wise collected on melatonin colloidal gold chromatographic detection card, start timing, after 3��5min, according to the detection T line of colloidal gold strip and the colour developing controlling C line, it is judged that testing sample is positive or negative;
3) step 2) described in colloidal gold strip on control C line and detection the equal displaing amaranth of T line, then be judged as feminine gender, i.e. undoped p melatonin chemical composition in testing sample;Control line C displaing amaranth on colloidal gold test, detection line T does not develop the color, then be judged as the positive, namely doped with melatonin chemical composition in testing sample; Control line C on colloidal gold test and detection line T does not all develop the color, or detection line T colour developing only occurs, then illustrative experiment is invalid, applies new colloidal gold test and again detects.
3. melatonin according to claim 2 detection card method for quick, it is characterised in that step 1) described in organic solvent be alcohols or chloroform.
4. the method for quick of melatonin according to claim 3 detection card, it is characterised in that described alcohols is ethanol.
5. melatonin according to claim 2 detection card method for quick, it is characterised in that step 1) described in slow releasing agent be PBS.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310698818.0A CN103969454B (en) | 2013-12-18 | 2013-12-18 | Melatonin method for quick and detection thereof block and preparation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310698818.0A CN103969454B (en) | 2013-12-18 | 2013-12-18 | Melatonin method for quick and detection thereof block and preparation method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103969454A CN103969454A (en) | 2014-08-06 |
| CN103969454B true CN103969454B (en) | 2016-06-08 |
Family
ID=51239203
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310698818.0A Expired - Fee Related CN103969454B (en) | 2013-12-18 | 2013-12-18 | Melatonin method for quick and detection thereof block and preparation method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103969454B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104237230B (en) * | 2014-10-14 | 2017-12-26 | 厦门奥亚仪器有限公司 | The quick determination method of epiphysin and its operation vessel combination |
| CN106370847A (en) * | 2016-08-22 | 2017-02-01 | 广州达元食品安全技术有限公司 | Rapid detection kit of lemon yellow in food |
| CN106248928A (en) * | 2016-08-22 | 2016-12-21 | 广州达元食品安全技术有限公司 | A kind of hydrochlorothiazide rapid detection card and detection method and preparation method thereof thereof |
| CN107290517A (en) * | 2017-06-28 | 2017-10-24 | 南通大学 | A kind of measuring method of human hair melatonin content |
| CN110632240B (en) * | 2019-11-08 | 2021-11-02 | 亳州职业技术学院 | Identification kit and identification method for traditional Chinese medicine ground beetles |
| CN112225795A (en) * | 2020-10-14 | 2021-01-15 | 湖南苏阳医疗科技有限公司 | 6-hydroxy sulfate melatonin derivative and preparation method and application of immunogen and specific antibody thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101718787A (en) * | 2009-11-20 | 2010-06-02 | 广东省惠州市药品检验所 | Method for quickly detecting doping of benzodiazepine compound in food, medicine or health-care product |
| CN103278499A (en) * | 2013-05-28 | 2013-09-04 | 无锡中德伯尔生物技术有限公司 | Method for testing melatonin |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TWI401063B (en) * | 2011-05-25 | 2013-07-11 | Nat Univ Tsing Hua | Melatonin suppression extent measuring device |
-
2013
- 2013-12-18 CN CN201310698818.0A patent/CN103969454B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101718787A (en) * | 2009-11-20 | 2010-06-02 | 广东省惠州市药品检验所 | Method for quickly detecting doping of benzodiazepine compound in food, medicine or health-care product |
| CN103278499A (en) * | 2013-05-28 | 2013-09-04 | 无锡中德伯尔生物技术有限公司 | Method for testing melatonin |
Non-Patent Citations (2)
| Title |
|---|
| 水样中左旋18-甲基炔诺酮的胶体金免疫层析试剂条的研制;王俊卿，等;《化学研究与应用》;20110531;第23卷(第5期);第623页左栏第10-15行，第623页左栏第28行-右栏第26行，图1和2 * |
| 褪黑素单克隆抗体的制备、鉴定及初步应用;王建和;《中国优秀博硕士学位论文全文数据库（硕士） 基础科学辑》;20030915(第3期);第3页最后1行，第12页第1行-第21页第15行，第37页第6行-第39页第17行 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103969454A (en) | 2014-08-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103969454B (en) | Melatonin method for quick and detection thereof block and preparation method | |
| WO2018174733A1 (en) | Method for determining bisphenol a in biological material, diagnostic device for detection of bisphenol a in biological material, diagnostic kit for detection of bisphenol a in biological material | |
| US9005986B2 (en) | Method for rapidly measuring melatonin adulteration of Chinese patent medicine or healthcare food | |
| CN102879495B (en) | Antidotal capsule of Tibetan medicine compound and quality detection method of preparation of antidotal capsule | |
| CN106124759B (en) | A kind of silaenafil colloidal gold test | |
| CN102078403A (en) | Detection method of Chinese traditional medicine preparation of oral liquid for treating infant cough with lung heat | |
| CN106153954A (en) | A kind of method for quick of phenytoin Sodium | |
| CN105136966B (en) | A kind of quality determining method of Liang Fu Wan class preparation | |
| CN101408519B (en) | Method for detecting sibutramine hydrochloride and application thereof | |
| CN104237230B (en) | The quick determination method of epiphysin and its operation vessel combination | |
| CN109239063A (en) | A kind of rapid detection method of melatonin in health products | |
| CN109596768B (en) | Detection method of infantile indigestion removing granules | |
| CN101718787A (en) | Method for quickly detecting doping of benzodiazepine compound in food, medicine or health-care product | |
| CN101865855B (en) | Fast detection method of nitrazepam doped in medicine and health care food | |
| CN104777244B (en) | A kind of quality determining method of Xingqi Tannikaer capsule | |
| CN106645748A (en) | Mammalian differential serum protein and differentially expressed protein system of penile smooth muscle tissue of abnormal and phlegmatic type of impotence syndrome and screening method and application | |
| AU2014257328B2 (en) | Drug concentration from an oral fluid | |
| CN103018361B (en) | Method for detecting related substance in naftopidil dispersible tablet | |
| CN103278498A (en) | Detecting method and kit of sibutramine hydrochloride | |
| CN205333654U (en) | Early detection test strip of kidney damage based on biomarker | |
| CN205982292U (en) | Phenytoin sodium rapid detection cards | |
| CN204101565U (en) | A kind of colloidal gold test paper card simultaneously detecting lincomycin and nitroimidazoles medicine | |
| CN106370846A (en) | Detection method for rapidly detecting illegally-added tadalafil in health product | |
| CN104483445B (en) | A kind of thiazolidinedione quick detection kit and detection method thereof | |
| CN107748220B (en) | Sichuan blackberry lily flavone capsule and detection method of Sichuan blackberry lily flavone extract |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20160608 Termination date: 20211218 |