CN110082523A - A kind of quantum dot-preparation method of myoglobins antibody immune complex and the preparation method of test strips - Google Patents
A kind of quantum dot-preparation method of myoglobins antibody immune complex and the preparation method of test strips Download PDFInfo
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Abstract
The present invention relates to a kind of quantum dot-myoglobins antibody immune complex preparation methods, it reacts quantum dot with 1- ethyl -3- (3- DimethylAminopropyl) carbodiimide hydrochloride and n-hydroxysuccinimide or its thio object, controls quantum dot, the molar ratio of 1- ethyl -3- (3- DimethylAminopropyl) carbodiimide hydrochloride, n-hydroxysuccinimide or its thio object is 1:4000 ~ 10000:5000 ~ 10000；The pH for adjusting reaction solution is 6 ~ 7；Myoglobins antibody is added into reaction solution, the molar ratio for controlling quantum dot and myoglobins antibody is 1:30 ~ 70.Coupling effect of the present invention is good, and preparation method is simple, low in cost.The standard curve of test strips produced by the present invention is good, and related coefficient is greater than 0.97, and detection sensitivity is high, can be realized the accurate quantitative analysis detection of myoglobins antigen.
Description
Technical field
The invention belongs to biomarker fields, and in particular to a kind of system of quantum dot-myoglobins antibody immune complex
The preparation method of Preparation Method and test strips.
Background technique
Quantum dot (Quantum dots, QDs) is mainly by II B race ~ VI A race element (such as CdSe, CdTe, CdS, ZnSe
Deng) or III A race ~ V A race element (such as InP, InAs) composition semiconductor nanoparticle, three dimensions are in 100nm
Hereinafter, appearance is just like a pointing object.The quanta point biological compatibility of synthesis in water is good, is readily incorporated functional groups.Quantum
Point has special fluorescent characteristic compared with the organic conventional fluorescent material of tradition: 1) emission spectrum can be by changing size
It is controlled with composition, Color tunable is, it can be achieved that quantum dot polychrome of the same race marks；2) organic dyestuff fluorescent molecule exciting light is single,
Emission spectrum is wide, there is a hangover, and quantum dot is due to its special quantum confined effect, exciting light spectrum width, and emission spectrum is narrow and right
Claim, no long wave hangover；3) biggish Stokes shift avoids excitation spectrum overlapping with emission spectrum, is conducive to fluorescence spectrum
Detection；4) photochemical stability is high, and resistance to photobleaching can be subjected to repeated multiple times excitation；5) fluorescence efficiency is high；6) bio-compatible
Property is good, after various chemical modifications, can carry out specific connection；7) fluorescence lifetime is long, when exciting light close several nanoseconds with
Afterwards, quantum dot fluorescence still has, and can get the fluorescence signal without background interference.
And quantum dot is as novel nano material, the preparation method of biological fluorescent labelling is divided into non-covalent linking and altogether
Valence connection.Wherein being not covalently linked mainly has Electrostatic Absorption, the connection of specific biological target, streptomysin-biotin connection etc.；Altogether
Valence connection includes quantum dot surface functional group carboxyl, amino, hydroxyl etc., is divided respectively with biology under the activation of different activator
Amino, sulfydryl of son etc. are covalently attached.Wherein quantum dot surface carboxyl connect with biomolecule amino covalence and is most widely used.
But prepare that quantum dot-capture antibody complex method is different, and purification process needs the instrument of Large expensive at present
It completes, limits quantum dot-capture antibody complex study condition, coupling efficiency can not be calculated, limit quantum dot in life
Application in terms of substance markers.Secondly, the not no verification method of Applied economy, affects the detection performance of Related product.
Myoglobins is a kind of binding protein, be intramuscular storage oxygen protein, in extremely low oxygen atmosphere or
Lead to muscle anoxic when strenuous exercise, the releasable oxygen of myoglobins is at this time for contraction of muscle.Myoglobins molecular weight is smaller, when
It when myocardium muscle damage, can be leaked into blood circulation from musculature, increase serum myoglobin concentration, pass through inspection
Content of hemoglobin in serum is surveyed to judge whether that muscle damage occurs.To the clinical meaning of acute myocardial infarction AMI detection are as follows: in urgency
Myoglobin concentration rises rapidly in blood in dozens of minutes after the onset of property heart infarction, 7 hours or so reach to peak values, in 12 hours almost
Myoglobin concentration has raising in all AMI blood samples of patients, therefore can be used as the early diagnosis marker of AMI.Immune examination
The detection of paper slip belongs to instant detection, can complete the quantitative detection to Troponin I, in a short period of time to meet clinic
Detect the requirement of myoglobins.
CN104280545A disclose a kind of quantum dot-labeled test strips for synchronizing quantitative Applications of Cardiac Markers multi objective and its
Method, still, inventor has found that being unable to get the mark of corresponding Applications of Cardiac Markers only with the content of the patent disclosure
Directrix curve, to cannot achieve the detection of corresponding Applications of Cardiac Markers.
Summary of the invention
Technical problem to be solved by the invention is to provide a kind of good quantum dot-myoglobins of calibration curve coefficient correlation
The preparation method of antibody immune complex and the preparation method of test strips.
In order to solve the above technical problems, the present invention adopts the following technical scheme that:
It is an object of the present invention to provide a kind of quantum dot-myoglobins antibody immune complex preparation method, features
It is: includes the following steps:
(1), make quantum dot and 1- ethyl -3- (3- DimethylAminopropyl) carbodiimide hydrochloride and n-hydroxysuccinimide
Or its thio object is reacted, and the quantum dot, described 1- ethyl -3- (3- DimethylAminopropyl) carbodiimides are controlled
The molar ratio of hydrochloride, the n-hydroxysuccinimide or its thio object is 1:4000 ~ 10000:5000 ~ 10000；
(2), the pH of set-up procedure (1) treated reaction solution is 6 ~ 7；
(3), myoglobins antibody is added into step (2) treated reaction solution, and that described quantum dot-myoglobins is made is anti-
Body immune complex, the molar ratio for controlling the quantum dot and the myoglobins antibody is 1:30 ~ 70.
In the present invention, the surface of the quantum dot is carboxyl modified, and the quantum dot can be hud typed or non-core
Shell type quantum point.
Preferably, in step (1), the average grain diameter of the quantum dot is 10 ~ 30nm, launch wavelength range be 560nm ~
650nm。
Preferably, in step (1), the quantum dot, 1- ethyl -3- (3- DimethylAminopropyl) carbonization two are sub-
The molar ratio of amine hydrochlorate, the n-hydroxysuccinimide or its thio object be 1:4000 ~ 6000:8000 ~
10000。
Preferably, the reaction condition of step (1) is ice-bath ultrasonic, and the reaction time is 20 ~ 40min.
According to a specific and preferred embodiment, the specific embodiment of step (1) are as follows: it is 5 ~ 6 that quantum dot, which is dissolved in pH,
In the borate buffer solution of 0.005 ~ 0.015M, it is sub- that 1- ethyl -3- (3- DimethylAminopropyl) carbonization two is then added
Amine hydrochlorate and the n-hydroxysuccinimide or its thio object carry out priming reaction.
Preferably, step (1) after the reaction was completed, removes excessive 1- ethyl -3- (3- dimethyl amine third using ultrafiltration
Base) carbodiimide hydrochloride and n-hydroxysuccinimide or its thio object, the borate buffer for being then 6 ~ 7 by addition pH
The pH of liquid adjustment reaction solution.
Preferably, in step (3), the molar ratio of the quantum dot and the myoglobins antibody be 1:50 ~
70, further preferably 1:60 ~ 70.The present invention passes through the dosage of strict control antibody and quantum dot, ensure that quantum dot and resists
The joint efficiency of body.
Preferably, the reaction condition of step (3) is ice-bath ultrasonic, and the reaction time is 2 ~ 5h.
It is a further object to provide a kind of preparation methods of test strips, include the following steps:
(1) using treatment fluid processing sample pad and bonding pad；
(2) using quantum dot-made from above-mentioned preparation method, treated for myoglobins antibody immune complex processing step (1)
Bonding pad；
(3) detection line and Quality Control are coated on nitrocellulose filter using myoglobins antibody and sheep anti-mouse igg polyclonal antibody
Line, then using the nitrocellulose filter after confining liquid closing coating；
(4) by step (1) treated sample pad, step (2) treated bonding pad, step (3) treated cellulose nitrate
Plain film, water absorption pad and carrier stack gradually, and the test strips are made.
Preferably, in step (1), based on mass fraction, the treatment fluid be containing 1 ~ 5% bovine serum albumin(BSA), 1 ~
3% polysorbas20,0.5 ~ 1.5% NaCl, 1 ~ 5% sucrose and 1 ~ 3% polyethylene glycol 2000 pH be 7 ~ 9 borate buffer
Liquid；In step (3), based on mass fraction, the confining liquid is the bovine serum albumin(BSA) for being 0.5 ~ 2% containing mass fraction
The borate buffer solution that pH value is 7 ~ 9.
Preferably, in step (3), use the myoglobins antibody-solutions that concentration is 0.5 ~ 2mg/ml with 0.5 ~ 1 μ l/cm's
Detection line is made in speed of crossing on the nitrocellulose filter, uses concentration for more grams of the sheep anti-mouse igg of 0.5 ~ 1 mg/ml
Nature controlling line is made with the scribing line speed of 0.5 ~ 1 μ l/cm in grand antibody-solutions on nitrocellulose filter.
It is further preferred that the myoglobins antibody-solutions and the sheep anti-mouse igg Anti-TNF-α liquid solution are adopted
It dilutes to obtain with borate buffer.
It is further preferable that the borate buffer of myoglobins antibody described in dilution is pH6.5, ionic strength: 0.05M's
Borate buffer.
It is further preferable that the borate buffer of sheep anti-mouse igg polyclonal antibody described in dilution is pH5.5, ionic strength:
The borate buffer of 0.01M.
Further, front of the detection line in the immunochromatography direction of nature controlling line when described stroke of film.
Further, detection line and nature controlling line are close to water absorption pad side when described stroke of film, and purpose is when reserving enough
Between allow antibody antigen carry out specific reaction.
Due to the implementation of above technical scheme, the invention has the following advantages over the prior art:
The coupling effect of quantum dot of the invention and myoglobins antibody is good, and preparation method is simple, low in cost.The present invention is made
Test strips standard curve it is good, related coefficient is greater than 0.97, and detection sensitivity is high, can be realized the accurate of myoglobins antigen
Quantitative detection.
Detailed description of the invention
Fig. 1 is that quantum dot control (left-hand bar band) and quantum dot-myoglobins antibody mediated immunity of the embodiment of the present invention 1 are compound
The agarose gel electrophoresis figure of object (right side band)；
Fig. 2 is in coupling reaction using quantum dot-myoglobins antibody immune complex gel electrophoresis made from different pH value
Figure；
Fig. 3 is solidifying using quantum dot-myoglobins antibody immune complex made from different activator levels in coupling reaction
Gel electrophoresis figure；
Fig. 4 is in coupling reaction using quantum dot-myoglobins antibody immune complex gel made from different antibodies dosage
Electrophoretogram；
Fig. 5 is that quantum dot control and quantum dot-myoglobins antibody immune complex particle size of the embodiment of the present invention 1 detect
Figure；
Wherein, control of the Fig. 1 into Fig. 5 is the result that the quantum dot being directly commercially available is detected；
Fig. 6 is that quantum dot control is ultraviolet after loading gradient myoglobins antigen concentration with the test strips of the embodiment of the present invention 1
Figure；
Fig. 7 is that quantum dot control and the test strips of the embodiment of the present invention 1 are obtained in loading gradient myoglobins antigen concentration
Canonical plotting.
Specific embodiment
The present invention will be further described in detail combined with specific embodiments below, but the present invention is not limited to following implementations
Example.Implementation condition used in the examples can do further adjustment according to specifically used different requirements, the implementation being not specified
Condition is the normal condition in the industry, the commercially available acquisition of reagent in the present invention.In following, unless otherwise specified, " % " is equal
For mass percentage.
Embodiment 1
A kind of preparation of quantum dot-myoglobins antibody immune complex:
5ul quantum dot (average grain diameter is 18 nm, and emission peak is 625nm ± 5nm, concentration 8uM, is purchased from Wuhan Ka source) is molten
In 500ul, pH 5.5 in 0.01M borate buffer solution, is vortexed and mixes 30s.Now prepare 0.01M 1- ethyl -3- (3- dimethyl
Amine propyl) carbodiimide hydrochloride and 0.01M N- hydroxy thiosuccinimide, solvent is pH 5.5,0.01M borate
Buffer.20 ul, 0.01M 1- ethyl -3- (3- DimethylAminopropyl) carbodiimides hydrochloric acid are added in quantum dot solution
Salt is vortexed and mixes, and 40ul is added after 5min, and 0.01M N- hydroxy thiosuccinimide is vortexed and mixes.Ice-bath ultrasonic 30min.
After the completion of activation, priming reaction liquid is taken out, is placed in super filter tube (100kd), is centrifuged in refrigerated centrifuge, revolving speed 3500rpm,
1ml is added after the completion in 7min in reaction solution, and pH 6.5,0.01M borate buffer solution, repeated centrifugation is primary, takes out reaction
Liquid.6.3ul myoglobins capture antibody (anti-Myo-7C3,7.3mg/ml) is added into reaction solution, is vortexed and mixes, ice bath is super
Sound 3h is to get quantum dot-myoglobins antibody immune complex.
The quantum dot-myoglobins antibody immune complex agarose gel electrophoresis figure is as shown in Figure 1, particle size is examined
Mapping is as shown in Figure 5, wherein the average grain diameter of quantum dot is 18nm, and quantum dot-myoglobins antibody immune complex is averaged
Partial size is 136nm, from Fig. 1 and Fig. 5 as it can be seen that embodiment 1 has successfully obtained quantum dot-myoglobins antibody immune complex.
The preparation of test strips:
(1) sample pad and bonding pad are respectively placed in treatment fluid (containing 1% bovine serum albumin(BSA), 1% polysorbas20,0.85%
The borate buffer of the pH8.5 of NaCl, 1% sucrose and 2% polyethylene glycol 2000) in, it impregnates 2 hours, then in 37 DEG C of perseverances
It is taken out after drying in warm incubator spare.
(2) quantum dot-myoglobins antibody mediated immunity made from the above method is sprayed on step (1) treated bonding pad
Compound and drying, control spray rate are 0.8 μ L/cm.
(3) myoglobins antibody (HyTest company) is dissolved in the borate buffer of pH6.5, ionic strength: 0.05M and is matched
The myoglobins antibody-solutions that concentration is 1mg/ml are made, are then drawn on nitrocellulose filter with the scribing line speed of 0.5 μ L/cm
The dry obtained detection line of line；Sheep anti-mouse igg polyclonal antibody (HyTest) is dissolved in the boric acid of pH5.5, ionic strength: 0.01M
It is configured to the mouse that concentration is 0.5 mg/ml in buffer and resists how anti-solution, then with the scribing line speed of 0.5 μ l/cm in nitric acid fibre
Tie up the dry obtained nature controlling line of scribing line on plain film.
(4) use containing mass fraction for 1% bovine serum albumin(BSA), pH value be 8.5 borate buffer to draw have detection
The nitrocellulose filter of line and nature controlling line impregnates two hours, after dry in 37 DEG C of constant incubators take out.
(5) by step (1) treated sample pad, step (2) treated bonding pad, step (4) treated nitric acid
Cellulose membrane, water absorption pad and carrier stack gradually, and test strips are made.
(6) the myoglobins antigenic solution containing various concentration is added drop-wise to the sample pad surface of test strips, obtained purple
Outer figure is shown in that Fig. 6, canonical plotting are shown in Fig. 7.
Embodiment 2
Substantially same as Example 1, the difference is that: when preparing quantum dot-myoglobins antibody immune complex, respectively
The pH of reaction solution after activation is adjusted using the borate buffer solution of different pH, agarose gel electrophoresis figure is shown in Fig. 2, wherein 1 is pair
According to band, 2 for pH5.5 borate buffer solution made from quantum dot-myoglobins antibody immune complex band, 3 are
Quantum dot made from the borate buffer solution of pH6.5-myoglobins antibody immune complex band, 4 be the borate of pH7.5
Quantum dot made from buffer-myoglobins antibody immune complex band, 5 be made from the borate buffer solution of pH8.5
Quantum dot-myoglobins antibody immune complex band, 6 be quantum dot-flesh red eggs made from the borate buffer solution of pH9.5
The band of white antibody immune complex.
It analyzes since the isoelectric point of different antibody is different, in different pH, the electrification of antibody is different, affects antibody
With the coupling effect of quantum dot, quantum dot is coupled with different antibodies, that is, has different best coupling pH.
Made from the borate buffer solution that condition is pH6.5 other than test strips, test strips obtained under the conditions of other pH
It is unable to get standard curve.
Embodiment 3
Substantially same as Example 1, the difference is that: when preparing quantum dot-myoglobins antibody immune complex, respectively
Add the quantum dot (QDs) and 1- ethyl -3- (3- DimethylAminopropyl) carbodiimide hydrochloride of different mol ratio example
(EDC), agarose gel electrophoresis figure is shown in Fig. 3, wherein 1 is the band of control；2 be quantum dot and 1- ethyl -3- (3- dimethyl
Amine propyl) molar ratio of carbodiimide hydrochloride is the band of 1:40,3 be quantum dot and 1- ethyl -3- (3- dimethyl amine
Propyl) molar ratio of carbodiimide hydrochloride is the band of 1:200,4 be quantum dot and 1- ethyl -3- (3- dimethyl amine
Propyl) molar ratio of carbodiimide hydrochloride is the band of 1:1000,5 be quantum dot and 1- ethyl -3- (3- dimethyl amine
Propyl) molar ratio of carbodiimide hydrochloride is the band of 1:5000,6 be quantum dot and 1- ethyl -3- (3- dimethyl amine
Propyl) carbodiimide hydrochloride molar ratio be 1:25000 band.
Analysis is since QDs is different from the molar ratio of EDC, the activation degree difference of QDs surface carboxyl groups, then with ammonia in antibody
The case where base reacts is different, causes to be coupled success rate difference.
Embodiment 4
Substantially same as Example 1, the difference is that: when preparing quantum dot-myoglobins antibody immune complex, respectively
The quantum dot and myoglobins for adding different mol ratio example capture antibody, and agarose gel electrophoresis figure is shown in Fig. 4, wherein 1 is quantum
The band for being 1:10 with the molar ratio of myoglobins capture antibody is selected, 2 capture mole of antibody for quantum dot with myoglobins
Ratio is the band of 1:30, and 3 be that quantum dot and myoglobins capture the band that the molar ratio of antibody is 1:50, and 4 be quantum dot
The band that molar ratio with myoglobins capture antibody is 1:70,5 capture the molar ratio of antibody for quantum dot with myoglobins
Example is the band of 1:90.
The present invention is described in detail above, its object is to allow the personage for being familiar with this field technology that can understand this
The content of invention is simultaneously implemented, and it is not intended to limit the scope of the present invention, all Spirit Essence institutes according to the present invention
The equivalent change or modification of work, should be covered by the scope of protection of the present invention.
Claims (10)
1. a kind of quantum dot-myoglobins antibody immune complex preparation method, characterized by the following steps:
(1), make quantum dot and 1- ethyl -3- (3- DimethylAminopropyl) carbodiimide hydrochloride and n-hydroxysuccinimide
Or its thio object is reacted, and the quantum dot, described 1- ethyl -3- (3- DimethylAminopropyl) carbodiimides are controlled
The molar ratio of hydrochloride, the n-hydroxysuccinimide or its thio object is 1:4000 ~ 10000:5000 ~ 10000；
(2), the pH of set-up procedure (1) treated reaction solution is 6 ~ 7；
(3), myoglobins antibody is added into step (2) treated reaction solution, and that described quantum dot-myoglobins is made is anti-
Body immune complex, the molar ratio for controlling the quantum dot and the myoglobins antibody is 1:30 ~ 70.
2. quantum dot according to claim 1-myoglobins antibody immune complex preparation method, it is characterised in that:
In step (1), the average grain diameter of the quantum dot is 10 ~ 30nm, and launch wavelength range is 560nm ~ 650nm.
3. quantum dot according to claim 1-myoglobins antibody immune complex preparation method, it is characterised in that:
It is the quantum dot, described 1- ethyl -3- (3- DimethylAminopropyl) carbodiimide hydrochloride, described in step (1)
The molar ratio of n-hydroxysuccinimide or its thio object is 1:4000 ~ 6000:8000 ~ 10000.
4. quantum dot according to claim 1-myoglobins antibody immune complex preparation method, it is characterised in that:
The reaction condition of step (1) is ice-bath ultrasonic, and the reaction time is 20 ~ 40min.
5. quantum dot according to claim 1-myoglobins antibody immune complex preparation method, it is characterised in that:
Step (1) removes excessive 1- ethyl -3- (3- DimethylAminopropyl) carbodiimides hydrochloric acid after the reaction was completed, using ultrafiltration
Then salt and n-hydroxysuccinimide or its thio object adjust the pH of reaction solution by the borate buffer that addition pH is 6 ~ 7.
6. quantum dot according to claim 1-myoglobins antibody immune complex preparation method, it is characterised in that:
In step (3), the molar ratio of the quantum dot and the myoglobins antibody is 1:50 ~ 70.
7. quantum dot according to claim 1-myoglobins antibody immune complex preparation method, it is characterised in that:
The reaction condition of step (3) is ice-bath ultrasonic, and the reaction time is 2 ~ 5h.
8. a kind of preparation method of test strips, characterized by the following steps:
(1) using treatment fluid processing sample pad and bonding pad；
(2) using quantum dot-myoglobins antibody mediated immunity made from the preparation method as described in any one of claims 1 to 7
Compound processing step (1) treated bonding pad；
(3) detection line and Quality Control are coated on nitrocellulose filter using myoglobins antibody and sheep anti-mouse igg polyclonal antibody
Line, then using the nitrocellulose filter after confining liquid closing coating；
(4) by step (1) treated sample pad, step (2) treated bonding pad, step (3) treated cellulose nitrate
Plain film, water absorption pad and carrier stack gradually, and the test strips are made.
9. preparation method according to claim 8, it is characterised in that: in step (1), based on mass fraction, the place
Manage liquid be containing 1 ~ 5% bovine serum albumin(BSA), 1 ~ 3% polysorbas20,0.5 ~ 1.5% NaCl, 1 ~ 5% sucrose and 1 ~ 3% it is poly-
The borate buffer that the pH of ethylene glycol 2000 is 7 ~ 9；In step (3), based on mass fraction, the confining liquid is to contain quality
The borate buffer solution that the pH value for the bovine serum albumin(BSA) that score is 0.5 ~ 2% is 7 ~ 9.
10. preparation method according to claim 8, it is characterised in that: in step (3), use concentration for 0.5 ~ 2mg/ml
Myoglobins antibody-solutions with the scribing line speed of 0.5 ~ 1 μ l/cm on the nitrocellulose filter be made detection line, use
The sheep anti-mouse igg Anti-TNF-α liquid solution that concentration is 0.5 ~ 1 mg/ml is with the scribing line speed of 0.5 ~ 1 μ l/cm in nitrocellulose
Nature controlling line is made on film.
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| MIHAELA SAVIN 等: ""A quantum dot-based lateral flow immunoassay for the sensitive detection of human heart fatty acid binding protein (hFABP) in human serum"", 《TALANTA》 * |
| 褚春旭 等: ""心肌损伤标志物肌酸激酶同工酶荧光检测方法的建立"", 《长春理工大学学报》 * |
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Application publication date: 20190802 |