CN102759628B - Microfluidic protein chip for detecting genetically modified crops and kit of microfluidic protein chip - Google Patents
Microfluidic protein chip for detecting genetically modified crops and kit of microfluidic protein chip Download PDFInfo
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Abstract
The invention discloses a microfluidic protein chip for high-through detecting genetically modified crops. The microfluidic protein chip comprises a nucleic acid chip which is synthesized on the basis of mu ParafloTM microfluidic protein chip and contains six nucleotide sequences and 6 types of antibody-oligonucleotide composite probes, thereby simultaneously detecting 6 transgenetic proteins of trans-bacillus thuringiensis Bt insecticidal crystal protein (Cry1Ac), trans-bacillus thuringiensis Bt insecticidal crystal protein (Cry1c), trans-bacillus thuringiensis Bt insecticidal crystal protein (Cry1F), trans-bacillus thuringiensis Bt insecticidal crystal protein (Cry1Ab), soybean trypsin (SBTI) and neomycin phosphate transferase II gene (NPTII). The invention further provides a transgenic assay kit which comprises a mu ParafloTM microfluidic deoxyribose nucleic acid (DNA) chip which contains six types of probes, six types of antibody-oligonucleotide composite probes, six types of detection antibodies and the like.
Description
Technical field
The present invention relates to the technical field of detection of GMOs and microfluid protein-chip, be specifically related to a kind of high flux microfluid protein-chip and related kit for detection of multiple transgene component.
Background technology
Nearly ten years, transgenic technology obtains broad development.The kind that researchist utilizes transgenic technology to cultivate antiweed, drought-resistant, Salt And Alkali Tolerance, preventing from heavy metal and disease and insect resistance, be of high nutritive value, to improving grain yield, reduce after results loss and increase agricultural product nutritive value and have a vital role.Along with people are to genetically modified continuous research, absorption, popularization, the diversity of genetically modified crops is got over abundant.A plurality of countries and regions have in succession been ratified genetically modified crops and have been entered and commercially produce, and the cultivated area of genetically modified crops also significantly increases simultaneously.The society that develops into of genetically modified crops has brought huge economic benefit.
Yet transgenic technology has simultaneously also been brought some potential threats to the mankind's life, as some gene can make food produce toxicity after importing host, transgenosis food produces anaphylactogen, and people is developed immunity to drugs, and nutritive value of food changes etc.People pass different judgements on to genetically modified attitude.Therefore; carrying out enetically modified food research and development and business-like while; for the security to enetically modified food gives comprehensive assessment; make consumer can distinguish fast enetically modified food and wholefood; set up suitable method and transgene component in enetically modified food is identified and detected, can promote agriculture genetically modified organism safety management, ensure the safety of humans and animals and microorganism; can preserve the ecological environment, promote the further research of agriculture genetically modified organism technology simultaneously.For Protection of consumer, freely select the rights and interests of food, the whole world has more than 40 country to set the upper limit of transgenosis content, and in European Union, its standard is 0.9%, and Korea S is 3%, and Germany is 0.5%, and Japan is 5%.
Transgene marking protein is the important object of transgenosis monitoring, the outstanding feature thing that it is not only measured as transgenosis, and can reflect the functional characteristic of genetically modified organism, therefore the detection of transgene protein is controlled and had great importance Transgene-safty.It is enzyme linked immunosorbent assay (ELISA) that transgenic protein detects conventional method at present, but this method has the limitation of small throughput, can not detect multiple protein simultaneously.Along with the development of transgenic technology and the diffusion of technology application, the kind that contains multiple transgene component increases gradually, and in the process of circulation of transgenic product, engenders " the unknown " sample, derives from field and pollutes or plant in violation of rules and regulations.For example on China Duo Sheng market, find not allow business-like genetically modified crops product, in the product in China's export Europe, be also detected transgene component.Can not greatly have been damaged trade prestige by the existence of the unknown transgenic product that detects in time and find, and people healthy and safe brought to threat.Therefore, the high-flux detection method to transgene protein, realizes the transgene protein composition that detects in same single test and judge all " hiding ", and Chinese crops trade and the healthy and safe of the mankind are had great importance.
Microfluid protein-chip is advanced person's a biochip technology both at home and abroad, and it adopts special fluid structure, and the reaction in biochip can be carried out in liquid environment.Micro-fluid chip system generally comprises the reaction chamber, stream of pump, sealing etc., is responsible for sensing, conveying, detection and the control of micro fluid, and in conjunction with imaging systems such as scanners, reaction result is carried out quantitatively, comprehensively analyzed.Microfluid formula compare a pattern chip have advantages of numerous, as have stable, be difficult for to pollute, detect the characteristics such as amount of samples is little, miniature, robotization.
Summary of the invention
The technical problem to be solved in the present invention is to provide a kind of microfluid protein-chip and kit thereof that detects genetically modified crops.
In order to solve the problems of the technologies described above, the invention provides the microfluid protein-chip that a kind of high flux detects transgene protein, comprise the Paraflo based on μ
tMnucleic acid chip and 6 kinds of antibody-oligonucleotide complex probes containing six kinds of nucleotide sequences that microfluid biochip synthesizes, thus realize turning Su Yun gold bud bubble bacillus Bt insecticidal crystal protein (Cry1Ac), turn the golden bud bubble of Su Yun bacillus Bt insecticidal crystal protein (Cry1c), turn Su Yun gold bud bubble bacillus Bt insecticidal crystal protein (Cry1F), turning when Su Yun gold bud steeps bacillus Bt insecticidal crystal protein (Cry1Ab), soybean trypsin (SBTI) and this 6 kinds of transgene proteins of neomycin phosphoric acid based transferase II gene (NPT II) and detect.
As high flux of the present invention, detect the improvement of the microfluid protein-chip of transgene protein:
Six kinds of nucleotide sequences are:
A：3' CAC ACG CCT TTA CGA AGA CGA T 5'
B：3' CAG GTC AAA AGG GTC CTT AGG GA 5'
C：3' TAG TGT GTT TCC GTT GAA AAC A 5'
D：3' TAG TTG TCT GTA ATT AAC CCG CG 5'
E：3' AAA CCG TTA CCA TCT TGA GTG TGG C 5'
F：3' TTA ACG TGC CAT AGG TAG ACA T 5' ；
Nucleotide sequence in 6 kinds of antibody-oligonucleotide complex probes and the corresponding antibody that detects are:
A:5'GTG TGC GGA AAT GCT TCT GCT A 3', anti-Cry1Ac rabbit monoclonal antibody;
B:5'GTC CAG TTT TCC CAG GAA TCC CT 3', how anti-anti-Cry1c rabbit is;
C:5'ATC ACA CAA AGG CAA CTT TTG T 3', anti-Cry1Ab rabbit monoclonal antibody;
D:5'ATC AAC AGA CAT TAA TTG GGC GC 3', how anti-anti-Cry1F rabbit is;
E:5'TTT GGC AAT GGT AGA ACT CAC ACC G 3', how anti-anti-SBTI rabbit is;
F:5'AAT TGC ACG GTA TCC ATC TGT A 3', anti-NPT II rabbit monoclonal antibody.
As high flux of the present invention, detect the further improvement of the microfluid protein-chip of transgene protein:
Antibody-oligonucleotide complex probe, its synthetic method is according to SoluLink
tMcross-linking reagent box is cross-linked corresponding antibodies and oligonucleotide chain.
As high flux of the present invention, detect the further improvement of the microfluid protein-chip of transgene protein:
According to SoluLink
tMthe compound that cross-linking reagent box is cross-linked gained to corresponding antibodies and oligonucleotide chain possesses following characteristics: 1) 3 ' of nucleic acid end is connected with the free amino group end of lysine on antibody; 2) on average on each antibody, be connected with 2-4 bar oligonucleotides; 3) antibody has good activity.
The present invention also provides a kind of transgenosis detection kit simultaneously, comprises following composition:
1. the μ Paraflo that, contains 6 kinds of probes
tMmicrofluid DNA chip is (that is, based on μ Paraflo
tMthe nucleic acid chip containing six kinds of nucleotide sequences that microfluid biochip is synthetic);
2., 6 kinds of antibody-oligonucleotide complex probes;
3., 6 kinds are detected antibody;
4., sealing damping fluid;
5., lavation buffer solution;
6., reaction buffer;
5. 4. 2. 1. described composition be used for the instant microfluid protein-chip that high flux as above detects transgene protein that generates.
Improvement as transgenosis detection kit of the present invention:
Composition 3. 6 kinds detects antibody and is: how anti-the anti-Cry1Ac rabbit of biotin labeling is, the anti-Cry1c rabbit of biotin labeling monoclonal antibody, anti-Cry1Ab mouse-anti, anti-Cry1F mouse-anti, anti-SBTI mouse-anti and anti-NPT II mouse-anti.
Further improvement as transgenosis detection kit of the present invention:
The preparation method of sealing damping fluid is:
1. prepare 6 * SSPE damping fluid, regulating pH is 6.8;
2. 6 * SSPE damping fluid and the 25mL formamide got after the adjusting pH of the above-mentioned 1. gained of 75mL mix;
3. prepare BSA solution: the BSA powder that takes 0.2mg is dissolved in 1mL distilled water;
4. prepare 100 μ L sealing damping fluids: get 5 μ L steps 3. the BSA solution of gained add the 2. solution of gained of 95 μ L steps, mix;
5. conventional sterilizing;
The preparation method of lavation buffer solution is: sealing damping fluid and water are mixed according to the volume ratio of 1:1, obtain mixed liquor, add the SDS of 1mg evenly to mix in the mixed liquor of every 500ml; Conventional sterilizing;
The preparation method of reaction buffer is: the BSA solution that the mass concentration that adds 25 μ L in the PBS damping fluid (PH=7.4) of every 475 μ L is 20%; Conventional sterilizing.
The present invention also provides the using method of above-mentioned transgenosis detection kit simultaneously, comprises the steps:
1), immediately generate microfluid protein-chip;
2), on the protein-chip (being microfluid protein-chip) generating, carry out sample detection, concrete steps are as follows:
Transgenosis is detected to sample (genetically modified crops) and after protein extraction and purifying, obtain sample detection liquid; By 50 μ L sample detection liquid access miniflow systems, flow velocity is set to 20 μ L/min, night incubation under 4 ℃ of conditions; After reaction finishes, with PBST damping fluid, at 30 ℃, wash chip 20min, wash away the unconjugated sample of chip surface;
3), detect immobilized antigen:
Relate to 6 kinds and detect antibody;
Relating to 2 kinds of substance that show colors comprises: 1. Cy3 labelled streptavidin; 2. the sheep anti-mouse antibody of Cy3 mark;
Prepare to detect solution: get 500 μ L reaction buffers, by 6 kinds, detect antibody and with 5000-50000, doubly dilute and be mixed in reaction buffer respectively, 2 kinds of substance that show colors are all diluted in reaction buffer with 1000 times;
4), by the above-mentioned detection solution access circulation system, under room temperature, react 2 hours, under room temperature, with PBST damping fluid, chip is washed to 20min, thereby washes away unnecessary detection antibody and substance that show color;
5) scanning: take out chip, use chip scanner Genepix
tM4000B extracts chip results, and sweep parameter is selected PMT500, focal length 190; Observe the signal on each site of chip.
Improvement as the using method of transgenosis detection kit of the present invention:
Step 1) comprises the following steps:
The cleaning of I, the chip circulation system;
The sealing of II, chip surface:
Using as composition 1. containing the μ Paraflo of 6 kinds of probes
tMmicrofluid DNA chip is inserted the sealing of carrying out chip surface in microfluid hybridization chamber;
III, probe are fixed chip surface:
Using as composition 6 kinds of antibody-nucleic acid complexes probes 2. with each 50nM concentration mixed diluting in sealing damping fluid, connect into the circulation system of chip, with circulation in flow velocity 20 μ L/ minutes, hatch 2h at 30 ℃; After probe fixation reaction completes, lavation buffer solution is moved in sample hose, with 20 μ L/ minute circulation cleaning chip surface 20 minutes, the unnecessary unconjugated antibody probe of chip surface is washed away at 30 ℃.
First object of the present invention is to provide a kind of transgenosis microfluid protein-chip that can immediately generate, and this chip has high specificity, the feature that highly sensitive, flux is high, reagent dosage is few, easy to operate.
Another object of the present invention is the exploitation kit relevant to this chip detection technology, provides accurate and effective, is convenient to the method for the detection transgenosis sample of result judgement.
The microfluid system using in the present invention is based on μ Paraflo
tMchip system (LC SCIENCES, the U.S.), innovative point is to be chip part.
Microfluid protein-chip of the present invention, the formation of chip by by 6 kinds of antibody-oligonucleotide complex probes in the surface reaction of microfluid nucleic acid chip, by the hybridization of nucleic acid, antibody is fixed to the surface of nucleic acid chip.Instant synthetic before detection needs, to guarantee the activity of antibody probe and the high sensitivity of detection.Every kind of synthetic 6 of probe repeats site, to guarantee the reliability of testing result.The reagent needing on chip and the volume of response sample only need 20-50 μ L.
Advantage of the present invention is:
(1) provide the protein-chip detection technique of a kind of high flux (can simultaneously detect 6 kinds of transgenosis materials).
(2) adopt the method for instant synthetic protein chip, avoided protein-chip in the active decline of standing storage process middle probe, guaranteed the high sensitivity of chip.On kit basis, chip building-up process only needs 2 hours, and without any loaded down with trivial details step.
(3) in chip, comprise 6 repetitions, make testing result more reliable.
(4) the synthetic 50nM antibody-oligonucleotide complex probe that only needs 50-100 μ L of chip, reagent consumption is little.
(5) reaction only needs 20-50 μ L sample detection liquid.
(6) in conjunction with microfluidic device, easy to operate, saved detection labor capacity.
Accompanying drawing explanation
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described in further detail.
Fig. 1 is that microfluid protein-chip is for the testing result of single transgenosis material;
Left figure is Cry1Ac transgenosis blade testing result, and right figure is non-transgenic blade testing result.
Fig. 2 is that microfluid protein-chip is for the testing result of six kinds of transgenosis materials.
Embodiment
Remarks explanation: following various damping fluids used all need to carry out conventional sterilizing.
Embodiment 1, a kind of high flux detect the preparation method of the microfluid protein-chip of transgene protein, carry out successively following steps:
The first step: based on μ Paraflo
tMmicrofluid biochip original position is synthesized six kinds of not homotactic single stranded oligonucleotides, and 3 ' end of this nucleic acid is connected with chip bottom.A-F numbering is made respectively in every kind of probe (being single stranded oligonucleotide) position, and every kind of probe repeats 6 sites, and to each site numbering, for example A kind probe is counted A1, A2 ... A5, A6, other kind probes are roughly the same.Therefore one has 36 probe site on chip.The sequence of every kind of probe and numbering are as following table 1:
The sequence of table 1, probe and numbering
This being walked to synthetic nucleic acid chip and be placed in normal temperature preservation steady in a long-term (storage life is generally 2 years), is the 1. number material in following kit.
Second step: synthetic antibody-oligonucleotides chain cpd probe for 6 kinds of transgene proteins, step is specific as follows:
1), prepare the antibody probe kind of 6 kinds of genetically modified crops albumen: (1) Bt Cry1Ac rabbit monoclonal antibody; (2) how anti-Bt Cry1c rabbit is; (3) Bt Cry1Ab rabbit monoclonal antibody; (4) how anti-Bt Cry1F rabbit is; (5) how anti-soybean trypsin (SBTI) rabbit is; (6) neomycin phosphoric acid based transferase II gene (NPT II) rabbit monoclonal antibody.
Above-mentioned (1) ~ (6) for example can be purchased from Yi Kang (Hangzhou) Bioisystech Co., Ltd.
2), synthetic six kinds of nucleotide sequences, 3 ' end is made NH
2modify, synthetic by Shanghai Sheng Gong biotech firm, the sequence of nucleic acid and treat the antibody type crosslinked with it table 2 specific as follows:
Table 2
Remarks explanation: the A in sequence a and the first step is complementary, and b and B are complementary, and c-C is complementary, and d-D is complementary, and e-E is complementary, and f-F is complementary.
3), with reference to SoluLink
tMcross-linking reagent box is cross-linked corresponding antibodies (described in step 1)) and oligonucleotide chain (described in table 2); Being characterized as of compound: 1) 3 ' of nucleic acid end is connected with the free amino group end of lysine on antibody; 2) on average on each antibody, be connected with 2-4 bar oligonucleotides; 3) antibody has good activity.
For example can adopt step specific as follows:
SoluLink
tMcross-linking reagent box provides all kit damping fluids that react required, but antibody desalination (sloughing unnecessary S-HyNiC dressing agent), oligonucleotides desalination (sloughing unnecessary S-4FB dressing agent) and purifying (finally sloughing unnecessary oligonucleotides) need be used the ultra filtration membrane that Millipore company produces in addition, (molecule interception is 10000,3000,100000 kDa) and refrigerated centrifuge.Concrete steps are as follows:
1., antibody (described in step 1)) is modified with S-HyNiC according to the requirement of kit instructions; Must revise rear antibody;
2., antibody desalination:
Antibody after modification is transferred on molecule interception 10,000 centrifugal ultrafiltration films, added 300 μ L to be cross-linked damping fluid (in kit) on filtering membrane, 4 ℃, centrifugal 20 minutes of 5000 * g, is less than 100 μ L to liquid on film; Again add the crosslinked damping fluid of 300 μ L to carry out centrifugal.Repeat aforesaid operations repeatedly, filter liquor is carried out to UV/vis monitoring with NanoDrop 1000, until detect without any absorption peak in filter liquor.The liquid of film top (be desalination after modified antibodies) is transferred in clean centrifuge tube.
3., oligonucleotide chain (described in table 2) modifies with S-4FB according to the requirement of kit instructions, oligonucleotides after must modifying.
4., oligonucleotides desalination: oligonucleotides after modification is transferred on molecule interception 3,000 centrifugal ultrafiltration films, added 300 μ L exchange buffering liquid (in kit) on film, 4 ℃, 13000 * g is centrifugal is about 50 μ L to liquid on film.Repeat aforesaid operations repeatedly, and collect the filter liquor of chimney filter below, by the residual condition of dressing agent in UV/vis method monitoring filter liquor, until detect without any absorption peak in filter liquor.The liquid of film top (be desalination after modified oligonucleotide) is collected in clean centrifuge tube.
5., crosslinked: modified oligonucleotide after modified antibodies after desalination and desalination is cross-linked according to the requirement of kit instructions.
6., purifying (removing oligonucleotides): it is 100 that the product after crosslinked is moved to molecule interception, on 000 centrifugal ultrafiltration film, add 300 μ L PBS damping fluids (PH=7.4), with 4 ℃ of refrigerated centrifuges, centrifugal several minutes of 5000 * g, is no more than 50 μ L to liquid on film.Repeat aforesaid operations repeatedly, by the residual condition of dressing agent in UV/vis method monitoring filter liquor, until detect without any absorption peak in filter liquor.Now in clean centrifuge tube, collect the crosslinked of sloughing free oligonucleotides.
Remarks explanation 1: connect several oligonucleotides on each antibody and be the molar weight ratio decision that inserts while react with dressing agent S-HyNic by antibody, calculate (illustrating according to the introduction of kit) by UV/vis absorption peak result and Beer-Lambert law.N(S-HyNic in the present invention)/n(antibody)=20.
Cross-linking process is all at consistent n(S-HyNic)/n(antibody) carry out under condition, the fluctuation of the oligonucleotides number connecting in different antibodies may cause due to antibody individual difference XOR experimental implementation, does not just affect subsequent step but the present invention the experiment proved that in this scope of 2-4.
Remarks explanation 2: experiment of the present invention shows that just can obtain antibody according to the requirement operation of kit has excellent activity, determines if wish, available ELISA method is carried out activity to the antibody after crosslinked and detected (for conventional method).
6 kinds of antibody-oligonucleotide complex probes that generate are placed in respectively-20 ℃ of short-term preservations (1 year) or-80 ℃ of long-term preservations (storage life is generally 2-3), are the 2. number material in following kit.
The 3rd step: immediately generate microfluid protein-chip, " instantaneity " refers to before detection is carried out synthetic protein chip again, and object is in order to guarantee the optimality of probe activity.Concrete steps following (first step " generation protein chip " of use kit is identical therewith):
1) cleaning of the chip circulation system
Before using chip system, the chip circulation system is cleaned.In microfluid hybridization chamber, pack an old chip (chip generating in the non-first step) into, (1%SDS(with 1mL, to be preheating to the cleansing solution of 95 ℃, the lauryl sodium sulfate of 1% mass ratio), using distilled water as solvent) pack sample hose into and access the circulation system, high rotating speed wash cycles is 30 minutes at 40 ℃; With 3mL deionized water normal temperature, rinse again.Then use the deionized water that 1mL is preheating to 95 ℃ instead, high rotating speed circulation cleaning system is 10 minutes at 40 ℃; Then by 3mL deionized water at normal temperature, rinse.
Remarks explanation: the object that old chip is packed into microfluid hybridization chamber is to connect whole miniflow system, so just can make cleansing solution flow it is washed in system.After cleaning finishes, old chip is taken out, by there being the synthetic chip of correspondent probe to be placed in hybridization chamber in kit, then carry out step 2 below).
2) sealing of chip surface
(be composition in kit 1.---the 1. number material) chip generating in the first step is inserted in microfluid hybridization chamber, get 100 μ L 6 * SSPE damping fluids access circulating systems, circulation rinse chip surface, catches up with bubble in most chip.Get 100 μ L sealing damping fluids (1%BSA, pH 6.8 for 6 * SSPE, 25% formamide) and move into sample hose, flow velocity 100 μ L/ minute, 30 ℃ of circular flows were through chip 30 minutes.
The preparation method of sealing damping fluid:
4. prepare 6 * SSPE damping fluid, regulating pH is 6.8.
5. 6 * SSPE damping fluid and the 25mL formamide got after the adjusting pH of the above-mentioned 1. gained of 75mL mix.
6. prepare BSA solution: the BSA powder that takes 0.2mg is dissolved in 1mL distilled water.
4. prepare 100 μ L sealing damping fluids: get 5 μ L steps 3. the BSA solution of gained add the 2. solution of gained of 95 μ L steps, mix.
5. conventional sterilizing.
3) probe fixing at chip surface
6 kinds of antibody-oligonucleotide complex probes prepared by second step (be composition in kit 2.---2. number material) with the concentration mixed diluting of each 50nM in the sealing damping fluid of 100 μ L, connect into the circulation system of chip, with circulation in flow velocity 20 μ L/ minutes, hatch 2h at 30 ℃.While reacting initial, alternately change flow direction several times, guarantee the bubble of chip surface to catch up with and remove, in order to avoid cause blind spot in chip.The switch of controlling flow direction is beaten to the shelves to " AUTO ", the liquid in the circulation system every 2 minutes automatic transition flow to, make sample can fill whole chip surface and each reaction chamber of flowing through.In this process, the probe that cross-linking antibody probe matches on DNA chip by DNA complementary strand is combined, and is respectively fixed to the relevant position of chip.After probe fixation reaction completes, by 1mL lavation buffer solution, (volume ratio of 1:1 is mixed sealing damping fluid and water, 0.2%SDS) move in sample hose, 20 μ L/ minute circulation cleaning chip surface is 20 minutes at 30 ℃, and the unnecessary unconjugated antibody probe of chip surface is washed away.
The preparation method of above-mentioned lavation buffer solution is: sealing damping fluid and water are mixed according to the volume ratio of 1:1, obtain mixed liquor, add the SDS of 1mg evenly to mix in the mixed liquor of every 500ml; Conventional sterilizing.
4) complete after above step, the surface-probe of synthetic protein-chip is distributed as:
A1-A6: inspection Cry1Ac albumen
B1-B6: inspection Cry1c albumen
C1-C6: inspection Cry1Ab albumen
D1-D6: inspection Cry1F albumen
E1-E6: inspection SBTI albumen
F1-F6: inspection NPT II albumen.
Embodiment 2, a kind of transgenosis microfluid protein-chip detection kit, it is used for synthesizing transgenosis microfluid protein-chip and carries out transgenosis detection; This detection kit comprises following composition:
1. the microfluid DNA chip that, contains 6 kinds of probes; According to the first step preparation of embodiment 1, obtain.
2., 6 kinds of antibody-nucleic acid complexes probes; According to the second step preparation of embodiment 1, obtain.
3., 6 kinds are detected antibody:
Comprise: how anti-the anti-Cry1Ac rabbit of biotin labeling is; The anti-Cry1c rabbit of biotin labeling monoclonal antibody; Anti-Cry1Ab mouse-anti; Anti-Cry1F mouse-anti; Anti-SBTI mouse-anti; Anti-NPT II mouse-anti.
Wherein rabbit is anti-for example can be purchased from Yi Kang (Hangzhou) Bioisystech Co., Ltd; Mouse-anti for example can be purchased from Huaan (Hangzhou) Bioisystech Co., Ltd.
7., sealing damping fluid (with embodiment 1);
8., lavation buffer solution (with embodiment 1);
9., reaction buffer:
Reaction buffer is: 20% (mass concentration) BSA solution that adds 25 μ L in the PBS damping fluid (PH=7.4) of every 475 μ L; Conventional sterilizing.
Embodiment 3, transgenosis microfluid protein-chip detection kit using method as described in Example 2, carry out successively following steps:
1), immediately generate microfluid protein-chip (with the 3rd step of embodiment 1);
2), on the protein-chip generating, carry out sample detection, concrete steps are as follows:
Transgenosis is detected to sample and after protein extraction and purifying, obtain sample detection liquid.By 50 μ L sample detection liquid access miniflow systems, flow velocity is set to 20 μ L/min, and hatch under 4 ℃ of conditions, spend the night (16 hours).After reaction finishes, with PBST damping fluid, at 30 ℃, wash chip 20min, wash away the unconjugated sample of chip surface.
The preparation method of PBST damping fluid is: take 8g NaCl, 0.2g KCl, 1.44g Na
2hPO
4, 0.24g KHPO
4, with 800mL distilled water, dissolve, add 2mL Tween-20, regulate pH to 7.2, add distilled water to be settled to 1L, conventional sterilizing.
Remarks explanation: protein extraction and purifying are known technology, for example, can extract method and the efficiency > > of Bt toxalbumin according to the < < being published in < < journal of Zhejiang university (agricultural and life science version) > > 02 phase of calendar year 2001 from transgenic paddy rice.
3), detect immobilized antigen:
Relate to 6 kinds and detect antibody (in kit, composition 3.)
The potpourri that relates to 2 kinds of substance that show colors comprises: 1. Cy3 labelled streptavidin (KPL company); 2. the sheep anti-mouse antibody of Cy3 mark (Proteintech Group company).
Prepare to detect solution: get 500 μ L reaction buffers, by 6 kinds, detect antibody and with 5000-50000, doubly dilute and be mixed in reaction buffer respectively, 2 kinds of substance that show colors are all diluted in reaction buffer with 1000 times.
That is, the preparation method of detection solution is:
In the reaction buffer of 500 μ L, add the anti-Cry1Ac rabbit of biotin labeling how anti-, the anti-Cry1c rabbit of biotin labeling monoclonal antibody, anti-Cry1Ab mouse-anti, anti-Cry1F mouse-anti, anti-SBTI mouse-anti and anti-NPT II mouse-anti; The sheep anti-mouse antibody that adds again Cy3 labelled streptavidin and Cy3 mark.
Remarks explanation: the volume that adds that detects each antibody in solution is determined by antibody titer, specific as follows:
Cry1Ac, Cry1c:0.1 μ L (diluting 5000 times)
Cry1F, Cry1Ab, SBTI, NPT II: after stoste is diluted to 10 times, get 0.1 μ L (diluting 50000 times);
Note: the antibody titer that different batches generates may some difference, should measure after tiring and suitably adjust.
Cy3 labelled streptavidin (total 100 μ L): get 0.1 μ L
Cy3 mark sheep anti-mouse antibody (total 100 μ L): get 0.1 μ L.
4), by the above-mentioned detection solution access circulation system, the lower reaction of room temperature (25 ℃) 2 hours, washs 20min with PBST damping fluid to chip under room temperature, thereby washes away unnecessary detection antibody and substance that show color.
5) scanning: take out chip, use chip scanner Genepix
tM4000B extracts chip results, and sweep parameter is selected PMT500, focal length 190.Observe the signal on chip A, B, C, D, E, each site of F.
When there is signal on certain site, interpret sample detects in liquid and contains the corresponding transgene protein composition in this site;
Otherwise interpret sample detects in liquid and does not contain the corresponding transgene protein composition in this site.
Test 1, according to the method for embodiment 3, utilize protein chip to detect the transgenosis material in Cry1Ac rice leaf, carry out successively following steps:
1. get 5g transgenic paddy rice blade (determine and contain Cry1Ac gene in advance) and non-transgenic rice leaf and be placed in respectively different centrifuge tubes, carry out the processing of protein extraction and purifying, for example be mainly: add 1mL PBS damping fluid (PH=7.4), with rifle head, gently press the about 2min of blade, to leachate, be green; Separating clarifying liquid after centrifugal 5min, is placed in 4 ℃ of 30min; Obtain sample detection liquid;
Remarks explanation: 2 kinds of sample detection liquid are to carry out respectively the operation of following steps 2 ~ 7.
2. use kit to generate high flux transgenosis microfluid protein-chip;
3. by 50 μ L sample detection liquid access miniflow systems, flow velocity is set to 20 μ L/min, night incubation under 4 ℃ of conditions.Reaction finishes the rear chip 20min that washs at 30 ℃ with PBST damping fluid, washes away the unconjugated sample of chip surface.
4. prepare to detect solution: get 500 μ L reaction buffers, by in kit 6 kinds detect antibody and with 5000-50000, doubly dilute and be mixed in reaction buffer respectively, by 2 kinds of substance that show colors: the sheep anti-mouse antibody of Cy3 labelled streptavidin (KPL company), Cy3 mark (Proteintech Group company) is all diluted in reaction buffer with 1000 times.
Cry1Ac, Cry1c:0.1 μ L (diluting 5000 times)
Cry1F, Cry1Ab, SBTI, NPT II: after stoste is diluted to 10 times, get 0.1 μ L (diluting 50000 times)
Cy3 labelled streptavidin (total 100 μ L): get 0.1 μ L
The sheep anti-mouse antibody of Cy3 mark (total 100 μ L): get 0.1 μ L
5. will detect the solution access circulation system, the lower reaction of room temperature (25 ℃) 2 hours, washs 20min with PBST damping fluid to chip under room temperature, washes away unnecessary detection antibody and substance that show color.
6. scanning: take out chip, use chip scanner Genepix
tM4000B reads chip results.Select PMT500, focal length 190.
7. testing result: Fig. 1 shows the testing result of Cry1Ac transgenosis blade and non-transgenic blade, and result shows, transgenosis sample obtains clear signal (A1:5587, A2:5153, A3:4508, A4:5186, A5:6558, A6:6098 on A site; Mean value: 5515; Standard deviation: 733), detect Cry1Ac protein ingredient; 6 groups of repetition consistance on chip are good.No signal in non-transgenic testing result.
Test 2, according to the method for embodiment 3, utilize protein chip to detect the multiple transgenosis material in analog sample:
1. with the preparation of PBS damping fluid, contain the mixed solution of Cry1Ac, Cry1c, Cry1F, Cry1Ab, NPT II, 6 kinds of transgene proteins of SBTI, the concentration of every kind of albumen is 1ng/mL, and simulation is containing the sample of multiple transgenosis material; As sample detection liquid;
2. use kit to generate high flux transgenosis microfluid protein-chip;
3. by 50 μ L sample detection liquid access miniflow systems, flow velocity is set to 20 μ L/min, night incubation under 4 ℃ of conditions.Reaction finishes the rear chip 20min that washs at 30 ℃ with PBST damping fluid, washes away the unconjugated sample of chip surface.
4. prepare to detect solution: get 500 μ L reaction buffers, 6 kinds of detection antibody in kit are doubly diluted and are mixed in reaction buffer with 5000-50000 respectively, the sheep anti-mouse antibody of 2 kinds of substance that show color Cy3 labelled streptavidin (KPL company), Cy3 mark (Proteintech Group company) is all diluted in reaction buffer with 1000 times.
Cry1Ac, Cry1c:0.1 μ L (diluting 5000 times)
Cry1F, Cry1Ab, SBTI, NPT II: after stoste is diluted to 10 times, get 0.1 μ L (diluting 50000 times)
Cy3 labelled streptavidin (total 100 μ L): get 0.1 μ L
Cy3 mark sheep anti-mouse antibody (total 100 μ L): get 0.1 μ L
5. will detect the solution access circulation system, the lower reaction of room temperature (25 ℃) 2 hours, washs 20min with PBST to chip under room temperature, washes away unnecessary detection antibody and substance that show color.
6. scanning: take out chip, use chip scanner Genepix
tM4000B reads chip results.Select PMT500, focal length 190.
7. the testing result that testing result: Fig. 2 is analog sample.All there is signal (as shown in table 3) in the A-F site of result display chip, proves that various transgenosis materials are all detected; Repeating signal consistance on chip is good.The intensity difference of the detection signal that transgene protein not of the same race obtains is relevant with tiring of antibody.
Table 3
Finally, it is also to be noted that, what more than enumerate is only several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, can also have many distortion.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention, all should think protection scope of the present invention.
Claims (5)
1. high flux detects the microfluid protein-chip of transgene protein, it is characterized in that: comprise the Paraflo based on μ
tMnucleic acid chip and 6 kinds of antibody-oligonucleotide complex probes containing six kinds of nucleotide sequences that microfluid biochip synthesizes, thus realize turning Su Yun gold bud bubble bacillus Bt insecticidal crystal protein (Cry1Ac), turn the golden bud bubble of Su Yun bacillus Bt insecticidal crystal protein (Cry1c), turn Su Yun gold bud bubble bacillus Bt insecticidal crystal protein (Cry1F), turning when Su Yun gold bud steeps bacillus Bt insecticidal crystal protein (Cry1Ab), soybean trypsin (SBTI) and this 6 kinds of transgene proteins of neomycin phosphoric acid based transferase II gene (NPT II) and detect;
Described six kinds of nucleotide sequences are:
A：3′CAC ACG CCT TTA CGA AGA CGA T5′
B：3′CAG GTC AAA AGG GTC CTT AGG GA5′
C：3′TAG TGT GTT TCC GTT GAA AAC A5′
D：3′TAG TTG TCT GTA ATT AAC CCG CG5′
E：3′AAA CCG TTA CCA TCT TGA GTG TGG C5′
F：3′TTA ACG TGC CAT AGG TAG ACA T5′；
Nucleotide sequence in described 6 kinds of antibody-oligonucleotide complex probes and the corresponding antibody that detects are:
A:5 ' GTG TGC GGA AAT GCT TCT GCT A3 ', anti-Cry1Ac rabbit monoclonal antibody;
B:5 ' GTC CAG TTT TCC CAG GAA TCC CT3 ', how anti-anti-Cry1c rabbit is;
C:5 ' ATC ACA CAA AGG CAA CTT TTG T3 ', anti-Cry1Ab rabbit monoclonal antibody;
D:5 ' ATC AAC AGA CAT TAA TTG GGC GC3 ', how anti-anti-Cry1F rabbit is;
E:5 ' TTT GGC AAT GGT AGA ACT CAC ACC G3 ', how anti-anti-SBTI rabbit is;
F:5 ' AAT TGC ACG GTA TCC ATC TGT A3 ', anti-NPT II rabbit monoclonal antibody.
2. high flux according to claim 1 detects the microfluid protein-chip of transgene protein, it is characterized in that:
Described antibody-oligonucleotide complex probe, its synthetic method is according to SoluLink
tMcross-linking reagent box is cross-linked corresponding antibodies and oligonucleotide chain.
3. high flux according to claim 2 detects the microfluid protein-chip of transgene protein, it is characterized in that: described according to SoluLink
tMthe compound that cross-linking reagent box is cross-linked gained to corresponding antibodies and oligonucleotide chain possesses following characteristics: 1) 3 ' of nucleic acid end is connected with the free amino group end of lysine on antibody; 2) on average on each antibody, be connected with 2-4 bar oligonucleotides; 3) antibody has good activity.
4. a transgenosis detection kit, is characterized in that comprising following composition:
1. the μ Paraflo that, contains 6 kinds of probes
tMmicrofluid DNA chip;
2., 6 kinds of antibody-oligonucleotide complex probes;
3., 6 kinds are detected antibody;
4., sealing damping fluid;
5., lavation buffer solution;
6., reaction buffer;
5. 4. 2. 1. described composition be used for immediately generating the microfluid protein-chip of the high flux detection transgene protein as described in claim 1,2 or 3;
Described composition 3. 6 kinds detects antibody and is: how anti-the anti-Cry1Ac rabbit of biotin labeling is, the anti-Cry1c rabbit of biotin labeling monoclonal antibody, anti-Cry1Ab mouse-anti, anti-Cry1F mouse-anti, anti-SBTI mouse-anti and anti-NPT II mouse-anti;
The preparation method of sealing damping fluid is:
1. prepare 6 * SSPE damping fluid, regulating pH is 6.8;
2. 6 * SSPE damping fluid and the 25mL formamide got after the adjusting pH of the above-mentioned 1. gained of 75mL mix;
3. prepare BSA solution: the BSA powder that takes 0.2mg is dissolved in 1mL distilled water;
4. prepare 100 μ L sealing damping fluids: get 5 μ L steps 3. the BSA solution of gained add the 2. solution of gained of 95 μ L steps, mix;
5. conventional sterilizing;
The preparation method of lavation buffer solution is: sealing damping fluid and water are mixed according to the volume ratio of 1:1, obtain mixed liquor, add the SDS of 1mg evenly to mix in the mixed liquor of every 500ml; Conventional sterilizing;
The preparation method of reaction buffer is: the BSA solution that the mass concentration that adds 25 μ L in the PBS damping fluid of the PH=7.4 of every 475 μ L is 20%; Conventional sterilizing.
5. the using method of transgenosis detection kit as claimed in claim 4, is characterized in that comprising the steps:
1), immediately generate microfluid protein-chip;
Comprise the following steps:
The cleaning of I, the chip circulation system;
The sealing of II, chip surface:
Using as composition 1. containing the μ Paraflo of 6 kinds of probes
tMmicrofluid DNA chip is inserted the sealing of carrying out chip surface in microfluid hybridization chamber;
III, probe are fixed chip surface:
Using as composition 6 kinds of antibody-oligonucleotide complex probes 2. with each 50nM concentration mixed diluting in sealing damping fluid, connect into the circulation system of chip, with circulation in flow velocity 20 μ L/ minutes, hatch 2h at 30 ℃; After probe fixation reaction completes, lavation buffer solution is moved in sample hose, with 20 μ L/ minute circulation cleaning chip surface 20 minutes, the unnecessary unconjugated antibody probe of chip surface is washed away at 30 ℃;
2), on the protein-chip generating, carry out sample detection, concrete steps are as follows:
Transgenosis is detected to sample and after protein extraction and purifying, obtain sample detection liquid; By 50 μ L sample detection liquid access miniflow systems, flow velocity is set to 20 μ L/min, night incubation under 4 ℃ of conditions; After reaction finishes, with PBST damping fluid, at 30 ℃, wash chip 20min, wash away the unconjugated sample of chip surface;
3), detect immobilized antigen:
Relate to 6 kinds and detect antibody;
Relating to 2 kinds of substance that show colors comprises: 1. Cy3 labelled streptavidin; 2. the sheep anti-mouse antibody of Cy3 mark;
Prepare to detect solution: get 500 μ L reaction buffers, by 6 kinds, detect antibody and with 5000-50000, doubly dilute and be mixed in reaction buffer respectively, 2 kinds of substance that show colors are all diluted in reaction buffer with 1000 times;
4), by the above-mentioned detection solution access circulation system, under room temperature, react 2 hours, under room temperature, with PBST damping fluid, chip is washed to 20min, thereby washes away unnecessary detection antibody and substance that show color;
5) scanning: take out chip, use chip scanner Genepix
tM4000B extracts chip results, and sweep parameter is selected PMT500, focal length 190; Observe the signal on each site of chip.
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| CN103060458A (en) * | 2013-01-17 | 2013-04-24 | 中国检验检疫科学研究院 | Primer, probe, kit and method for detecting transgenic rice strain T1c-19 |
| CN103160533B (en) * | 2013-03-19 | 2015-01-07 | 中国检验检疫科学研究院 | Standard molecule for specifically detecting transgenic rice strain Kefeng No.6 and application thereof |
| CN105092853A (en) * | 2014-05-06 | 2015-11-25 | 中国检验检疫科学研究院 | Liquid chip for detecting various Bt proteins and detection method |
| CN117144054B (en) * | 2023-10-27 | 2024-06-11 | 莱肯生物科技(海南)有限公司 | Nucleic acid detection method and application thereof |
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