CN109884021A - A kind of quantum dot-labeled THC envelope antigen, preparation method and the method for detecting hemp content in food using it - Google Patents
A kind of quantum dot-labeled THC envelope antigen, preparation method and the method for detecting hemp content in food using it Download PDFInfo
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Abstract
A kind of quantum dot-labeled THC envelope antigen, preparation method and the method for detecting hemp content in food using it, belong to technical field of food safety detection, the preparation process includes the following steps: the preparation of (1) THC haptens: weighing succinic anhydride and THC, under the catalytic action of potassium carbonate, 55 ~ 65 DEG C of heating stirrings are reacted to fully reacting, TLC separation, gained succinyl THC are THC haptens；(2) preparation of THC envelope antigen: using EDC and NHS as catalyst, THC haptens and ovalbumin are reacted to obtain THC envelope antigen；(3) preparation of quantum dot-labeled THC envelope antigen: CdSe/ZnS QDs is dissolved in borate buffer, under EDC and NHS effect, THC envelope antigen is added dropwise in the borate buffer of CdSe/ZnS QDs, after reaction, with PBS buffer solution dialyse to get.
Description
Technical field
The invention belongs to field of detection of food safety, and in particular to the quantum dot-labeled THC envelope antigen of one kind, its preparation
Method and the method for detecting hemp content in food using it.
Background technique
Hemp (Cannabis), also known as fiery fiber crops etc., the chemical analysis of hemp is extremely complex, is mainly the following chemical combination
Object is constituted: the big phenol of lipides, flavone compound, terpenes, hydrocarbon, other than ring type, alkaloid, silver citrate and annular
The content of cannabinol (such as tetrahydrocannabinol), every kind of compound component is different in the different parts content of same strain plant.
Bioactive ingredients in hemp plant are the compounds such as tetrahydrocannabinol, cannabinol, cannabidiol, wherein tetrahydro
Hemp phenol content is higher, easily detects.
Food is to maintain one of human survival and protection human health essential condition, is the important guarantor of social harmonious development
Barrier, and improve one of the important material base of human quality.Food safety has attracted more and more attention from people and payes attention to, non-food
Abuse of the product additive hemp in food, it has also become the outstanding problem of food safety.At present to feature alkaloid in hemp shell
Most common detection method has instrumental method, immunoassay etc..Immunoassay is sensitive with its, specific and simple and quick
The features such as be used widely enzyme linked immunosorbent assay (ELISA) (ELISA) technology be immunological technique important composition, by success
Applied to food inspection, the immune chromatography test paper technology based on the principle is preferably developed, in addition to enzyme or substrate are marked
Object, common there are also radioactive substance, fluorescent materials etc., such as pervious radioimmunoassay technology, but due to radioactive material
Confrontation human body and environment have great injury, so no longer common.Fluorescence immunoassay is due to quick, sensitive, the pollution spies such as small at present
Point is concerned, and keeps its development rapider as fluorescent marker especially with quantum dot, fluorescence quantum is excellent due to its
Optical property and stability are widely used in bioanalysis detection field, mutually tie quantum dot-labeled with immuno analytical method
It closes, the novel detection method of exploitation is significant to food-safety problem is solved.The present invention will surround novel fluorescence quantum dot
Label biological antibody establishes the new method of feature alkaloid THC and application in detection hemp and conducts a research work, is expected that by this
Invention, the novel fluorescence immunologic detection method of foundation are able to satisfy detection demand currently growing in hemp detection field.This
Invention mainly combines fluorescence quantum point mark envelope antigen with immuno analytical method, establishes the quick detection side to hemp
Method reaches simplified operating procedure, saves the operating time, improves the purpose of detection efficiency, and the method established is used for chafing dish
The quick detection of hemp is illegally added in bottom material.
Summary of the invention
It is an object of the invention to be directed to sensitivity, the feature of environmental protection and the simplicity of current enzyme linked immunosorbent assay (ELISA), provide
It is a kind of based on the quick of quantum dot-labeled immunoassay, in real time, highly sensitive hemp detection technique, the method sensitivity
Height greatly reduces the leak detection rate of food safety detection, improves working efficiency, has significant application value.
The present invention is based on the hemp of quantum dot-labeled immunoassay detection method, be by artificial synthesized THC haptens, with
Ovalbumin (OVA) coupling forms complete envelope antigen, using THC antibody, while adsorbing and surveying by indirect competition fluorescence immunoassay
Determine method detection THC.It is anti-with THC coating using band carboxyl water-soluble quantum dot CdSe/ZnS QDs using EDC/NHS as crosslinking agent
Protozoa covalent coupling is characterized at fluorescent labeled antigen, while to coupled complex, and verifying shows fluorescence probe success
Good specificity is prepared and had, the fluorescence immune analysis method of detection THC is further established.It is carried by solid phase of 96 orifice plates
Body establishes fluorescence immunoassay competitive Adsorption analytic approach using the fluorescence envelope antigen immunological probe of preparation and quickly detects THC, and passes through
Research various concentration THC and fluorescent marker envelope antigen competitive binding THC antibody cause the variation of fluorescence intensity to be realized to THC's
Qualitative, quantitative judge detection.
Based on above-mentioned purpose, the technical solution adopted by the present invention is as follows:
A kind of preparation method of quantum dot-labeled THC envelope antigen, includes the following steps:
(1) preparation of THC haptens: weighing succinic anhydride and THC, and under the catalytic action of potassium carbonate, 55 ~ 65 DEG C of heating are stirred
Reaction is mixed to fully reacting, TLC separation, gained succinyl THC is THC haptens；
(2) preparation of THC envelope antigen: using EDC and NHS as catalyst, THC haptens and ovalbumin react
To THC envelope antigen (THC-OVA)；
(3) preparation of quantum dot-labeled THC envelope antigen (CdSe/ZnS QDs-THC-OVA):
CdSe/ZnS QDs is dissolved in borate buffer, under EDC and NHS effect, THC envelope antigen is added dropwise to
In the borate buffer of CdSe/ZnS QDs, after reaction, with PBS buffer solution dialyse to get.
Further, detailed process is as follows for the step (1): taking 1 mg THC to be dissolved in 250 μ L methylene chloride, sufficiently
Concussion is uniformly mixed, and 138 mg potassium carbonate is then added, 60 DEG C of 20 min of heating stirring add 19.1 mg succinic anhydrides and stir
Mix heating reaction 4 h, it is post-treated to obtain the final product.
Further, the process of the post-processing is as follows: by reaction system be fully transferred in centrifuge tube be added DCM from
The heart takes supernatant liquor, to reduce loss as far as possible, a small amount of DCM is added every time, centrifugation is repeated 3 times, utilize thin-layer chromatography again
Method separates succinyl THC with other reactive materials；Solvent is methylene chloride and petroleum ether when THC is separated with succinyl THC
(v/v=1/1) mixing, when succinyl THC is separated with other reactive materials solvent be methylene chloride and petroleum ether (v/v=
2/1) it mixes.
Further, the process of the step (2) is as follows: 1.3 mg being purified to dried succinyl THC, are dissolved in 0.2
The distilled water of mL adds 1.7 mg EDC HCl, is denoted as solution A；1.2 mg ovalbumins are weighed, the distilled water of 0.3 mL is dissolved in,
With the HCl tune pH to 4.0 of 0.1 mol/L, it is denoted as B solution；After two solution are mixed, pH to 3.5 ~ 4.5 is adjusted, is adjusting pH process
In, solution gradually becomes clarification, adds the NHS of 1 mg, and 4 DEG C of stirrings are to fully reacting after stirring 30 min at room temperature, with 10 mM
PBS buffer solution dialysis.
Further, the process of the step (3) is as follows:
Under the conditions of being protected from light, 20.3 μ L CdSe/ZnS QDs are diluted to 1 μM with the borate buffer of 10 mM, pH=7.4,
It is stirred at room temperature down, takes in step (2) 17 μ L THC envelope antigens to be added drop-wise in CdSe/ZnS QDs solution and be uniformly mixed；
After stirring 10 min, under stirring, 13 μ L, 10 mg/mL EDC solutions are added drop-wise to step20 are reacted in solution
min；
Under stirring, then into reaction solution 15 μ L are added dropwise, 10 mg/mL NHS solution are stirred to react 4 ~ 5 h at room temperature；
24 h of THC envelope antigen PBS buffer solution dialysis that CdSe/ZnS QDs is marked after reaction remove excessive
CdSe/ZnS QDs；
After dialysis, solution is concentrated into 1/10th of original volume with 12000 rpm with pipe is concentrated by ultrafiltration, final product redissolves
In PBS buffer solution, 12000 rpm are centrifuged 3 min except reuniting after taking-up, and the THC envelope antigen of CdSe/ZnS QDs label is protected
It is stored in spare under 4 DEG C of dark conditions.
Further, the CdSe/ZnS QDs quantum dot concentration is 8 μM；StepMiddle EDC solution and stepIn
The solvent of NHS solution is the borate buffer solution of 10 mM, pH=7.4.
Quantum dot-labeled THC envelope antigen made from above-mentioned preparation method.
The application of above-mentioned quantum dot-labeled THC envelope antigen (such as soup for chafing dish) hemp content in detection food.
It is described to be detected as fluorescence immunoassay adsorption analysis method, when detection, with the THC coating of CdSe/ZnS QDs label
Antigen is fluorescence probe, and the THC envelope antigen of CdSe/ZnS QDs label is exempted from analyte THC direct competitive with specificity
The THC antibody of epidemic disease reaction.
The process for preparation of borate buffer solution (10 mM, pH=7.4) is as follows: taking 45 mL boric acid solution (0.2 mol/
L it) is mixed into the borate buffer solution of 0.2 M with the borate solution of 5 mL (0.05 mol/L), being diluted 20 times is 10
The borate solution of mM, room temperature preservation；4 DEG C of 5 × PBS solution (0.05 mol/L, pH=7.4) preservations, are diluted to 1 before use
×PBS。
The method of hemp content, includes the following steps: in a kind of detection food
(1) the transparent each hole of 96 hole elisa Plates first is cleaned with 10 mM PBS buffer solution, takes THC antibody-solutions that 96 hole polyphenyl are added
Ethylene ELISA Plate don't cry and the experimental port for detecting sample to be tested in, make every hole containing 1 μ g THC antibody, be incubated for 2 at 37 DEG C ~
4h adds non-specific sites on the casein solution blocking antigen that mass fraction is 4%, is incubated for 2 ~ 4h at 37 DEG C, then uses
PBST solution washs every hole to remove excess reagent；
(2) then the THC sample of known concentration is added in colorimetric hole according to the gradient of setting, adds claim 1 and is made
Quantum dot-labeled THC envelope antigen as tracer, obtain colorimetric sample；
(3) sample to be tested is added in the 96 orifice plate experimental ports of step (1), is added quantum dot-labeled made from claim 1
THC envelope antigen as tracer；PBS solution constant volume is finally used, is incubated for 2 ~ 4h at 37 DEG C, it is strong by range estimation fluorescence after washing
Spend and and colorimetric hole in fluorescence intensity compare so that it is determined that in sample to be tested hemp concentration.
Further, THC sample gradient concentration is successively are as follows: 2 × 103 μg/mL、1×103 μg/mL、0.5×103 μg/
mL、0.1×103 μg/mL、0.1×102μg/mL、1 μg/mL、0.1 μg/mL、0.1×10-2 μg/mL、0.1×10-3 μg/
ML is successively labeled as 1 ~ No. 9 respectively, and the blank control without THC ingredient is No. 0 hole.
Detection when quantum dot-labeled THC fluorescence immunoassay adsorption analysis method is detected THC is limited to 1 ng/mL.
Envelope antigen measures fluorescence intensity after placing 30 days.Freshly prepared quantum dot-labeled THC envelope antigen with put
Quantum dot-labeled THC envelope antigen simultaneously detects THC standard solution after setting one month, and fluorescence signal intensity is almost without obvious
Difference, the results showed that, solution fluorescence intensity and specificity are after placing one month almost without significant difference, to the detection side of THC
For method without influence, the fluorescent marker envelope antigen stability of the method preparation is preferable.
The present invention is prepared for one kind based on 96 orifice plate fluorescence immunoassay competition analysis for THC, compares the quick of fluorescence intensity
Determination method.Pass through a series of experiment, it was therefore concluded that, the 96 orifice plate fluorescence immunoassay competition laws established by with compare
The comparison of group fluorescence intensity can effectively realize the fast qualitative detection to THC, about 1 h of detection time, and effectively can quickly sieve
The soup for chafing dish of choosing addition THC.It is strong that the analysis method can be observed visually fluorescent containing determinand under hand-held ultraviolet lamp
The power of the fluorescence intensity compared with standard sample is spent, this method is suitble to the soup for chafing dish of the illegal addition hemp of the quick screening in scene,
Technical support can be provided for food safety field quick detection.
Advantages of the present invention:
(1) the method for the invention is easy to operate, operating procedure is simple, save the time, signal is easy to collect, and use is fast on site
Speed screening be the costly and time consuming laborious instrumental method of substitution based on 96 orifice plate fluorescence immunoassay competition laws and more complicated is immunized
Measuring method, form provide a kind of THC detection method simply with low cost.
(2) THC antibody of the present invention and carboxylated water-soluble quantum dot CdSe/ZnS QDs preparation method are mature,
It has been be commercialized that, purchase is convenient, and the fluorescent labeled antibody holding time of preparation is long, and the dosage detected each time is few, is a kind of
Very economical detection method.
(3) high sensitivity of fluorescent labeled antibody obtained by the method for the present invention, greatly reduces the minimum detection limit of THC.
Detailed description of the invention
Fig. 1 is the fluorescence emission spectrogram of compound of ovalbumin Yu THC envelope antigen；
Fig. 2 is the fluorescence emission spectrogram of compound of the envelope antigen of CdSe/ZnS QDs and CdSe/ZnS QDs label；
Fig. 3 is the THC solution fluorescence spectrum that CdSe/ZnS QDs marks envelope antigen and various concentration；
Fig. 4 is that CdSe/ZnS QDs marks the fluorescence intensity of maximum emission wavelength in envelope antigen and THC solution with THC solution
Variation.
Specific embodiment
Below in conjunction with drawings and examples, further details of the technical solution of the present invention, but is not to the present invention
The limitation of protection scope.
Embodiment 1
A kind of preparation method of quantum dot-labeled THC envelope antigen, includes the following steps:
(1) preparation of THC haptens:
Take 1 mg THC to be dissolved in 250 μ L methylene chloride, fully shake uniformly mixed, be then added 138 mg potassium carbonate, 60 DEG C plus
20 min of thermal agitation adds 19.1 mg succinic anhydride, 60 DEG C of heating stirrings and reacts 4 h, and gained succinyl THC is THC half
Antigen.
The separating-purifying of succinyl THC: it is clear that reaction system is fully transferred to addition DCM centrifuging and taking upper layer in centrifuge tube
Liquid can repeat 2 ~ 3 cleaning reaction systems to reduce loss as far as possible, when transfer and be centrifuged with DCM, utilize thin-layer chromatography again
Method separates succinyl THC with other reactive materials；
It is that methylene chloride and petroleum ether are mixed by volume 1:1 using solvent used in thin-layered chromatography separation THC and succinyl THC
It closes；It is methylene chloride and petroleum ether by volume that succinyl THC, which is separated solvent with other reactive materials, using thin-layered chromatography
2:1 mixing.
(2) preparation of THC envelope antigen；It is carrier protein use using succinylation THC and ovalbumin (OVA)
EDC/NHS is coupled into envelope antigen carrier protein couplet object；
It is taken in step (1) and purifies dried succinyl THC1.3 mg, be dissolved in the distilled water of 0.2 mL, add 1.7 mg EDC
HCl remembers solution A；1.2 mg OVA are weighed, the distilled water of 0.3 mL is dissolved in, with the HCl tune pH to 4.0 of 0.1 mol/L, are denoted as B
Solution.After two solution are mixed, with the HCl tune pH to 4.0 or so of 0.1 mol/L, during adjusting pH, solution gradually becomes clear
Clearly, the NHS of 1 mg is added, at room temperature 4 DEG C of magnetic stirrer over night after 30 min of magnetic agitation.With 1 × PBS, (10 mM PBS are slow
Fliud flushing) dialysing three days removes unreacted small molecule THC haptens, and every 24 h replaces a dialyzate (10 mM PBS buffering
Liquid)；Up to succinyl THC-OVA solution, which is THC envelope antigen.(10000 D MWCO) is managed with being concentrated by ultrafiltration
5000 r/min centrifugal concentratings are concentrated into 125 μ L by 500 μ L, obtain being protected from light storage at 4 DEG C of THC envelope antigen for use.
The characterization of THC envelope antigen: reaction system in step (2) is taken out completely and is settled to 500 μ L fluorescence spectrophotometer degree
Meter measures fluorescence intensity under the excitation wavelength of 230 nm, the scanning speed of 240 nm/min, under the conditions of same measured with etc. it is dense
The isometric pure OVA solution fluorescence intensity of degree compares；Pure OVA solution and succinyl THC-OVA used in Fig. 1 as shown in Figure 1:
Solution concentration is 2.4 mg/mL, 500 μ L.The succinyl prepared compared with pure 328 nm of OVA solution maximum emission wavelength
THC-OVA has obvious blue shift at maximum emission peak, and this blue shift is caused by being successfully coupled due to succinyl THC and OVA.
(3) preparation of CdSe/ZnS quantum dot-labeled THC envelope antigen (abbreviation THC-OVA-QDs): carboxylated is water-soluble
Property CdSe/ZnS QDs by EDC/NHS in borate buffer solution (pH=7.4,10 mM) with step (2) preparation THC packet
It is coupled to form tracer by antigen, specific preparation process is as follows:
Under the conditions of being protected from light, (10 mM, pH=7.4) are diluted to by 20.3 μ L CdSe/ZnS QDs with borate buffer
1 μM, in round-bottomed flask, is stirred at room temperature down, and 17 μ L THC envelope antigens in step (2) is taken to be added drop-wise to CdSe/ZnS QDs solution
In be uniformly mixed (stirring is unsuitable too fast several times, and 800 ~ 1000 r/min are subject to and do not generate bubble)；
After stirring 10 min, 500 ~ 700 r/min of mixing speed is turned down, the EDC solution of 13 μ L (concentration is 10 mg/mL,
Using the boric acid of 10 mM, pH=7.4 salt buffer as solvent) it is added drop-wise in solution and reacts 20 min；
Under magnetic stirring, (concentration is 10 mg/mL to 15 μ L NHS solution of dropwise addition, with the borate of 10 mM, pH=7.4
Buffering is solvent) 4 ~ 5 h are stirred at room temperature；
The THC-OVA that CdSe/ZnS QDs is marked after reaction 1 × PBS buffer solution dialysis, 24 h remove excessive
CdSe/ZnS QDs；
After dialysis, with 12000 rpm solution is concentrated into 1/10th of original volume with pipe is concentrated by ultrafiltration, with 1 × PBS from
The heart washs 3 min.Product after washing redissolves in 1 × PBS buffer solution, and 12000 rpm are centrifuged 3 min except reunion.CdSe/
ZnS QDs label envelope antigen is stored in spare under 4 DEG C of dark conditions；
The quantum dot is the quantum dot for being 8.0 μM by the concentration that Wuhan Jia Yuan technology of quantum dots development corporation, Ltd. buys
Solution, totally 200 μ L；Borate buffer solution (10 mM, pH=7.4): 45 mL boric acid solutions (0.2 mol/L) and 5 mL are taken
Borate solution (0.05 mol/L) be mixed into the borate buffer solution of 0.2 M, diluted 20 times be 10 mM boric acid
Salting liquid, room temperature preservation；4 DEG C of 5 × PBS solution (0.05 mol/L, pH=7.4) preservations, are diluted to 1 × PBS before use.
The characterization of CdSe/ZnS QDs label envelope antigen: the excitation wavelength of CdSe/ZnS QDs labelled antibody is fixed on
365 nm, scan its solution with determine maximum emission wavelength in 520 nm, as shown in Figure 2；As shown in Figure 2, with CdSe/ZnS
520 nm of QDs maximum emission wavelength has obvious red shift compared to the fluorescent marker coating THC antigen of preparation at maximum emission peak, this
Kind red shift may be as caused by quantum size effect: the surface of quantum dot and antibody coupling are modified, and nonradiative transition can be reduced
Probability.After coupling, quantum dot dipole acts on enhancing and stoke shift to increase.It is made compared with the emission peak of quantum dot
The maximum half-peak breadth of standby fluorescent labeled antibody does not change significantly.Also it could be assumed that, preparation process does not cause quantum dot
Aggregation, dispersion and irregular distribution.
The optimum ratio of the carboxylated water-soluble CdSe/ZnS QDs:EDC:NHS is studied according to Sheng-Mei Wu etc.
Choose the ratio between different amount of substance carboxylated water-soluble CdSe/ZnS QDs:EDC:NHS (1:2000:8000,1:4000:
8000,1:6000:8000,1:8000:8000,1:4000:4000,1:4000:6000 and 1:4000:10000), in other phases
With under coupling condition, CdSe/ZnS QDs and envelope antigen are coupled, it is glimmering with multi-function microplate reader measurement different proportion conjugate
Luminous intensity observes fluorescent stability, using the THC of FLISA detection various concentration, determines EDC/NHS optimal proportion, works as QDs:
When the ratio of the amount of the substance of EDC:NHS is 1:2000:8000, conjugate relative intensity of fluorescence is maximum, the object of QDs:EDC:NHS
Conjugate is more stable when the ratio of the amount of matter is 1:4000:8000 when ratio 1:2000:8000, more steady using FLISA detection THC
Fixed, sensitivity is higher.Therefore, the conjugate of 1:4000:8000 is chosen for testing.It is surveyed after fluorescent labeled antibody is placed 30 days
Determine fluorescence intensity.Freshly prepared fluorescent labeled antigen and place one month after fluorescent labeled antigen simultaneously to THC standard solution
Detection, fluorescence signal intensity almost no significant difference, the results showed that, solution fluorescence intensity and specificity be almost after placing one month
It is not significantly different, on the detection method of THC without influence, the fluorescent marker envelope antigen stability of the method preparation is preferable.
Step (4): fluorescence immunoassay adsorption measurement (FLISA) qualitative and quantitative detection THC and determining minimum detection limit；
The transparent each hole of 96 hole elisa Plates first is cleaned with 10 mM PBS buffer solution, takes 10 holes μ L/, the THC antibody of 0.1 μ g/ μ L
96 holes hole polystyrene ELISA Plate A1 ~ A10 are added in solution, are incubated for 2 h at 37 DEG C, THC antibody is adsorbed on polystyrene surface, removes
Remove antibody-solutions, be fixed on 96 orifice plates, hereafter A1 ~ A8 be added mass fraction be 4% 30 hole μ L/ of casein solution, 37
3h is incubated at DEG C；Then every hole is washed 3 times with PBST (10 mM PBS pH=7.4+0.05% Tween20), to remove excess
Reagent；Then by the THC sample (2 × 10 of known concentration3 μ g/ml) multiple dilution are as follows: 2 × 103 μg/mL、1×103 μg/mL、
0.5×103 μg/mL、0.1×103 μg/mL、0.1×102μg/mL、1 μg/mL、0.1 μg/mL、0.1×10-2 μg/mL、
0.1×10-3THC sample is not added in μ g/mL, 0 μ g/mL(), THC-OVA- made from 30 μ L steps (3) is added in every hole later
QDs is as tracer；100 μ L finally are settled to the every hole of 1 × PBS solution, 2 h are incubated at 37 DEG C, in corresponding washing step
After obtain colorimetric sample, when detection, by comparing the fluorescence intensity signals in the experimental port for detecting sample to be tested and measurement
Fluorescence intensity in every hole, THC content is higher in determinand, and it is fiercer to compete with fluorescent marker envelope antigen, by with antibody
Fluorescent marker envelope antigen on 96 orifice plates is fewer, and measured fluorescence signal is weaker in conjunction with being adsorbed on, and achievees the purpose that detect THC,
A kind of novel quick fluorescence immunoassay is established using THC-OVA-QDs label THC antibody as fluorescence probe combination immunoassay
Analysis method；
Described THC antibody (the Tetrahydrocannabinol Monoclonal Antibody) solution passes through the U.S.
CalBioreagents supplier is commercially available, and concentration is 4.9 mg/mL(Lot:MA1166, Catalog:M469), it is being added
96 orifice plate is cleaned 2 ~ 3 times with PBS before antibody-solutions, and THC-OVA-QDs is that step (3) are prepared, and concentration is 9.6 mg/mL；
A series of THC sample (such as 1000,100,10,1,0.1,0.01,0.001,0.0001, μ g/mL) of various concentrations is separately added into
THC sample is not added as blank control group in the hole A8 in the hole A1 ~ A7；First 365 after reacting and terminating corresponding washing step
The fluorescence intensity in each hole is observed under nm uv analyzer, and the power of fluorescence signal in each hole, fluorescence mark are judged by estimating
Testing concentration in the fluorescence intensity and sample of the coating antigen binding antibody of note is negatively correlated.
The fluorescence signal in each hole is being measured with fluorescent sub-photometer, establishes standard curve；
It repeats step (4) in further test to operate, measurement relative standard deviation is 6.85%, changes the dense of sample to be tested
Gradient is spent, minimum detection limit is determined, establishes more accurate standard curve.As shown in Figure 3；Sample to be tested concentration from bottom to up in figure
Gradient (c) it is respectively 0.01,0.1,1,10,100, μ g/ml；Gained calibration curve equation is the -40.2x of y=1280, is such as schemed
4；THC content is higher in sample to be tested, more in conjunction with antibody competition, fluorescent labeled antigen and the antibody being adsorbed on 96 orifice plates
Fewer in conjunction with antigen, measured fluorescence signal is stronger, achievees the purpose that detect THC, using quantum dot-labeled THC antigen as glimmering
Light probe combination immunoassay establishes a kind of novel quick fluorescence immune analysis method.
Step (5): the detection in actual sample；
Transparent 96 hole elisa Plates by 1 ~ 10 hole of A row according to step (4) THC sample concentration successively are as follows: 2 × 103 μg/mL、1×103
μg/mL、0.5×103 μg/mL、0.1×103 μg/mL、0.1×102μg/mL、1 μg/mL、0.1 μg/mL、0.1×10-2 μ
g/mL、0.1×10-3μ g/mL, preparation colorimetric sample are successively labeled as 1 ~ No. 9, and THC sample is not added in 0 μ g/mL() it is labeled as No. 0；
100g chafing dish bottom flavorings are taken, adds water to be settled to 1L and is cooled to room temperature after heating is boiled, takes 0.5 mL tracer and 0.5 mL
Soup for chafing dish prepare liquid mixes, and the mixed liquor of 100 ~ 200 μ L is added dropwise in 96 orifice plate experimental ports, stands 2h at 37 DEG C, washs
Afterwards by range estimation fluorescence intensity and and colorimetric hole in fluorescence intensity compare so that it is determined that in sample to be tested hemp concentration.
The pretreated 96 orifice plate processing method be by the 10 diluted THC antibody of μ L PBS, make include in every bore region
1 μ g THC antibody, pays attention to avoiding liquid reagent horizontal proliferation.37 DEG C of incubation 2h are the closing of 4% casein solution with mass fraction
Non-specific sites on antigen, 37 DEG C of 2 ~ 4h of incubation, are washed three times, 37 DEG C of dryings, 4 DEG C of kept dries with PBST；
50 μ L are detected liquid (the CdSe/ZnS QDs in step (3) marks envelope antigen solution) point by the quantitative determination analysis
It does not mix and is added in each reacting hole of 96 good orifice plates of above-mentioned pretreatment with 50 μ L soup for chafing dish samples, washed after 2 h with PBST
It washs, is observed under ultraviolet light in dark.THC envelope antigen on 96 orifice plates is performed the competitive determination with free THC, for
Each conversion zone, CdSe/ZnS QDs mark THC envelope antigen it is identical with the Percentage bound of antibody and free THC, dissociate THC with
CdSe/ZnS QDs marks the specific position of the THC antibody on 96 orifice plate of envelope antigen conjugate competitive binding, to reach
Detection of the fluorescent labeled antibody to THC, PBST thoroughly wash unbonded free THC and CdSe/ZnS QDs label envelope antigen
Conjugate, the determinand by visual determination, in the fluorescence intensity and sample of the THC antibody that the envelope antigen of fluorescent marker combines
Concentration is negatively correlated.The fluorescence intensity change in observing response region under ultraviolet light in dark, compared with control group reacting hole,
The fluorescence intensity of conversion zone increases in succession with the reduction of THC concentration, and the visual detection for measuring THC is limited to 1 ng/mL, is
The visual minimum detection limit for determining 96 orifice plate fluorescence immunoassay determining adsorption THC is 1000 μ g/mL and 1 ng/mL in THC concentration
The fluorescence intensity shown on region, compared with control point region (being free of THC), the fluorescence intensity of conversion zone is with THC concentration
Increase and continuously reduce.There is notable difference in fluorescence intensity of the THC concentration between 1 ng/mL and 10 ng/mL, and reacts
Occur in control zone and THC concentration to be that 0.1 ng/mL and the fluorescence intensity difference in 1 region ng/mL are smaller, it is more difficult to distinguish.Cause
This, determines that the lowest detection level of 96 orifice plate fluorescence immunoassay competition assay THC is 1 ng/mL.
Embodiment 2
(1) preparation of tracer:
The preparation and preparation CdSe/ZnS QDs in step (3) in embodiment 1 for detecting liquid mark envelope antigen reactor solution phase
Together；
(2) preparation of standard solution:
Used 96 orifice plate recycling addition THC concentration will make phase in each hole in 1 step of preparation, that is, embodiment (4) of standard solution
It should mark, be 2 × 10 by THC concentration3 μg/mL、1×103 μg/mL、0.5×103 μg/mL、0.1×103 μg/mL、0.1×
102μg/mL、1 μg/mL、0.1 μg/mL、0.1×10-2 μg/mL、0.1×10-3μ g/mL is successively labeled as 1 ~ No. 9 respectively,
Blank control without THC ingredient is No. 0 hole, and 4 DEG C of dryings are fixed under the conditions of being protected from light to be saved, for use；
(3) colorimetric determination:
It include following step with the hemp component in the legal quantitative determination soup for chafing dish of above-mentioned 96 orifice plate fluorescence immunoassay competitive Adsorption
It is rapid:
96 pretreated orifice plates are laid flat, sample to be tested soup for chafing dish adds water constant volume to boil according to the ratio of embodiment 1,
It is cooled to room temperature, takes 0.5 mL detection liquid to mix with 0.5 mL soup for chafing dish prepare liquid, be added dropwise 100 in 96 orifice plate, one hole
The mixed liquor of ~ 200 μ L stands 0.5 h at 37 DEG C,
It is visually observed under hand-held ultraviolet lamp after being washed with PBST, testing sample fluorescence intensity is No. 5 and 6 in 96 orifice plates
Between number fluorescence intensity, i.e., THC concentration range should be between 10 μ g/mL and 1 μ g/mL.
With 230 nm of Hitachi F-2600 luminoscope excitation wavelength, the standard items fluorescence intensity of THC, linear fit are measured
Obtain the linear equation of Fig. 4.Fire is calculated after bringing linear equation into the fluorescence intensity that sample to be tested is measured under similarity condition
The concentration of THC in pot liquor material is 5.6 μ g/mL.
For the authenticity for further verifying detection, the method that analysis is used in conjunction according to national standard THC makings in sample to be tested is used
GC-MS is analyzed, and measuring in soup for chafing dish THC content is 5.63 μ g/mL, consistent with result measured by the present invention.
Using detection 2
The present embodiment be applied to relevant detection liquid, tracer, standard solution with application detection 1 in it is identical, difference exists
In the brand of soup for chafing dish, taste and the time boiled and temperature；The uniform GC-MS testing result of institute's testing result is consistent.
The above is only preferable implementation example of the invention, is not intended to limit the present invention in any form.
Anyone skilled in the art, without departing from the scope of the technical proposal of the invention, all using the disclosure above
Methods and technical content makes many possible changes and modifications to technical solution of the present invention, or is revised as the equivalent of equivalent variations
Implement example.Therefore, anything that does not depart from the technical scheme of the invention according to the technical essence of the invention show the above implementation
Any simple modifications, equivalents, and modifications that example is done, all of which are still within the scope of protection of the technical scheme of the invention.
Claims (9)
1. a kind of preparation method of quantum dot-labeled THC envelope antigen, which comprises the steps of:
(1) preparation of THC haptens: using methylene chloride or acetone as solvent, a certain amount of succinic anhydride and THC are weighed, in carbonic acid
Under the catalytic action of potassium, 55 ~ 65 DEG C of heating stirrings to fully reacting, TLC separation, gained succinyl-THC is THC half
Antigen；
(2) preparation of THC envelope antigen: using EDC and NHS as catalyst, THC haptens and ovalbumin react
To THC envelope antigen；
(3) preparation of quantum dot-labeled THC envelope antigen: CdSe/ZnS QDs is dissolved in borate buffer, THC packet
It is added dropwise to by antigen in the borate buffer of CdSe/ZnS QDs, under EDC and NHS catalytic action, after reaction, uses PBS
Buffer dialysis to get.
2. the preparation method of quantum dot-labeled THC envelope antigen according to claim 1, which is characterized in that the step
(1) detailed process is as follows: THC being dissolved in methylene chloride or acetone, potassium carbonate, 55 ~ 65 DEG C of heating are added after being completely dissolved
Stir 10 ~ 30 min, add succinic anhydride agitating and heating and react to fully reacting, it is post-treated to obtain the final product, potassium carbonate, THC and
The molar ratio of succinic anhydride is 1:(0.18 ~ 0.19): 0.003；The process of the post-processing is as follows: reaction system is all shifted
DCM centrifugation is added in centrifuge tube, takes supernatant liquor, utilizes thin-layered chromatography by succinyl THC and other reactive materials again
Separation；When THC is separated with succinyl THC solvent be methylene chloride mixed with petroleum ether by volume 1:1, succinyl THC and its
Solvent is that methylene chloride is mixed with petroleum ether by volume 2:1 when his reactive material separation.
3. the preparation method of quantum dot-labeled THC envelope antigen according to claim 1, which is characterized in that the step
(2) process is as follows: 1.3 mg being purified to dried succinyl THC, are dissolved in the distilled water of 0.2 mL, add 1.7 mg EDC
HCl is denoted as solution A；1.2 mg ovalbumins are weighed, the distilled water of 0.3 mL is dissolved in, extremely with the HCl tune pH of 0.1 mol/L
4.0, it is denoted as B solution；After two solution are mixed, pH to 3.5 ~ 4.5 is adjusted, during adjusting pH, solution gradually becomes clarification, adds
The NHS of 1 mg, after stirring 30 min at room temperature, 4 DEG C of stirrings to fully reacting are dialysed with 10 mM PBS buffer solution.
4. the preparation method of quantum dot-labeled THC envelope antigen according to claim 1, which is characterized in that the step
(3) process is as follows:
Under the conditions of being protected from light, 20.3 μ L CdSe/ZnS QDs are diluted to 1 μM with the borate buffer of 10 mM, pH=7.4, room
Under temperature stirring, takes in step (2) 17 μ L THC envelope antigens to be added drop-wise in CdSe/ZnS QDs solution and be uniformly mixed；
After stirring 10 min, under stirring, the EDC solution of 13 μ L, 10 mg/mL are added drop-wise to step20 are reacted in solution
min；
Under stirring, then into reaction solution 15 μ L are added dropwise, 10 mg/mL NHS solution are stirred to react 4 ~ 5 h at room temperature；
The THC that CdSe/ZnS QDs is marked after reaction removes excessive CdSe/ZnS with 24 h of PBS buffer solution dialysis
QDs；
After dialysis, solution is concentrated into 1/10th of original volume with 12000 rpm with pipe is concentrated by ultrafiltration, final product redissolves
In PBS buffer solution, 12000 rpm are centrifuged 3 min except reuniting after taking-up, and the THC envelope antigen of CdSe/ZnS QDs label is protected
It is stored in spare under 4 DEG C of dark conditions；Wherein, the CdSe/ZnS QDs quantum dot concentration is 8 μM；StepEDC solution and step
SuddenlyThe solvent of NHS solution is the borate buffer solution of 10 mM, pH=7.4.
5. quantum dot-labeled THC envelope antigen made from any preparation method of Claims 1-4.
6. the application of the hemp content in detection food of quantum dot-labeled THC envelope antigen described in claim 5.
7. application according to claim 6, which is characterized in that described to be detected as fluorescence immunoassay adsorption analysis method, detection
When, using the THC envelope antigen of CdSe/ZnS QDs label as fluorescence probe, the THC coating of CdSe/ZnS QDs label is anti-
The former THC antibody with analyte THC direct competitive with specific immune response.
8. a kind of method of hemp content in detection food, which comprises the steps of:
(1) the transparent each hole of 96 hole elisa Plates first is cleaned with 10 mM PBS buffer solution, takes THC antibody-solutions that 96 hole polyphenyl are added
Ethylene ELISA Plate don't cry and the experimental port for detecting sample to be tested in, make every hole containing 1 μ g THC antibody, be incubated for 2 at 37 DEG C ~
4h adds non-specific sites on the casein solution blocking antigen that mass fraction is 4%, is incubated for 2 ~ 4h at 37 DEG C, then uses
PBST solution washs every hole to remove excess reagent；
(2) then the THC sample of known concentration is added in colorimetric hole according to the gradient of setting, adds claim 1 and is made
Quantum dot-labeled THC envelope antigen as tracer, obtain colorimetric sample；
(3) sample to be tested is added in the 96 orifice plate experimental ports of step (1), is added quantum dot-labeled made from claim 1
THC envelope antigen as tracer；PBS solution constant volume is finally used, is incubated for 2 ~ 4h at 37 DEG C, it is strong by range estimation fluorescence after washing
Spend and and colorimetric hole in fluorescence intensity compare so that it is determined that in sample to be tested hemp concentration.
9. the method for hemp content in detection food described in claim 8, which is characterized in that THC sample gradient concentration is successively are as follows:
2×103μg/mL、1×103 μg/mL、0.5×103μg/mL、0.1×103μg/mL、0.1×102μg/mL、1 μg/mL、0.1
μg/mL、0.1×10-2μg/mL、0.1×10-3μ g/mL is successively labeled as 1 ~ No. 9 respectively, and the blank control without THC ingredient is
No. 0 hole.
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