CN108535475A - Ternary system Immune competition method detects the quantum dot immune chromatography detection card and detection method of sulindac - Google Patents
Ternary system Immune competition method detects the quantum dot immune chromatography detection card and detection method of sulindac Download PDFInfo
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- 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
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- 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
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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Abstract
The present invention relates to quantum dot immune chromatography detection cards and detection method that a kind of ternary system Immune competition method detects sulindac.The detection card includes liner plate, detection antigen release pad, quantum dot probe release pad, chromatographic film and water absorption pad；Detection antigen release pad includes to detect antigen made of oralbumin and sulindac coupling；Quantum dot probe release pad includes to be marked with the quantum dot probe of anti-oralbumin antibody；Chromatographic film is equipped with detection line and nature controlling line, and detection line fixes anti-sulindac antibody, and nature controlling line fixes oralbumin antigen.Detection antigen dissolves in detection architecture from release pad, forms ternary system immune complex with detection antibody and labelled antibody, generates fluorescent assay signal.Under the detection reference effect of nature controlling line, whether sulindac is contained in judgement sample according to the fluorescence signal in detection line, the signal strength and stability of detection is increased, improves detection sensitivity and quantitative precision.
Description
Technical field
The present invention relates to food and drug safety detection field more particularly to it is a kind of by immunochemistry detection technique be applied to protect
The detection of the illegal addition chemicals of strong product, Chinese patent drug, the especially quantum dot of ternary system Immune competition method detection sulindac
Immunochromatographydetection detection card and detection method.
Background technology
Entitled (Z) -2- methyl-1s-[(4- methylsulfinyls phenyl) methylene] the fluoro- 1H- indenes -3- of -5- of chemistry of sulindac
Acetic acid belongs to non-steroidal anti-inflammatory drugs, have it is anti-inflammatory, analgesia and refrigeration function, for rheumatic arthritis, ankylosing spondylitis,
Pain caused by non-inflammatory arthralgia, arthritis, nonarticular rheumatism, non-non-articular inflammatory, various neuralgias, cancer pain
Bitterly, post-traumatic pain and the caused fever of various inflammation etc., but have certain side effect, clinically belong to prescription medicine, it is necessary to
It is taken under the guidance of doctor.
Rheumatic disease is using pain such as joint, muscle, soft tissue, nerves as cardinal symptom, and the course of disease is in chronic and anti-more
Recurrence is made.There is the tradition by " dietotherapy " or herbal treatment chronic disease in China, and in chemosynthesis medicine, there are many side effects
Background under, favor in recent years by market for health food (based on health liquor), the Chinese patent drug of rheumatic disease.
Sulindac analgesic effect is rapid and cheap, then has illegal producer is illegal in health food, Chinese patent drug to add
Add sulindac, illegitimate benefits is tryed to gain to heighten the effect of a treatment.It is improper take sulindac there may be nausea, headache or allergic rash
Equal adverse reactions, cause to damage to alimentary canal, liver and kidney, serious to even result in anaphylactic shock and acute renal declines
It exhausts.Patient is ignorant and takes the health food containing sulindac for a long time, and harmfulness is very serious, it is necessary to be reinforced to illegal
Add the supervision and check of sulindac product.Currently, the confirmation method of the illegal addition sulindac of detection is high performance liquid chromatography, liquid matter
Combination detection.But these method equipment investments are big, operating cost is high, sample pretreatment is complicated, can not scene be detected,
It is difficult to that screening is unfolded to illegal addition phenomenon on a large scale.
Existing sulindac rapid detection method is mainly chemical detection method and thin-layered chromatography detection method, the spirit of detection
The needs that sensitivity and anti-interference ability are improved.Immunological detection method is sensitive, special, quick and inexpensive, in environmental monitoring and
Field of food safety has been widely used.The immunological method for the detection micromolecular compound reported at present, it is anti-to be all based on detection
It is former and detection antibody " binary system Immune competition method ", semiochemicals are marked on detection antibody, and it is solid in chromatographic film
Fixed detection antigen forms immune complex, generates detection signal." binary system Immune competition method " has the following disadvantages：1, it needs
Marking signal substance is distinguished to each detection antibody, causes semiochemicals that can not unify to prepare；2, detection antibody is stablized in liquid phase
Property it is insufficient；3, it detects signal strength and sensitivity is relatively low.
Invention content
Based on this, the object of the present invention is to provide the quantum dots that a kind of ternary system Immune competition method detects sulindac
Immunochromatographydetection detection card and detection method have the advantages that increase detection signal strength and stability, improve detection sensitivity.
The purpose of the present invention is what is be achieved through the following technical solutions：
Ternary system Immune competition method detects the quantum dot immune chromatography detection card of sulindac, including liner plate and successively
Be adhered on liner plate and between adjacent overlapping portions detection antigen release pad, quantum dot probe release pad, chromatographic film and water suction
Pad；The detection antigen release pad includes to detect antigen made of oralbumin and sulindac coupling；The quantum dot is visited
Needle release pad includes to be marked with the quantum dot probe of anti-oralbumin antibody；The chromatographic film is equipped with detection line and nature controlling line,
The detection line fixes anti-sulindac antibody, and the nature controlling line fixes oralbumin antigen.
Compared to traditional " binary system Immune competition method ", detection card of the invention is utilized that " ternary system is immunized competing
Strive method " principle increases except " detection antigen " and " detection antibody " the two immune detection elements for detection antigen load
" labelled antibody " of body protein.The present invention is covalently attached detection substance (sulindac) on carrier protein (oralbumin) and synthesizes
Antigen is detected, labelled antibody (anti-oralbumin antibody) is marked on quantum dot, by detection antibody (anti-sulindac antibody)
It is fixed in chromatographic film, to detect the detection substance being coupled on antibody capture detection antigen, labelled antibody combines detection antigen
The carrier protein of upper coupling forms ternary system immune complex, generates fluorescent assay signal.When the inspection that detection antibody is dissociated
It surveys substance to combine, fluorescence intensity will be suppressed, and inhibit detection signal to generate.
Specifically, dripping to sample solution in detection antigen release pad when detection, sample solution will inspection during moving
Antigen and quantum dot probe dissolving are surveyed, and is carried to detection line and nature controlling line, the anti-egg white for the quantum dot probe label being carried
Albumin (OVA) antibody, detection antigen and the anti-sulindac antibody in detection line form ternary system immune conjugate, make detection
Line generates fluorescence signal；The anti ova antibody for the quantum dot probe label being carried and fixed OVA antigen bindings on nature controlling line,
Accumulation forms fluorescence signal on nature controlling line.Reaching a certain concentration when there is free sulindac in sample, being immunized in detection line
Reaction will be blocked by competition, and fluorescence signal is suppressed；And the fluorescence signal on nature controlling line, it is not influenced by sulindac concentration.
The relevant report that sulindac is detected currently with fluorescence quantum immune chromatography method is less, and the present invention is food medicine
The work of product safety detection provides new tool.Quantum dot is the nanometer half that three diameter of Spherical Volume are collected at by 200-10000 atoms
Conductor crystal, spherical nucleus diameter 2-8nm, in order to increase the water solubility and biocompatibility of quantum dot, spherical nucleus outer layer is logical
It can often be coated by organic molecule and introduce functional group, diameter can be increased to 15-30nm, have good colloidal property and dynamics
Characteristic is suitable as the Nanoparticle labeling material of Biological Detection.Compared with the nanoparticle of traditional organic fluorescent dye filling, amount
Son point is with width and in continuously distributed excitation spectrum, narrow and symmetrical emission peak；Stability is strong, anti-light Bleachability strong；Light ability
Transformation efficiency height, brightness are that the decades of times of organic dyestuff is even more.
Compared with the existing technology, detection fixture of the invention has the advantage that：1, it is uniformly marked with anti-carrier protein antibodies
Quantum dot generate detection signal, only need centralized system for it is a kind of be used as fluorescence probe, be conducive to detect work scale and mark
Standardization；2, detection antibody is fixed in chromatographic film, by the closing and drying to film, is more had to the active protection effect of antibody
Profit increases Antibody stability；3, steric hindrance smaller when combining is immunized, improves detection signal strength and sensitivity；4, specific
By force, detection time is short (5-10 minutes), can execute-in-place, and can excite fluorescence signal by portable 360nm light sources, visually sentence
It reads as a result, testing cost is low, easy to operate, suitable base testing staff operates.
Further, the detection antigen release pad is absorbed using glass fibre element film containing detection antigen, surface-active
Agent, mannitol, the PBS solution of sucrose are obtained.Preferably, use thickness for the glass fibre element film of 0.85mm fully absorb containing
The PBS solution of 10 μ g/mL detections antigens, 50 μ g/mL surfactants, 30mg/mL mannitol, 50mg/mL sucrose.Wherein, sweet
Dew alcohol is as freeze-drying holder for ensuring that detecting antigen in detection process dissolves rapidly；Sucrose is for adjusting detection solution viscosity control
Preparative layer analyses development rate；Surfactant is used to eliminate the non-specific adsorption of detection process, preferably polyethylene glycol octyl phenyl
Ether (Triton X-100)；Detection antigen synthesize by OVA and sulindac covalent coupling, can on envelope the combination of detection antibody and
It can be combined by the labelled antibody on quantum dot.
Further, the quantum dot probe release pad using artificial cellulose's film absorb containing quantum dot fluorescence probe,
Polyethylene glycol, mannitol, sucrose, the PBS solution of glycine are obtained.Preferably, use thickness for the artificial cellulose of 0.34mm
Film is absorbed containing 20 μ g/mL quantum dot fluorescence probes, 60 μ g/mL polyethylene glycol (PEG-500), 10mg/mL mannitol, 60mg/
The PBS solution of mL sucrose, 10mg/mL glycine.Wherein, mannitol is used to ensure quantum dot in detection process as freeze-drying holder
Probe dissolves rapidly；Polyethylene glycol, sucrose are for adjusting detection solution viscosity control chromatography development rate；Glycine is for eliminating
The non-specific adsorption of detection process；Quantum dot fluorescence probe is made of quantum dot surface label anti ova antibody, can combine quilt
The OVA antigens for capturing the detection antigen in detection line and being fixed on nature controlling line, generate fluorescent assay signal respectively.
Further, the chromatographic film uses nitrocellulose filter, and the detection line is by containing anti-sulindac antibody and sugarcane
The PBS solution of sugar is sprayed on nitrocellulose filter and is made.Preferably, the anti-sulindac antibody of 0.3mg/mL and 10mg/ will be contained
The 0.05M PBS solutions (pH 7.4) of mL sucrose, are sprayed on the amount of 1.0~4.0 μ g/cm on nitrocellulose filter.Detection line
The basis that fluorescence signal is formed is the immune response of antibody and sulindac coupled by detection antigen, can be detected the Shu Lin to dissociate in product
Sour Competitive assays.
Further, the nature controlling line is sprayed on nitrocellulose filter by the PBS solution containing oralbumin and sucrose
It is upper to be made.Preferably, by the 0.05M PBS solutions (pH 7.4) containing 0.2mg/mL OVA and 10mg/mL sucrose, with 1.0~
The amount of 3.0 μ g/cm is sprayed on nitrocellulose filter.The basis that nature controlling line fluorescence signal is formed is the anti ova antibody of quantum dot
It is unrelated with sulindac inspection product with the immune response of Quality Control OVA antigens, the sulindac Competitive assays to dissociate in product will not be detected.
The quantum dot immune that ternary system Immune competition method detects sulindac chromatographs detection method, includes the following steps：
S1：Prepare detection antigen, quantum dot probe, detection line and nature controlling line；The detection antigen by oralbumin and
Sulindac is coupled, and the quantum dot probe is marked with anti-oralbumin antibody, and it is anti-that the detection line fixes anti-sulindac
Body, the nature controlling line fix oralbumin antigen；
S2：Sample solution, detection antigen and quantum dot probe are mixed, are delivered to detection line and nature controlling line together, according to
Fluorescence signal in detection line and nature controlling line comes in judgement sample whether contain sulindac.
Sulindac is micromolecular compound, the immune detection principle of conventional detection micromolecular compound be " detection antigen " with
" the binary system Immune competition method " that " detection antibody " is formed, for deficiency of this method in the lateral immunochromatography of quantum dot,
" labelled antibody " formation " ternary system Immune competition is added on the basis of " detection antigen " is with " detection antibody " in the present invention
Method ", and combination of each immune response element in tomographic system is also adjusted, detection antibody is fixed on detection line,
Label anti ova antibody constitutes fluorescence probe over the qds, with detection antigen (OVA- sulindacs) " bridging " formation " triplet
System " immune complex, fluorescence probe accumulate to form detection signal, increase the signal strength and stability of detection, improve inspection
Sensitivity is surveyed, when the detection substance that detection antibody is dissociated combines, fluorescence intensity will be suppressed, and inhibit detection letter to generate
Number.Be 2.0ng/mL to the minimum detectability degree of sulindac in sample solution using the detection method of the present invention, to health products, in
The minimum detectability degree of illegal addition sulindac and the like is 2.0 μ g/kg in patent medicine, and can be completed in 5 minutes quickly
Detection.
Further, in step S2, judged by the following method：Detection line and nature controlling line all show fluorescence signal,
Conclude that result is feminine gender, sulindac is free of in sample；Detection line does not show that fluorescence signal, nature controlling line show fluorescence signal, conclude
As a result it is the positive, contains sulindac in sample.Wherein, nature controlling line be for the method for inspection effectively whether and set, nature controlling line is aobvious
Show that fluorescence signal shows that method is effective, nature controlling line does not show that fluorescence signal shows that method itself is invalid；And detection line forms ternary
Fluorescence signal is generated when system immune complex, when there is free sulindac in sample, the immune response in detection line is just
It can be blocked by competition, to which fluorescence signal disappears.
Further, the quantum dot probe generates the transmitting light of 630nm under 365nm light source activations.
In order to better understand and implement, the invention will now be described in detail with reference to the accompanying drawings.
Description of the drawings
Fig. 1 is the structure that the ternary system Immune competition method of embodiment detects the quantum dot immune chromatography detection card of sulindac
Schematic diagram.
Fig. 2 a are the schematic diagram that immune response occurs in detection line, generates fluorescence signal.
Fig. 2 b are the schematic diagram that immune response is blocked by competition, fluorescence signal disappears in detection line.
Fig. 3 is the schematic diagram that immune response occurs on nature controlling line, generates fluorescence signal.
Fig. 4 is the judgment principle of the principle that fluorescence immunoassay testing result generates and testing result.
Specific implementation mode
Referring to Fig. 1, it detects the quantum dot immune chromatography of sulindac for the ternary system Immune competition method of the present embodiment
Detection card, including PVC liner plates and be adhered to successively on PVC liner plates and the detection antigen release pad of overlapping portions between adjacent,
Quantum dot probe release pad, nitrocellulose filter (NC films) and water absorption pad；The detection antigen release pad includes by OVA and Shu Lin
Antigen is detected made of acid coupling；The quantum dot probe release pad includes to be marked with the quantum dot probe of anti ova antibody；It is described
Nitrocellulose filter is equipped with detection line and nature controlling line, and the detection line fixes anti-sulindac antibody, and it is anti-that the nature controlling line fixes OVA
It is former.
Specifically, the preparation method of detection antigen release pad is as follows：Thickness is used to be filled for the glass fibre element film of 0.85mm
Divide and absorbs the PBS containing 10 μ g/mL detections antigens, 50 μ g/mL Triton X-100,30mg/mL mannitol, 50mg/mL sucrose
Solution, freeze-dried back.
Specifically, the preparation method of quantum dot probe release pad is as follows：First, using active ester method to carboxylated quantum dot
It is marked, 1.0mL carboxylated quantum dots is taken to be diluted in 20mL ultra-pure waters, the EDC solution of 0.05mL Fresh is added
(10mg/mL), being stirred at room temperature 20 minutes makes activated carboxylic；0.45mg anti ova antibody is added in pre-activate microballoon, is stirred at room temperature
The amino of activated carboxyl and antibody is set within 30 minutes to connect；Solution in 16000rpm low-temperature centrifugations 30 minutes, discard supernatant liquid with point
From unbonded antibody；Quantum dot probe is resuspended with the PBS solution of the X-100 containing 10%Triton, is protected from light 4 DEG C and is saved backup.So
Afterwards, thickness is used to be absorbed containing 20 μ g/mL quantum dot fluorescence probes, 60 μ for 85 artificial cellulose's films of Whatman of 0.34mm
The PBS solution of g/mL PEG-500,10mg/mL mannitol, 60mg/mL sucrose, 10mg/mL glycine, freeze-dried back.
Specifically, the preparation method of detection line is as follows：The anti-sulindac specific antibodies of 0.3mg/mL and 10mg/mL will be contained
The 0.05M PBS solutions (pH 7.4) of sucrose, are sprayed on the amount of 1.00 μ g/cm on nitrocellulose filter, form detection line.
Specifically, the preparation method of nature controlling line is as follows：By the 0.05M containing 0.2mg/mL OVA and 10mg/mL sucrose
PBS solution (pH 7.4) is sprayed on the amount of 1.00 μ g/cm on nitrocellulose filter, forms nature controlling line.
Specific antibody induction production method, anti ova antibody induction production method, the Yi Ji of sulindac and the like
Anti ova antibody, the method for preparing quantum dot fluorescence probe is marked to be all made of the prior art on quantum dot.
The quantum dot immune chromatography detection method of the ternary system Immune competition method detection sulindac of the present embodiment is as follows：
0.2g (or 0.2mL) sample is taken, is dissolved in 2.0mL absolute ethyl alcohols and fully extracts target inspection product, extract is static
So that impurity is fully precipitated within 10 minutes, 0.2mL supernatants is taken to be added to 20mL sample buffers, fully shakes up as sample solution；
3-4 drop sample solutions are added dropwise in detection antigen release pad with dropper, during sample solution is moved to nitrocellulose filter
So that detection antigen and quantum dot probe is discharged, and successively cross detection line and nature controlling line, according to glimmering in detection line and nature controlling line
Optical signal comes in judgement sample whether contain sulindac.
The anti ova antibody marked on anti-sulindac antibody, quantum dot probe in nitrocellulose filter detection line, respectively
With detection antigen binding, ternary system immune conjugate is formed, so that detection line is generated fluorescent assay signal, as shown in Figure 2 a.When
There is free sulindac in sample and reach a certain concentration, immune response will be by competition blocking, fluorescence signal meeting in detection line
It disappears, as shown in Figure 2 b.Anti ova antibody of the quantum dot probe by label and fixed OVA on nitrocellulose filter nature controlling line
Antigen binding accumulates on nature controlling line and forms fluorescence signal, as shown in Figure 3.Nature controlling line be method of inspection itself effectively whether and
Setting, colour developing is effective, does not develop the color and shows that method itself is invalid.
As shown in figure 4, quantum dot probe under 365nm light source activations, generates the transmitting light of 630nm, testing result is sentenced
Disconnected principle is：Detection line and nature controlling line all show fluorescence signal, conclude that result is negative (A), sulindac is free of in sample；Detection
Line does not show that fluorescence signal, nature controlling line show fluorescence signal, concludes that result is positive (B and C), contains sulindac in sample,
In, sulindac concentration is higher than 2.0ng/mL in a concentration of 1.0ng/mL of sulindac in positive findings B, positive findings C.
Compared with the existing technology, " the ternary system Immune competition method " that the present invention uses has the following advantages：1, it uniformly uses
The quantum dot of anti-carrier protein antibodies label generates detection signal, only needs centralized system to be used as fluorescence probe for a kind of, is conducive to examine
Survey the scale and standardization of work；2, detection antibody is fixed in chromatographic film, by the closing and drying to film, to antibody
Active protection acts on advantageously, increases Antibody stability；3, steric hindrance smaller when combining is immunized, improves detection signal strength
And sensitivity.It is right using the detection method of the present invention to being 2.0ng/mL to the minimum detectability degree of sulindac in sample solution
The minimum detectability degree of illegal addition sulindac and the like is 2.0 μ g/kg in health products, Chinese patent drug, and can be in 5 minutes
Complete quickly detection.
Several embodiments of the invention above described embodiment only expresses, the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the protection of the present invention
Range.
Claims (8)
1. ternary system Immune competition method detects the quantum dot immune chromatography detection card of sulindac, it is characterised in that：Including liner plate,
And it is adhered to successively on liner plate and the detection antigen release pad, quantum dot probe release pad of overlapping portions between adjacent, chromatography
Film and water absorption pad；The detection antigen release pad includes to detect antigen made of oralbumin and sulindac coupling；It is described
Quantum dot probe release pad includes to be marked with the quantum dot probe of anti-oralbumin antibody；The chromatographic film be equipped with detection line and
Nature controlling line, the detection line fix anti-sulindac antibody, and the nature controlling line fixes oralbumin antigen.
2. the quantum dot immune chromatography detection card of ternary system Immune competition method detection sulindac according to claim 1,
It is characterized in that：The detection antigen release pad is absorbed using glass fibre element film containing detection antigen, surfactant, sweet dew
Alcohol, the PBS solution of sucrose are obtained.
3. the quantum dot immune chromatography detection card of ternary system Immune competition method detection sulindac according to claim 1,
It is characterized in that：The quantum dot probe release pad is absorbed using artificial cellulose's film containing quantum dot fluorescence probe, poly- second two
Alcohol, mannitol, sucrose, the PBS solution of glycine are obtained.
4. the quantum dot immune chromatography detection card of ternary system Immune competition method detection sulindac according to claim 1,
It is characterized in that：The chromatographic film uses nitrocellulose filter, and the detection line is by the PBS containing anti-sulindac antibody and sucrose
Solution spraying is made on nitrocellulose filter.
5. the quantum dot immune chromatography detection card of ternary system Immune competition method detection sulindac according to claim 4,
It is characterized in that：The nature controlling line is sprayed on nitrocellulose filter by the PBS solution containing oralbumin and sucrose and is made.
6. the quantum dot immune that ternary system Immune competition method detects sulindac chromatographs detection method, it is characterised in that：Including with
Lower step：
S1：Prepare detection antigen, quantum dot probe, detection line and nature controlling line；The detection antigen is by oralbumin and Shu Lin
Acid is coupled, and the quantum dot probe is marked with anti-oralbumin antibody, and the detection line fixes anti-sulindac antibody, institute
It states nature controlling line and fixes oralbumin antigen；
S2：Sample solution, detection antigen and quantum dot probe are mixed, detection line and nature controlling line are delivered to together, according to detection
Fluorescence signal on line and nature controlling line comes in judgement sample whether contain sulindac.
7. the quantum dot immune of ternary system Immune competition method detection sulindac according to claim 6 chromatographs detection side
Method, it is characterised in that：In step S2, judged by the following method：Detection line and nature controlling line all show fluorescence signal, conclude
As a result it is feminine gender, sulindac is free of in sample；Detection line does not show that fluorescence signal, nature controlling line show fluorescence signal, conclude result
For the positive, contain sulindac in sample.
8. the quantum dot immune of ternary system Immune competition method detection sulindac according to claim 6 chromatographs detection side
Method, it is characterised in that：The quantum dot probe generates the transmitting light of 630nm under 365nm light source activations.
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