CN105954513A - {0><}0{>Preparation and application of Flow-through film carrier qunatum dot detection morphine test paper - Google Patents
{0><}0{>Preparation and application of Flow-through film carrier qunatum dot detection morphine test paper Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- 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
- G01N33/531—Production of immunochemical test materials
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Abstract
{0><}0{>The invention discloses preparation and application of Flow-through film carrier qunatum dot detection morphine test paper and belongs to a film carrier qunatum dot fluorescence immunoblotting technology. <0}{0><}0{>The method comprises the following steps of: coating the surface of a nitro cellulose film serving as a solid phase carrier with morphine antigens by use of the imprinting effect of the nitro cellulose film; sealing non-specific sites with bovine serum albumin; coupling and marking a morphine specific antibody and carboxyl-containing water-soluble Cdte@ZnS qunatum dots under the effect of 1-(3-dimethylin propyl)-3-ethyl carbodiimide/N-hydroxy succinimide, thereby preparing a morphine fluorescent antibody marked with the qunatum dots; and achieving a detection effect by use of specific combination of the antibody and the antigen and the imprinting absorption effect of a Flow-through film carrier. <0}{0><}0{>The preparation process is simple; the prepared product is good in physical and chemical properties, and the method can be used for preparing rapid detection test paper which is good in physical and chemical properties and relatively high in detection sensitivity; by virtue of change of fluorescence intensity on the Flow-through film, the content of morphine in a sample is qualitatively detected. <0}{0><}0{>Compared with an ELISA rapid detection method, the method provided by the invention is relatively low in manufacturing cost, simple, convenient and rapid in detection method, and obvious and visual in detection effect.
Description
Technical field
The invention belongs to membrane carrier rapid detection technical field, particularly to one based on Flow-through membrane carrier quantum dot mark
Note immunoblot assay realizes method and the application thereof of quickly detection morphine.
Background technology
Some illegal businessmans add the violated raw materials of non-food stuff such as Pericarpium Papaveris in food processing, comprise morphine, small-mouthed jar in these raw materials
The alkaloid components such as pavine, codeine, thebaine, narcotine, wherein morphine in being Pericarpium Papaveris the most representational feature biological
Alkali, the easy addiction of human body Long Term Contact, also nerve system of human body can be caused damage.At present, there is no a kind of quick, sensitive,
Effectively illegal in detection flavoring agent adding Pericarpium Papaveris and the standard method of hydrolysate thereof, common method mainly has spectrophotography, thin
The analysis means such as layer chromatography and gas phase, liquid chromatograph and mass spectrometry.Spectrophotography and thin layer chromatography are capable of simply
Detection, but sensitvity constraint, have difficulties for the detection of trace banned substance in flavoring agent.Instrumental Analysis detection sensitivity is high,
Its bottleneck is instrument and equipment and the professional operator needing complex and expensive, sample pre-treatments loaded down with trivial details time-consuming, testing cost is high, difficult
To meet high flux, the needs of quickly detection.In recent years, the immuno analytical method combined based on antigen and antibody specific is fast because of it
Speed is sensitive and is widely used in food safety detection.
Inhale with enzyme linked immunological using fluorochrome labeled antibodies or antigen as a kind of new immuno analytical method of tracer, its principle
Attached dose of mensuration (ELISA) is similar, it is not necessary to add substrate colour developing, utilizes detector watch fluorescence phenomenon or measure fluorescence intensity, thus
Judge that antigen or the existence of antibody, location and distribution situation or detection are by antigen or the content of antibody in inspection specimen.Quantum dot
(quantumn dots, QDs) is a kind of semiconductor nanocrystal, and its spectrochemical property is more stable than organic dyestuff, and scattering is few,
Being not susceptible to fluorescent bleach, fluorescence duration time is long.Through the water-soluble quantum dot of chemical modification, there is good biocompatibility,
Biomarker and detection can be directly used in after coupling effective with biomolecule, there is preferable safety.Utilize functionalization water solublity
Itself and morphine biological antibody are carried out coupling labelling by fluorescence quantum CdTe@ZnS, are prepared as the fluorescence identifying element of a kind of morphine,
Establish Flow-through membrane carrier immunoblotting assay method with nitrocellulose filter for solid phase carrier and realize quick to morphine
Highly sensitive detection.
Summary of the invention
It is an object of the invention to fill up currently not yet have based on the immunity of quantum dot-labeled specific antibody Flow-through membrane carrier
Trace quickly detects the blank of morphine test strips.There is provided a kind of with nitrocellulose filter as solid phase carrier, by morphine specific antibody
Under EDC/NHS effect, carry out coupling labelling with band carboxyl water-soluble CdTe@ZnS quantum dot and prepare morphine fluorescent antibody, build
Found the method for quick of a kind of morphine, and this test strips preparation process is simple, good physical and chemical properties, detection sensitivity are higher.
For solving above-mentioned technical problem, the technical solution used in the present invention is:
Employing nitrocellulose filter (Millipore) is as solid phase carrier, by being coated at its pan coating a certain amount of morphine specificity
Antigen, with nonspecific binding site on BSA closing membrane carrier, utilizes the biocompatibility of water-solubility function quantum dot and glimmering
Photosensitiveness stability, by quantum dot-labeled morphine specific antibody, utilizes fluorescent antibody to react at membrane carrier with envelope antigen and morphine
Competitive binding on region, uses flow-through mode to react on caudacoria morphine content in the judgment sample of fluorescence power.
The preparation method of the Flow-through membrane carrier quantum dot fluorescence immunoblotting Rapid detection test strip of morphine, according to following
Step is carried out:
(1) pretreatment of Flow-through film:
Cellulose nitrate film pencil drawing a diameter of 5mm circular hole, soaks 15min by phosphate buffered saline(PBS) (PBS), takes out
Rinsing with distilled water, test strips is close to distilled water and drenches the filter paper of tiling, every hole uniformly drips 20 μ L morphine specific antigens,
37 DEG C of incubation 30min, with non-specific sites on bovine serum albumin (BSA) blocking antigen, 37 DEG C of incubation 30min, with containing
The phosphate buffer (PBST) of tween washs three times, and 37 DEG C are dried, 4 DEG C of kept dry.
(2) preparation of carboxyl water-soluble CdTe@ZnS quantum dot labelling morphine specific antibody:
Carboxylated CdTe@ZnS borate buffer is diluted to 1 μM in round-bottomed flask, magnetic agitation.Add 43 μ L concentration
For the morphine specific antibody of 3.73mg/mL, mix homogeneously (mixing speed to be difficult to too fast, not produce bubble be as the criterion).Stirring
After 5min, turn down mixing speed, add 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide (EDC) solution of 13 μ L, instead
15 μ L N-hydroxy-succinamide (NHS), room temperature reaction 2h is added after answering 20min.Reaction terminates, and uses refrigerated centrifuger
12000rpm is centrifuged 3min except reuniting.
(3) purification of carboxyl water-soluble CdTe@ZnS quantum dot labelling morphine specific antibody:
With super filter tube by Sample Purification on Single 5 times, end-product redissolves in PBS.After end-product takes out, 12000rpm is centrifuged
3min except reuniting, measure be stored in after concentration 4 DEG C standby.
In step (1), described PBS is 10mM, pH 7.4；Described PBS preparation morphine specific antigen concentration is
0.05 μ g/ μ L, the concentration of described BSA is 1-1.5% (W/V).
In step (2), described EDC concentration is with borate buffer solution the configuration 10mg/mL, NHS of 10mM, pH=7.4
Concentration is for configuring 10mg/mL with 10mM, pH=7.4 borate-buffered saline.
In step (2), the temperature of described coupling reaction is 25 DEG C, and the time is 2h.
In step (3), described each purification of liquid and borate-buffered saline volume ratio are not less than 10.
Morphine Flow-through membrane carrier quantum dot fluorescence immunoblotting quick detection test paper prepared by preparation method of the present invention
Bar.
Morphine Flow-through membrane carrier quantum dot fluorescence immune-blotting method test strips of the present invention is for quickly detecting fire
The feature alkaloid morphine of illegal additive Pericarpium Papaveris in flavoring food, is substantially dissolved in water addition stone by testing sample bottom material of chafing dish
After oil ether extracts, after taking out aqueous layer and the mixing of CdTe@ZnS quantum dot labelling morphine specific antibody, liquid is uniform
Dropping is in test strip.
Described sample and CdTe@ZnS quantum dot labelling morphine specific antibody fully react 30min.
It is blank reference by the test strip without morphine simultaneously.
The invention has the beneficial effects as follows:
(1) detection method is easy quickly, and testing result is substantially directly perceived, and detection sensitivity is higher, is not required to by complex instrument, is suitable for
Identify in on-the-spot rapid screening.
(2) preparation process is simple, with low cost.
(3) less pollution that preparation process produces.
Accompanying drawing explanation
Fig. 1 be Flow-through membrane carrier immune-blotting method morphine test strips prepare overhaul flow chart.
Fig. 2 is the detection schematic diagram of Flow-through membrane carrier immune-blotting method morphine test strips, with the increase of morphine concentration,
Fluorescence gradually weakens, and 1 is without morphine, and 2 is 0.01mg kg-1Morphine, 3 is 0.1mg kg-1Morphine, 4 is 1mg kg-1
Morphine.
Fig. 3 is that the non-coupling labelling quantum dot CdTe ZnS of Flow-through membrane carrier immune-blotting method morphine test strips inhales
Attached diagram, it can be seen that free CdTe@ZnS quantum dot is inhaled almost without electrostatic on Flow-through film
Attached, and with the increase of morphine concentration, fluorescence is unchanged, 1 is without morphine, and 2 is 0.01mg kg-1Morphine, 3 is 0.1mg
kg-1Morphine, 4 is 1mg kg-1Morphine.
Fig. 4 is that Flow-through membrane carrier immune-blotting method morphine ELISA test strip chaffy dish sample interpolation Pericarpium Papaveris boils difference
Time detecting schematic diagram, 1 is without morphine, and 2 for boiling 10min, and 3 for boiling 20min.
Fig. 5 is that Flow-through membrane carrier immune-blotting method morphine ELISA test strip chaffy dish sample interpolation Pericarpium Papaveris boils difference
Time detecting schematic diagram, 1 is without morphine, and 2 for boiling 0.5h, and 3 for boiling 1h, and 4 for boiling 2h.
Fig. 6 is the response rate of Flow-through membrane carrier immune-blotting method morphine ELISA test strip sample Different adding amount morphine
Detection schematic diagram, 1 is without morphine, and 2 is 0.01mg kg-1Morphine, 3 is 0.1mg kg-1Morphine, 4 is 1mg kg-1?
Coffee.
Detailed description of the invention
The present invention is to be combined with immunoassay technology by Flow-through film engram technology, prepares fluorescence quantum point mark morphine
Specific antibody, prepares immune-blotting method test strips on solid phase carrier nitrocellulose filter, comes below in conjunction with specific embodiment
The present invention is described, but protection scope of the present invention is not limited to this.
Fig. 1 be Flow-through membrane carrier immune-blotting method morphine test strips prepare overhaul flow chart.
Fig. 2 is the detection schematic diagram of Flow-through membrane carrier immune-blotting method morphine test strips, with the increase of morphine concentration,
Fluorescence gradually weakens, and 1 is without morphine, and 2 is 0.01mg kg-1Morphine, 3 is 0.1mg kg-1Morphine, 4 is 1mg kg-1
Morphine.
Fig. 3 is that the non-coupling labelling quantum dot CdTe ZnS of Flow-through membrane carrier immune-blotting method morphine test strips inhales
Attached diagram, it can be seen that free CdTe@ZnS quantum dot is inhaled almost without electrostatic on Flow-through film
Attached, and with the increase of morphine concentration, fluorescence is unchanged, 1 is without morphine, and 2 is 0.01mg kg-1Morphine, 3 is 0.1mg
kg-1Morphine, 4 is 1mg kg-1Morphine.
Fig. 4 is that Flow-through membrane carrier immune-blotting method morphine ELISA test strip chaffy dish sample interpolation Pericarpium Papaveris boils difference
Time detecting schematic diagram, 1 is without morphine, and 2 for boiling 10min, and 3 for boiling 20min.
Fig. 5 is that Flow-through membrane carrier immune-blotting method morphine ELISA test strip chaffy dish sample interpolation Pericarpium Papaveris boils difference
Time detecting schematic diagram, 1 is without morphine, and 2 for boiling 0.5h, and 3 for boiling 1h, and 4 for boiling 2h.
Fig. 6 is the response rate of Flow-through membrane carrier immune-blotting method morphine ELISA test strip sample Different adding amount morphine
Detection schematic diagram, 1 is without morphine, and 2 is 0.01mg kg-1Morphine, 3 is 0.1mg kg-1Morphine, 4 is 1mg kg-1?
Coffee.
Embodiment 1
The preparation of membrane carrier immunoblotting Flow-through morphine test strips:
The pretreatment of A.Flow-through film: cellulose nitrate film (Millipore) pencil drawing a diameter of 5mm circular hole, uses
PBS (10mM, pH 7.4) soaks 15min, and taking-up distilled water rinses, and test strips is close to distilled water and drenches the filter paper of tiling,
Every hole uniformly drips 20 μ L morphine specific antigens, 37 DEG C of incubation 30min, with non-specific sites on BSA blocking antigen,
37 DEG C of incubation 30min, wash three times with PBST, and 37 DEG C are dried, 4 DEG C of kept dry；
B. the preparation of carboxyl water-soluble CdTe@ZnS quantum dot labelling morphine specific antibody: by carboxylated CdTe@ZnS boron
Acid buffer (10mM, pH7.4) is diluted to 1 μM in round-bottomed flask, magnetic agitation.Add 43 μ L morphine specific antibodies
(3.73mg/mL), mix homogeneously (mixing speed to be difficult to too fast, not produce bubble be as the criterion).After stirring 5min, turn down stirring speed
Degree, adds the EDC solution of 13 μ L, adds 15 μ LNHS, room temperature reaction 2h after reaction 20min.Reaction terminates, with cold
Freeze centrifuge 12000rpm and be centrifuged 3min except reuniting.
C. the purification of carboxyl water-soluble CdTe@ZnS quantum dot labelling morphine specific antibody: with super filter tube by Sample Purification on Single 5 times,
End-product redissolves in PBS.After end-product takes out, 12000rpm is centrifuged 3min except reuniting, and is stored in after measuring concentration
4 DEG C standby.
In embodiment 1, the detecting step of immunoblotting test strips application actual sample is as follows:
Selecting the bottom material of chafing dish without morphine is sample, by adding a certain amount of Pericarpium Papaveris heated and boiled different time, with preparing
Morphine immunoblotting ELISA test strip at the Detection results of the feature alkaloid-morphine added in Pericarpium Papaveris sample.Weigh solid-like
Product bottom material of chafing dish 5.00g, Pericarpium Papaveris 1g, add 50mL warm water soaking and boil 10min, 20min, 0.5h, 1h, 2h,
Filtration washing filtering residue, distilled water is settled to 50mL.Accurately pipette 25mL filtrate in separatory funnel, add 25mL petroleum ether
Shaking oil removing fat, stratification, release lower floor and be liquid to be measured.Take 30 μ L CdTe@ZnS quantum dot traget antibody and 30 μ L
The sample boiling different time liquid to be measured mixing, react 30min, uniformly drip 30 μ L in the every hole of test strip, after washing
Fluorescence is observed under 365nm ultraviolet.
The test strips of embodiment 1 is easy and simple to handle, and testing result is the most easily differentiated.As shown in Figure 4, Figure 5, morphine immunoblotting
The negative chaffy dish sample (without Pericarpium Papaveris) of test strips and negative chaffy dish sample add Pericarpium Papaveris and boil 10min, 20min contrast fluorescence
Strength Changes is less obvious, compares fluorescence intensity change substantially to boiling 30min, 60min, 120min, and along with when boiling
Between increase Test paper fluorescence die down, illustrate that the stripping quantity of morphine increases along with the increase of boiling time.In the present embodiment together
Time have employed the mensuration demonstrating the feature alkaloid morphine contained in Pericarpium Papaveris with morphine biological antibody ELISA method, such as table 1
Shown in, the testing result of two kinds of methods has preferable concordance.
Table 1 bottom material of chafing dish adds Pericarpium Papaveris and boils the testing result (in terms of morphine) of different time
Note: to be not added with the negative bottom material of chafing dish sample of Pericarpium Papaveris (without morphine) as reference, (+) " positive ", speckle fluorescence ratio
With reference to weak, represent containing Pericarpium Papaveris feature alkaloid morphine；(-) " negative ", speckle fluorescence compares reference with living with reference to indistinction
Color is deep, represents without Pericarpium Papaveris feature alkaloid morphine, (±) " male/female ", speckle fluorescence is little with reference to difference.
Embodiment 2
In embodiment 2, the detecting step of morphine immunoblotting test strips application actual sample is as follows:
Selecting negative bottom material of chafing dish is actually detected sample, by adding the morphine of variable concentrations, with the morphine immunoblotting prepared
ELISA test strip effect in actual sample.
Sample adds recovery experiment 2: weigh three parts of 2g bottom material of chafing dish blank samples, adds 10mL water, add 0.25,0.5,
1mg kg-1Morphine, after heating for dissolving, filtration washing filtering residue, distilled water is settled to 10mL, adds the shaking of 10mL petroleum ether
Oil removing fat, releases lower floor and is liquid to be measured.Take 30 μ L CdTe@ZnS quantum dot traget antibodies and 30 μ L boil 0.5h, 1h,
2h liquid to be measured mixes, and reacts 30min, uniformly drips 30 μ L in the every hole of test strip, sees after washing under 365nm ultraviolet
Examine fluorescence.
The test strips using embodiment 2 is easy and simple to handle, and testing result is the most easily differentiated.As shown in Figure 6, morphine immunoblotting
Test strips is obvious to morphine variable concentrations addition fluorescence intensity change compared with the bottom material of chafing dish blank sample without adding, and along with
The increase fluorescence of addition weakens.The present embodiment have employed simultaneously with the tradition morphine biological antibody ELISA as recognition component
Method validation adds the mensuration of sample recovery rate, and as shown in table 2, testing result has preferable concordance.Result shows system
Standby morphine immunoblotting quickly detects Flow-through film test strips and can effectively realize the qualitative detection of morphine in sample.
Table 2 chaffy dish sample directly adds the recovery testing result of morphine
Illustrate: experimental data represents (mean ± SD with mean+SD；N=3), Flow-through membrane carrier ELISA test strip:
Be not added with Pericarpium Papaveris negative bottom material of chafing dish sample as reference, (+) " positive ", detection sample speckle fluorescence ratio with reference to weak,
Represent that sample contains Pericarpium Papaveris feature alkaloid morphine；(-) " negative ", the speckle fluorescence of detection sample fluorescence compared with reference
Strong or indistinction, represents without Pericarpium Papaveris feature alkaloid morphine, (±) " male/female ", the speckle fluorescence of detection sample
Inconspicuous with reference to difference.
Described embodiment be the present invention preferred embodiment, but the present invention is not limited to above-mentioned embodiment, without departing substantially from this
In the case of the flesh and blood of invention, any conspicuously improved, replacement or modification that those skilled in the art can make are equal
Belong to protection scope of the present invention.
Claims (8)
1. the preparation method of the Flow-through membrane carrier quantum dot fluorescence immunoblotting Rapid detection test strip of morphine, it is characterised in that carry out as steps described below:
The pretreatment of Flow-through film:
Cellulose nitrate film pencil drawing a diameter of 5mm circular hole, (15min is soaked by phosphate buffered saline(PBS) (PBS), taking-up distilled water rinses, test strips is close to distilled water and drenches the filter paper of tiling, every hole uniformly drips 20 μ L morphine specific antigens, 37 DEG C of incubation 30min, with non-specific sites on bovine serum albumin (BSA) blocking antigen, 37 DEG C of incubation 30min, three times are washed with (PBST) containing tween phosphate buffer, 37 DEG C are dried, 4 DEG C of kept dry；
(2) preparation of carboxyl water-soluble CdTe@ZnS quantum dot labelling morphine specific antibody:
Carboxylated CdTe@ZnS borate buffer is diluted to 1 μM in round-bottomed flask, magnetic agitation；Add the morphine specific antibody that 43 μ L concentration are 3.73mg/mL, mix homogeneously (mixing speed to be difficult to too fast, not produce bubble be as the criterion)；After stirring 5min, turn down mixing speed, add 1-(3-dimethylamino-propyl)-3-ethyl carbodiimide (EDC) solution of 13 μ L, after reaction 20min, add 15 μ LN-N-Hydroxysuccinimide (NHS), room temperature reaction 2h；Reaction terminates, and is centrifuged 3min except reuniting with refrigerated centrifuger 12000rpm；
(3) purification of carboxyl water-soluble CdTe@ZnS quantum dot labelling morphine specific antibody:
With super filter tube by Sample Purification on Single 5 times, end-product redissolves in PBS；After end-product takes out, 12000rpm is centrifuged 3min except reuniting, measure be stored in after concentration 4 DEG C standby.
The preparation method of the Flow-through membrane carrier quantum dot fluorescence immunoblotting Rapid detection test strip of morphine the most according to claim 1, it is characterised in that in step (1), described PBS is 10mM, pH 7.4；Described PBS preparation morphine specific antigen concentration is 0.05 μ g/ μ L, and the degree of described BSA is 1-1.5%(W/V).
The preparation method of the Flow-through membrane carrier quantum dot fluorescence immunoblotting Rapid detection test strip of morphine the most according to claim 1, it is characterized in that in step (2), described EDC concentration is for using 10mM, the borate buffering configuration 10mg/mL of pH=7.4, NHS concentration is for configuring 10mg/mL by 10mM, pH=7.4 borate buffering.
The preparation method of the Flow-through membrane carrier quantum dot fluorescence immunoblotting Rapid detection test strip of morphine the most according to claim 1, it is characterised in that in step (2), the temperature of described coupling reaction is 25 DEG C, and the time is 2h.
The preparation method of the Flow-through membrane carrier quantum dot fluorescence immunoblotting Rapid detection test strip of morphine the most according to claim 1, it is characterised in that in step (3), described each purification of liquid and borate buffer volume ratio are not less than 10.
6. according to the Flow-through membrane carrier quantum dot fluorescence immunoblotting morphine Rapid detection test strip prepared by claim 1-5 any one preparation method.
7. utilize the Flow-through membrane carrier quantum dot fluorescence immunoblotting Rapid detection test strip described in claim 6 for the method detecting morphine, it is characterized in that, testing sample is substantially dissolved in water after being placed in petroleum ether extraction, adds a certain amount of CdTe/ZnS quantum dot labelling morphine specific antibody mixing；Described liquid is uniformly dripped in test strip.
Detection method the most according to claim 7, it is characterised in that sample and CdTe/ZnS quantum dot labelling morphine specific antibody fully react 30min.
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| CN107515214A (en) * | 2017-07-27 | 2017-12-26 | 广东省药品检验所（广东省药品质量研究所、广东省口岸药品检验所） | Morphine, the quick determination method of codeine and detection kit in a kind of chafing dish food |
| CN108375679A (en) * | 2017-08-08 | 2018-08-07 | 济南德亨医学科技有限公司 | A kind of quantum dot fluorescence Western blot and Allergic skin test kit used |
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| CN1327158A (en) * | 2000-06-02 | 2001-12-19 | 北京大学 | Establishment of anti-morphine single anti-cell strain and method for producing double-side morphine test paper |
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| CN1327158A (en) * | 2000-06-02 | 2001-12-19 | 北京大学 | Establishment of anti-morphine single anti-cell strain and method for producing double-side morphine test paper |
| CN102539771A (en) * | 2011-12-29 | 2012-07-04 | 北京康美天鸿生物科技有限公司 | Immunofiltration assay fluorescent quantitative detection method based on high-sensitivity quantum dot |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107515214A (en) * | 2017-07-27 | 2017-12-26 | 广东省药品检验所（广东省药品质量研究所、广东省口岸药品检验所） | Morphine, the quick determination method of codeine and detection kit in a kind of chafing dish food |
| CN108375679A (en) * | 2017-08-08 | 2018-08-07 | 济南德亨医学科技有限公司 | A kind of quantum dot fluorescence Western blot and Allergic skin test kit used |
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Application publication date: 20160921 |