CN103792370A - Method for detecting CP4-EPSPs (5-enolpyruvylshikimate-3-phosphate synthase) protein by using QCM (Quartz Crystal Microbalance) sensor and special gold piece - Google Patents
Method for detecting CP4-EPSPs (5-enolpyruvylshikimate-3-phosphate synthase) protein by using QCM (Quartz Crystal Microbalance) sensor and special gold piece Download PDFInfo
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Abstract
The invention discloses a method for detecting CP4-EPSPs (5-enolpyruvylshikimate-3-phosphate synthase) protein by using a QCM (Quartz Crystal Microbalance) sensor and a special gold piece. The invention provides a method for preparing a QCM gold piece for detecting CP4-EPSPs, wherein the method comprises the following steps: (1) modifying a QCM gold piece together with 11-mercaptoundecanoic acid (11-MUA) and 3-mercapto propionic acid (3-MPA), so as to obtain a modified gold piece; (2) fixing a CP4-EPSPs antibody on the modified gold piece, thereby obtaining the QCM gold piece for detecting CP4-EPSPs. According to the method, shown by experiments, the detection on MCMV (Maize Chlorotic Mottle Virus) is researched by using a QCM biosensor; through comparing and analyzing modified QCM gold pieces of two kinds, namely QCM gold pieces independently modified by 11-MUA and QCM gold pieces together modified by 3-MPA and 11-MUA, the QCM gold pieces together modified by 3-MPA and 11-MUA are higher in detection sensitivity and better in specificity and can be applied to actual detection methods.
Description
Technical field
The present invention relates to biological technical field, relate in particular to a kind of method and special gold plaque that utilizes qcm sensor to detect CP4-EPSPs albumen.
Background technology
Glyphosate is not only a kind of for blanket wide spectrum, highy potent herbicide in annual and perennial weeds five, and has the characteristic that soil strong absorbent and mammal, birds, fish hypotoxicity etc. are conducive to environment.Be widely used in corn, soybean, cotton in orchard, bare place, no-tillage ground and broadcast front or broadcast aftertreatment, and directional process after emerging, be one of kind of whole world herbicide use amount maximum.5-enol acetone shikimic acid-3-phosphate synthase (EPSPs) catalysis penultimate shikimic acid pathway, in the time that substrate phosphoenolpyruvate carboxylate has high affinity, enzyme shows height glyphosate tolerant characteristic, catalyzing and synthesizing aromatic acid, when phenylalanine and tyrosine, this is committed step, also follows the biochemical compound of some heteroauxins, lignin and phytoalexin grade.It is the first-selected target enzyme of many microbiotic herbicides.Glyphosate can be combined and cause plant death with the EPSPs of plant.The EPSPs enzyme separating from natural Agrobacterium fungus strain CP4 has good resistance glyphosate function, claims CP4-EPSPs.
Whole world glyphosate resistant crops cultivated area is ascendant trend year by year.Since genetically modified crops come into the market, cause the generally vigilant of food safety and controlling.The long-term use of glyphosate can affect soil microorganism, strengthen soybean root disease probability, and the appearance of resistance glyphosate weeds most serious of all, therefore, grasp science fast detecting resistant weed method so that and the transgenic product that enters in a large number Chinese market to not being affixed by the situation that label stated detect and just seem very urgent and significant.Detect at present EPSPs genetic method and mainly contain the PCR method based on nucleic acid level and the euzymelinked immunosorbent assay (ELISA) based on protein level and immuno-chromatographic test paper strip method.The results of study such as Kan Guizhen show negative sample one band positive two bands in test strips method, and in PCR testing result, positive gene specific fragment is 146bp.Dong Feng double fastener heart euzymelinked immunosorbent assay (ELISA) result shows to detect and is limited to 80ppb.G.J.Rogan detects detecting of CP4-EPSPs albumen in soybean by euzymelinked immunosorbent assay (ELISA) and is limited to 210 μ g/g, and the coefficient of variation is less than 15%.
QCM (Quartz Crystal Microbalance) is a kind of simple, the height of tiring, and high resolving power, utilizes the mass sensitivity technology of piezoelectric effect principle.Immunological detection can detect has antigenic protein-based expression product, it is realized by Ag-Ab specific reaction, antigen-antibody reaction is a kind of non-covalent bond specific adsorption reaction, in the ordinary course of things, antigen only and the antibody of the antibody being produced by own induction or the antigen induction generation with same antigen determinant carry out specific reaction.So immunological response has the specificity of height.
Summary of the invention
The object of this invention is to provide a kind of method of the QCM gold plaque for the preparation of detection CP4-EPSPS.
Method provided by the invention, comprises the steps:
1) with sulfydryl undecanoic acid and the co-modified QCM gold plaque of 3-mercaptopropionic acid, obtain modifying rear gold plaque;
2) will resist CP4-EPSPS antibody to be fixed to gold plaque after described modification, obtain the QCM gold plaque for detection of described CP4-EPSPS.
In said method, above-mentioned QCM gold plaque is one-sided gold plating film, 12mm × 20mm, one-sided gold-plated 50nm, BioNavis company, article No. QSX301-Au-13052.
In step 1), the co-modified QCM gold plaque of described sulfydryl undecanoic acid and 3-mercaptopropionic acid is prepared as follows: described QCM gold plaque is immersed in the ethanolic solution that contains sulfydryl undecanoic acid and 3-mercaptopropionic acid.
In step 1), described immersion is 4 ℃ and soaks 12h;
In said method,
In step 1), described in contain sulfydryl undecanoic acid and 3-mercaptopropionic acid ethanolic solution in, the mol ratio of described sulfydryl undecanoic acid and described 3-mercaptopropionic acid is 1:10.
In said method,
The final concentration of described sulfydryl undecanoic acid in described solution is 0.9M;
The final concentration of described 3-mercaptopropionic acid in described solution is 9M.
In said method,
Step 2) in, it is that described anti-destination protein antibody is connected by covalent bond with sulfydryl undecanoic acid and 3-mercaptopropionic acid on gold plaque after described modification that anti-destination protein antibody is fixed to gold plaque after described modification.
Step 2) in, described gold plaque after resisting CP4-EPSPS antibody to be fixed to described modification is specifically prepared as follows: first drip gold plaque surface after described modification with the solution that contains EDC and Sulfo-NHS, normal temperature (25 ℃) is lower leaves standstill 2 hours; Drip described anti-CP4-EPSPS antibody, 37 ℃ are reacted 3 hours again; Dripping concentration is 1mg/mLBSA again, leaves standstill 1 hour at 25 ℃, obtains the QCM gold plaque for detection of described CP4-EPSPS;
Contain EDC(N-ethyl-N'-(dimethylamino-propyl) carbodiimide) and Sulfo-NHS(N-hydroxy thiosuccinimide) solution: EDC and Sulfo-NHS are dissolved in MES solution, make the final concentration 0.075M of EDC in solution, the final concentration of NHS in solution is 0.015M.
MES(fatty acid methyl ester sulfonate) solution: take 3.90g MES and be dissolved in 180mLddH
2o, regulates pH to 5.0, and constant volume is to 200mL.
In said method,
Described anti-destination protein antibody is monoclonal antibody or polyclonal antibody.
In said method,
Described destination protein is resistance glyphosate albumen, and described resistance glyphosate albumen is specially CP4-EPSPS;
Described anti-destination protein antibody is anti-CP4-EPSPS monoclonal antibody.
The QCM gold plaque for detection of target protein of being prepared by above-mentioned method is also the scope of protection of the invention; Described destination protein is specially resistance glyphosate albumen.
The above-mentioned QCM gold plaque for detection of target protein is also the scope of protection of the invention in the application detecting in target protein; Described destination protein is specially resistance glyphosate albumen.
The above-mentioned QCM gold plaque for detection of target protein is also the scope of protection of the invention in the application detecting in antiweed genetically modified plants; Described herbicide is specially glyphosate.
Of the present invention experimental results show that, the present invention is according to the principle of antigen and antibody specific combination, CP4-EPSPs protein monoclonal antibody is modified on gold plaque, to there being antigenic CP4-EPSPs protein-specific to detect, can be realized convenient and swift by QCM biology sensor, true and reliable, testing process, in two hours, facilitates actual sample to detect, and sensitivity is up to 0.01%, with other detection method comparisons, there is very strong practical application advantage.The present invention detects the method for CP4-EPSPs transgene protein with QCM, can be widely applied to and import and export in plant inspection quarantine, detects and has important using value for transgenosis.As specific detection glyphosate transgene protein chip, there is good stability and keeping quality, give play to commercial value.
Accompanying drawing explanation
Fig. 1 is the sensitivity and repeatability that QCM detects CP4-EPSPs albumen
Fig. 2 is the specificity that QCM detects CP4-EPSPs albumen
Fig. 3 is that the co-modified QCM gold plaque of 3-MPA and 11-MUA detects actual sample column diagram
Embodiment
The experimental technique using in following embodiment if no special instructions, is conventional method.
Material, reagent etc. used in following embodiment, if no special instructions, all can obtain from commercial channels, and part is listed below:
QCM (Quartz Crystal Microbalance) (Biolin Scientific AB company); CP4-EPSPS albumen (sequence 1) and anti-CP4-EPSPS monoclonal antibody (purchased from Beijing overpass quality inspection Science and Technology Ltd.); Bovine serum albumin (BSA) (Shanghai You Ran sensing Science and Technology Ltd.); N-ethyl-N'-(dimethylamino-propyl) diimine (EDC) (Sigma company); N-hydroxy thiosuccinimide (Sulfo-NHS) (Sigma company); Fatty acid methyl ester sulfonate (MES) (Shanghai covalent chemical Science and Technology Ltd.); Damping fluid PBS(Sigma company); Sulfydryl undecanoic acid (11-MUA) and 3-mercaptopropionic acid (3-MPA) (Shanghai covalent chemical Science and Technology Ltd.); Tis-HCl(pH6.5) (Shanghai covalent chemical Science and Technology Ltd.); 30% ammoniacal liquor and ammoniacal liquor (Beijing Han Longda development in science and technology company limited).Bt proteantigen, 10% transgenic corns standard items MON810(Ouyang Style standard substance and measurement research institute, ERM-BF-413gk) and non-transgenic corn.
The configuration of solution used in following embodiment:
(1) 3-mercaptopropionic acid (3-MPA) ethanolic solution (10mM): take 0.1061g3-MPA with analytical balance and be dissolved in 100mL absolute ethyl alcohol, mix.
(2) sulfydryl undecanoic acid (11-MUA) ethanolic solution (10mM): take 0.2183g11-MUA with analytical balance and be dissolved in 100mL absolute ethyl alcohol, mix.
(3) MES(fatty acid methyl ester sulfonate) solution: take 3.90g MES with analytical balance and be dissolved in 180mL ddH
2o, regulates pH to 5.0, and constant volume is to 200mL.
(4) contain EDC(N-ethyl-N'-(dimethylamino-propyl) carbodiimide) and Sulfo-NHS(N-hydroxy thiosuccinimide) solution: take a certain amount of EDC and Sulfo-NHS is dissolved in 100 μ L MES solution with analytical balance, vibration evenly, make the concentration 0.075M of EDC in solution, the concentration 0.015M of NHS in solution.Matching while using.
(5) phosphate buffer (PBS, pH7.4): take respectively 4g NaCl, 0.72g Na with analytical balance
2hPO
4, 0.12g KH
2pO
4be dissolved in the distilled water after 480mL sterilizing with 0.1g KCl, use acidity tester and HCl to regulate the pH to 7.4 of damping fluid; And then adding sterile purified water in bottle, constant volume is to 500mL.
(6) Tris-HCl(0.1M, pH6.0): the ddH that takes 1.211g Tris-base and be dissolved in 80mL with analytical balance
2o, with HCl adjusting pH to 6.0, then constant volume is to 100mL.
The preparation of the QCM gold plaque of embodiment 1, detection CP4-EPSPs albumen
1, QCM gold plaque pre-service
Take out QCM gold plaque (QSX301-Au-13052), first rinse surface with MilliQ water, afterwards gold plaque is put into the small beaker that mixed solution (mixed solution is by 30% ammoniacal liquor, 30% hydrogen peroxide and MilliQ water 1:1:5 mixed preparing by volume) is housed.Elder generation is 85 ℃ the Temperature Setting of water-bath and makes it to be warming up to established temperature, afterwards small beaker is put into water-bath (notice that beaker does not contact with water-bath heating tube, prevent that beaker from splitting broken), processes about 10 minutes.Take out beaker, press from both sides out gold plaque with blunt-ended forceps, clean gold plaque remained on surface liquid with MilliQ water, dry up surface with nitrogen; Continue to clean gold plaque surface with absolute ethyl alcohol afterwards, nitrogen dries up, for the QCM gold plaque after cleaning, stand-by.
2, modify QCM gold plaque
Contain 11-MUA and 3-MPA ethanolic solution is prepared as follows: the 3-MPA of the 11-MUA of 10mM and 10mM is dissolved in absolute ethyl alcohol, the solution obtaining, mix according to 1:10, wherein: the final concentration of 11-MUA in solution is 0.9M, the final concentration of 3-MPA in solution is 9mM.
The above-mentioned 1 pre-service QCM gold plaque obtaining is placed in the vial that contains 11-MUA and 3-MPA ethanolic solution is housed, and in order to prevent that solution evaporation sealed membrane from sealing a bottle, 4 ℃ are soaked 12h, obtain the co-modified QCM gold plaque of 11-MUA and 3-MPA.
3, the QCM gold plaque of fixing monoclonal antibody
By anti-CP4-EPSPS monoclonal antibody be fixed to modify after gold plaque be that 11-MUA and 3-MPA on anti-CP4-EPSPS monoclonal antibody and above-mentioned 2 11-MUA that obtain and the co-modified QCM gold plaque of 3-MPA are connected by covalent bond; Concrete grammar is as follows:
1) activation
Obtain 11-MUA and the co-modified QCM gold plaque surface of 3-MPA with absolute ethyl alcohol drip washing above-mentioned 2, then use nitrogen drying, again the solution of the Sulfo-NHS of the EDC that contains 0.075M and 0.015M is evenly dripped on QCM gold plaque surface, normal temperature (25 ℃) is lower leaves standstill 2 hours, be fixed on the 11-MUA on QCM gold plaque surface and the mercaptan carboxyl of 3-MPA in order to activation, obtain the gold plaque of activation.
2) fixing monoclonal antibody
Outwell through 1) solution of the EDC that contains 0.4M on the gold plaque of activation processed and the Sulfo-NHS of 0.1M, cleans gold plaque surface with absolute ethyl alcohol, and dries up surface with nitrogen; By Tris-HCl(0.1M, pH6.0) dilution CP4-EPSPS albumen monoclonal antibody concentration is 0.2mg/mL, then monoclonal antibody solution is dripped equably on QCM gold plaque surface, be placed on and mix up in (37 ℃) constant temperature oven of temperature reaction 3 hours, after 3 hours, dry up with the monoclonal antibody solution of MilliQ water cleaning QCM remained on surface and with nitrogen; Drip BSA on QCM gold plaque surface, concentration is 1mg/mL(PBS dilution), normal temperature (25 ℃) is lower leaves standstill 2 hours, reaches the object of the unconjugated NHS-ester group of sealing; Then clean gold plaque surface, dry up stand-by (depositing in PBS solution 4 ℃) with nitrogen, be fixed the QCM gold plaque of anti-CP4-EPSPS monoclonal antibody.
Quartz crystal microbalance sensor detection method: the gold plaque of having modified is fixed in qcm sensor, opening peristaltic pump, to start flow velocity be 200 μ l/min, sample hose passes into PBS, isobase is stablized flow velocity and is changed 50 μ l/min into, pass into antigen to be detected, PBS is diluted to aimed concn, by the middle F of Qsoft software and the variation of D, Real Time Observation reaction result.
1, QCM detects the sensitivity of CP4-EPSPS albumen
Determined antigen CP4-EPSPS albumen is diluted to 500ng/mL, 1 μ g/mL, 5 μ g/mL, 10 μ g/mL and 15 μ g/mL with PBS, and the CP4-EPSPS albumen of getting each concentration of 100 μ L carries out QCM detection according to above-mentioned method.
As shown in Figure 1, when about the about 20min of sample injection time, after second, antigenic solution is sent to gold plaque surface by peristaltic pump to result, causes that gold plaque frequency declines and dissipates and rise, and illustrates that the surperficial fixing antibody of antigen and QCM gold plaque combines specifically.Antigenic solution sample concentration is larger, and the antigen of being combined with gold plaque surface monoclonal antibody is more, causes that frequency declines more.Can reach 500ng/ml with the sensitivity that QCM detects CP4-EPSPs albumen as shown in Figure 1, its frequency declines and is respectively 1.39,1.55 and 1.72HZ.
2, QCM detects the repeatability of CP4-EPSPS albumen
Under same condition, respectively 15 QCM gold plaques are cleaned, modify the processes such as activation, immobilized monoclonal antibody, to fix the QCM gold plaque of monoclonal antibody for detection of the antigen of above-mentioned 5 kinds of concentration, each measurement of concetration three times, obtain result as table 1, calculated mean value, SD and the CV% of three measured values.The CV% obtaining is all less than 10% of corresponding mean value, illustrates that the repeatability of QCM gold plaque detection CP4-EPSPs albumen is better.
Result is as shown in table 1, and each detection has obvious reaction, and the response difference of same concentration is very little, with also corresponding the reducing of reduction reaction response value of protein concentration, shows that the repeatability of QCM detection CP4-EPSPS is better.
The repeatability of table 1 QCM gold plaque
3, QCM detects the specificity of CP4-EPSPS albumen
In order to verify that QCM detects the specificity of CP4-EPSPS albumen (sequence 1), select the Bt albumen cry1Ah1(sequence 2 by Bt gene code) in contrast, respectively with two kinds of PROTEIN C P4-EPSPS and cry1Ah separately and equal-volume compound sample detect, sampling volume is 1ml, concentration is 15 μ g/mL, carries out specific detection experiment according to above-mentioned QCM detection method.
Result, as Fig. 2, detects the sample solution of same concentration, and the antigenic solution that only contains CP4-EPSPs albumen has obvious response.Illustrate that the gold plaque gold plaque that the method is modified has good specificity.
4, QCM detects the CP4-EPSPS albumen in testing sample
Take 100mg transgenosis content and be respectively 0.1% transgenic corns (0.1%Mon810, the verified CP4-EPSPS albumen that contains) and 0.01% transgenic corns (0.01%Mon810, the verified CP4-EPSPS albumen that contains) (Joint Research Centre of European Union standard substance and the ERM-BF-413gk of quantitative study institute), use beveller that sample is pounded to powdered, by 1000ul PBS dissolution sample, room temperature (25 ℃) leaves standstill 10min, the centrifugal 10min of 12000r, after drawing supernatant, filter, detect with QCM according to the method described above.10% the positive contrast of positive, the negative contrast of not genetically modified negative sample, in triplicate, records experimental data and averages.
Result as shown in Figure 3, can find out, the transgenic corns solution of two concentration all has response, and therefore known the method can be used as detecting actual transgenosis sample.
Claims (10)
1. for the preparation of a method for the QCM gold plaque of testing goal albumen, comprise the steps:
1) with sulfydryl undecanoic acid and the co-modified QCM gold plaque of 3-mercaptopropionic acid, obtain modifying rear gold plaque;
2) will resist destination protein antibody to be fixed to gold plaque after described modification, obtain the QCM gold plaque for detection of described CP4-EPSPS.
2. method according to claim 1, is characterized in that:
In step 1), the co-modified QCM gold plaque of described sulfydryl undecanoic acid and 3-mercaptopropionic acid is prepared as follows: described QCM gold plaque is immersed in the ethanolic solution that contains sulfydryl undecanoic acid and 3-mercaptopropionic acid.
3. method according to claim 2, is characterized in that:
In step 1), described in contain sulfydryl undecanoic acid and 3-mercaptopropionic acid ethanolic solution in, the mol ratio of described sulfydryl undecanoic acid and described 3-mercaptopropionic acid is 1:10.
4. it is characterized in that according to the method in claim 2 or 3:
The final concentration of described sulfydryl undecanoic acid in described solution is 0.9M;
The final concentration of described 3-mercaptopropionic acid in described solution is 9M.
5. according to arbitrary described method in claim 1-4, it is characterized in that:
Step 2) in, it is that described anti-destination protein antibody is connected by covalent bond with sulfydryl undecanoic acid and 3-mercaptopropionic acid on gold plaque after described modification that anti-destination protein antibody is fixed to gold plaque after described modification.
6. according to arbitrary described method in claim 1-5, it is characterized in that:
Described anti-destination protein antibody is monoclonal antibody or polyclonal antibody.
7. according to arbitrary described method in claim 1-6, it is characterized in that:
Described destination protein is resistance glyphosate albumen, and described resistance glyphosate albumen is specially CP4-EPSPS;
Described anti-destination protein antibody is anti-CP4-EPSPS monoclonal antibody.
8. the QCM gold plaque for detection of target protein of being prepared by the arbitrary described method of claim 1-7; Described destination protein is specially resistance glyphosate albumen.
9. the QCM gold plaque for detection of target protein claimed in claim 8 is in the application detecting in target protein; Described destination protein is specially resistance glyphosate albumen.
10. the QCM gold plaque for detection of target protein claimed in claim 8 is in the application detecting in antiweed genetically modified plants; Described herbicide is specially glyphosate.
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