CN101551387A - Immunization grouping analysis and detection method based on quantum point - Google Patents
Immunization grouping analysis and detection method based on quantum point Download PDFInfo
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- CN101551387A CN101551387A CNA2009100507481A CN200910050748A CN101551387A CN 101551387 A CN101551387 A CN 101551387A CN A2009100507481 A CNA2009100507481 A CN A2009100507481A CN 200910050748 A CN200910050748 A CN 200910050748A CN 101551387 A CN101551387 A CN 101551387A
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Abstract
An immunization grouping analysis and detection method based on quantum point belongs to the nano-material field, comprises the following steps: (1) dispersing quantum points into borate buffer solution, adding sulfurated diimine and second antibody, mixing, reacting, centrifugating, washing, and obtaining second antibody compound with quantum point marks; wherein, the mole ratio of the quantum points, the sulfurated diimine and the second antibody is: 60: 15: 1; or (2) dispersing the quantum points into phosphoric acid buffer solution containing 1.25 % of glutaraldehyde by volume fraction, dispersing, reacting, adding second antibody, reacting, centrifugating, washing, and obtaining second antibody compound with quantum point marks; wherein, the mole ratio of the quantum points, the glutaraldehyde and the second antibody is: 60: 15: 1; then performing immunization reaction and observation according to the standard immunization grouping detection method. The method in the invention performs observation and judgement directly by fluorescence microscope, is simple and easy, can increase the fluorescence stability, and improve analysis accuracy and detection sensitivity.
Description
Technical field
The present invention relates to a kind of detection method of field of nanometer technology, is a kind of based on the immunohistochemical analysis detection method on the quantum dot basis specifically.
Background technology
SABC is applied immunology ultimate principle---antigen-antibody reaction, it is the principle that antigen combines with antibody specificity, make developer (fluorescein, enzyme, metallic ion, the isotope) colour developing of labelled antibody determine histocyte endoantigen (polypeptide and protein) by chemical reaction, to its position, qualitative and quantitative research.Because antibody combines the immune complex of back formation with antigen be colourless, therefore, also need the antigen-antibody reaction position be shown by means of histochemical method.By antigen-antibody reaction and color reaction, the chemical constitution in showed cell or the tissue can clearly be seen the antigen-antibody reaction product that takes place in the cell at microscopically, thereby can be determined distribution, the content of some chemical constitution in the cell or tissue original position.Everyly can make antigen or haptenic material in tissue or the cell, detect as all available corresponding specific antibodies such as protein, polypeptide, amino acid, polysaccharide, phosphatide, acceptor, enzyme, hormone, nucleic acid and pathogen.As a kind of simple to operate, detect fast, method of protein detection in the tissue samples of reliable results, SABC is applied in medical research and the clinical diagnosis just more and more.
What be used for pathological diagnosis mainly contains immunofluorescence technique and immunoenzyme.Immunofluorescence technique is one of method of widespread use in modern biology and the medical science, comprise fluorescence antibody and fluorescent antigen technology, have the specificity of antigen-antibody reaction, the rapidity of staining technique, the accuracy of on cell or tissue, locating, and the advantages such as sensitivity of fluorescent effect.But mostly fluorescein at present commonly used is organic dyestuff, exist fluorescence intensity in time prolongation and disappear gradually, the result is difficult for shortcomings such as long preservation, is subjected to certain limitation in popularization and application.
Quantum dot (QDs) refer in particular to radius less than or near the semiconductor nanoparticle of exciton Bohr radius, because it is narrow and have size " tuning " characteristic that it has emission spectrum, can excite different quantum dots with single wavelength, the fluorescence quantum yield height, good stability, advantages such as good biocompatibility, aspect the biological medicine sign, research and using value that quantum dot is huge progressively are expected.By quantum dot being connected on antigen or the antibody, form quantum dot-labeled antigen (antibody) compound, directly carry out chromogenic reaction by means of histochemical method, can showed cell or tissue in chemical constitution, under fluorescent microscope, can clearly see in the cell or the antigen-antibody reaction product that takes place in the tissue, thereby can determine the distribution and the content thereof of some chemical constitution in the cell or tissue original position.In addition, wide absorption peak and the narrow emission peak of quantum dot makes that carrying out the detection of polychrome multichannel analysis in a simple system becomes possibility.
Immunohistochemical analysis method on the quantum dot basis has the following advantages: light stability increases, and high sensitivity is more highly sensitive or suitable with existing chemiluminescence method; Linear strong, improve expressing quantity; Compatible with existing imaging system, without specialized equipment; Save time and cost; Polychrome detects, and can observe the existence of a plurality of antibody and determine their concentration at same detection window, detects than monochrome to reduce cost.With quantum dot-labeled method for immunohistochemical detection, can quicken and improve the sensitivity of detection and the accuracy of analysis greatly.
Find through literature search prior art, people such as Chen Honglei are at " Wuhan University's journal (medicine) " (2001,29 (5), P607～609) " application of quantum dot immune labelling technique on the cancerous lung tissue chip " literary composition of delivering on, this article mainly is that the characteristics of utilizing the Streptavidin compound (QDs-SA) of QDs mark to combine with biotinylation two anti-IgG are carried out albumen detection of expression on the cancerous lung tissue chip, and whether the location that is used for estimating albumen is accurate.Mainly be the detection of carrying out at the cancerous lung tissue chip that the lung cancer different phase is formed, but not directly on histotomy, carry out the detection of protein expression in the multiple tissue; Selected quantum dot is to have carried out the hydrophobic function modification, but not directly water-soluble and have a quantum dot of functional group.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art, provide a kind of based on the immunohistochemical analysis detection method on the quantum dot basis.Method of the present invention is directly observed judgement with fluorescent microscope, has saved dyeing, redyes, dehydration, operation such as transparent, and is more simple, saves time and cost; And the increase of fluorescence light stability, to compare with existing immunohistochemistry coloration method, the accuracy of analysis and the sensitivity of detection increase.
The present invention is achieved by the following technical solutions, the present invention includes following steps:
Step 1 has carboxyl or amino water-soluble quantum dot and two anti-connections with the surface,
(1) if the surface has the water-soluble quantum dot of carboxyl, then at first quantum dot is dispersed in the borate buffer solution, adding sulfuration diimine and two resists then, mixes, and 25 ℃ were reacted 3 hours down, and centrifugal, washing obtains two quantum dot-labeled anti-compounds;
Wherein, quantum dot: sulfuration diimine: two anti-mol ratios are 60: 15: 1;
(2) if the surface has amino water-soluble quantum dot, then at first quantum dot is dispersed in and contains in the phosphate buffer solution that volume fraction is 1.25% glutaraldehyde, ultrasonic making it disperseed fully, 25 ℃ of following shaking table priming reactions 3 hours, add two and resist, reacted 24 hours down at 4 ℃, centrifugal, washing obtains two quantum dot-labeled anti-compounds;
Wherein, quantum dot: glutaraldehyde: two anti-mol ratios are 60: 15: 1;
Step 2 is put into dimethylbenzene earlier with section preparation and is taken off cured 2 times, inserts gradient aquation in the absolute ethyl alcohol then, washing, hydrogen peroxide deactivation endogenous hydrogen peroxidase, the microwave antigen retrieval, the washing back is sealed with the normal goats NIS, dripping one resists, 4 ℃ of reactions are spent the night, and phosphate buffer solution is washed the back and added two quantum dot-labeled anti-compounds, the following 37 ℃ of reactions of lucifuge condition 30～60 minutes, the phosphate buffer solution rinsing, the glycerine mounting; Phosphate buffer solution replaces one to resist as negative control, observes sample under fluorescent microscope.
In the step 1, the pH of described borate buffer solution is 9.18.
In the step 1, the pH of described phosphate buffer solution is 7.4.
In the step 1, described surface has carboxyl or amino water-soluble quantum dot is InP, InAs, InCaAs, InAs/Cdse, InAs/ZnSe, InAs/InP, ZnS, CdS, HgS, ZnSe, CdSe, HgSe, PbSe, HgTe, CaAs, CdS/ZnS, CdS/Pbs, ZnS/CdS, ZnS/CdS, CdSe/CdS, CdSe/Cuse, CdSe/HgTe, CdSe/ZnSe, CdSe/HgSe, CdTe, MgS, Mgse, MgTe, CaS, the combination of one or more among Case or the CaTe.
In the step 1, described surface has carboxyl or amino water-soluble quantum dot, and emission wavelength is 520nm～680nm.
In the step 2, described one anti-freeze-dried powder one for phosphate buffer solution dilution in 1: 500 is anti-.
In the step 2, described two quantum dot-labeled anti-compounds are specially, and the two quantum dot-labeled anti-compounds that step 1 is obtained dilute 300 times.
The present invention utilizes the fluorescent characteristic of quantum dot, with itself and antibody (antigen) coupling, directly with cell or tissue in related antigen (antibody) reaction and colour developing, with determine cell or organize in chemical constitution and distribution thereof.The present invention is according to the difference of quantum dot radius, to occur the positive of various greens, yellow, orange-red fluorescence signal in the cell.
The present invention has following beneficial effect: the present invention is based on the immunohistochemical method basis of standard, utilize the fluorescence property of nano-quantum point, process color that need not be traditional, but directly observe judgement with fluorescent microscope, saved dyeing, redyed, dehydration, operation such as transparent, more simple, save time and cost; And the increase of fluorescence light stability, to compare with existing immunohistochemistry coloration method, the accuracy of analysis and the sensitivity of detection increase.
Embodiment
Below embodiments of the invention are elaborated: present embodiment is being to implement under the prerequisite with the technical solution of the present invention, provided detailed embodiment and concrete operating process, but protection scope of the present invention is not limited to following embodiment.
Embodiment
Detection method is analyzed the expression of BRCAA1 albumen in the kinds of tumors tissue, the one anti-IgG polyclonal antibody of selecting the neoantigen epi-position of the anti-people BRCAA1 of rabbit for use, two is anti-for quantum dot-labeled goat anti-rabbit igg monoclonal antibody, and phosphate buffer solution (PBS) substitutes one and resists and makes negative control.
The prescription of borate buffer solution is:
Sodium tetraborate decahydrate 3.81g adds the distilled water constant volume to 1000mL, and pH is 9.18.
The prescription of phosphate buffer solution is:
Sodium chloride 8g
Potassium chloride 0.2g
Sodium hydrogen phosphate 1.44g
Potassium dihydrogen phosphate 0.24g
Add distilled water to 1000ml, pH transfers to 7.4.
Step 1, the water-soluble CdTe quantum dots that the surface is had carboxyl is dispersed in the borate buffer solution (pH=9.18), add sulfuration diimine (EDC) and goat anti-rabbit igg monoclonal antibody (two is anti-), whirlpool mixes vibration evenly, and 25 ℃ were reacted the reaction solution high speed centrifugation 3 hours, with phosphate buffer solution (PBS, 0.1M pH=7.4) repeatedly washing obtains two quantum dot-labeled anti-compounds (QD1-antiibody);
Perhaps the surface being had amino CdSe@ZnS water-soluble quantum dot is dispersed in and contains in the phosphate buffer solution (pH=7.4) that volume fraction is 1.25% glutaraldehyde concentration, ultrasonic making it disperseed fully, 25 ℃ of reactions of shaking table activation 3 hours, add goat anti-rabbit igg monoclonal antibody (two is anti-), reacted 24 hours down at 4 ℃, reaction solution is centrifugal, repeatedly washs two quantum dot-labeled anti-compounds (QD2-antibody) with phosphate buffer solution (pH=7.4).
Step 2 is chosen multiple different tumor tissues sample, and the immunohistochemical method of using on the quantum dot basis comes qualitative analysis to detect the expression of BRCAA1 in tumor tissues not of the same race, and analyzes in conjunction with relevant clinical data.
Tumor sample is chosen: 60 routine cancer of the stomach, 19 routine cancers of the esophagus, 18 routine lung cancer, 19 routine intestinal cancer, 20 routine urinary system tumours; 19 routine thyroid cancers, 18 routine cervical carcinomas, 11 routine lymthomas, 51 routine breast cancer, 29 routine mammary gland benign tumor samples, 30 routine normal galactophore tissues.
Above-mentioned histotomy sample is put into dimethylbenzene earlier to be taken off cured twice; Insert gradient aquation in the absolute ethyl alcohol then; Washing; Hydrogen peroxide deactivation endogenous hydrogen peroxidase, the microwave antigen retrieval; The washing back is sealed with the normal goats NIS; Choose one of freeze-dried powder state and resist, with phosphate buffer solution dilution in 1: 500, drip the IgG polyclonal antibody of the neoantigen epi-position of the anti-people BRCAA1 of an anti-rabbit after diluting, 4 ℃ of reactions are spent the night; Phosphate buffer solution is washed the quantum dot-labeled goat anti-rabbit igg monoclonal antibody (two is anti-) (QD1-antiibody or QD2-antiibody) of usefulness that the back adds dilution in 1: 300, hatches under the lucifuge condition, and 37 ℃ were reacted 30～60 minutes; Phosphate buffer solution rinsing 3 times, the glycerine mounting, Direct observation sample under fluorescent microscope, BRCAA1 is positive, and colour developing is red fluorescence, is positioned cytoplasm, is positioned nucleus on a small quantity, phosphate buffer solution replaces one to resist as negative control.
Carry out finding that the BRCAA1 positive expression rate is 38.3% (23/60) in 60 routine stomach organizations after immunohistochemical analysis on the quantum dot basis detects BRCAA1 protein expression situation for all samples, do not express (0/60) and in normal gastric mucosa, have; Positive rate is 68.4% in cancer of the esophagus; Positive rate in lung cancer is 66.7%; Positive rate in the intestinal cancer is 63.2%; Positive rate in the urinary system cancer is 35%; Positive rate in the thyroid gland cancer is 47.4%; Positive rate is 38.9% in the cervical carcinoma; Positive rate is 54.5% in the lymthoma; Positive rate is 27.6% in the mammary gland benign lesion (fibroma and lobular hyperplasia); Positive rate is 70.6% in the breast cancer, does not express (0/30) and have in normal galactophore tissue.
Detect the back discovery by carry out the BRCAA1 proteantigen in the kinds of tumors tissue specimen, BRCAA1 has expression in the kinds of tumors tissue, do not express and have in normal tissue.
Present embodiment can quicken and improve the sensitivity of detection and the accuracy of analysis greatly to be connected with two quantum dots that resist as immunohistochemical good fluorescence imaging agent.
Claims (7)
1, a kind ofly it is characterized in that, comprise the steps: based on the immunohistochemical analysis detection method on the quantum dot basis
Step 1 has carboxyl or amino water-soluble quantum dot and two anti-connections with the surface,
(1) if the surface has the water-soluble quantum dot of carboxyl, then at first quantum dot is dispersed in the borate buffer solution, adding sulfuration diimine and two resists then, mixes, and 25 ℃ were reacted 3 hours down, and centrifugal, washing obtains two quantum dot-labeled anti-compounds;
Wherein, quantum dot: sulfuration diimine: two anti-mol ratios are 60: 15: 1;
(2) if the surface has amino water-soluble quantum dot, then at first quantum dot is dispersed in and contains in the phosphate buffer solution that volume fraction is 1.25% glutaraldehyde, ultrasonic making it disperseed fully, 25 ℃ of following shaking table priming reactions 3 hours, add two and resist, reacted 24 hours down at 4 ℃, centrifugal, washing obtains two quantum dot-labeled anti-compounds;
Wherein, quantum dot: glutaraldehyde: two anti-mol ratios are 60: 15: 1;
Step 2 is put into dimethylbenzene earlier with section preparation and is taken off cured 2 times, inserts gradient aquation in the absolute ethyl alcohol then, washing, hydrogen peroxide deactivation endogenous hydrogen peroxidase, the microwave antigen retrieval, the washing back is sealed with the normal goats NIS, dripping one resists, 4 ℃ of reactions are spent the night, and phosphate buffer solution is washed the back and added two quantum dot-labeled anti-compounds, the following 37 ℃ of reactions of lucifuge condition 30～60 minutes, the phosphate buffer solution rinsing, the glycerine mounting; Phosphate buffer solution replaces one to resist as negative control, observes sample under fluorescent microscope.
2, according to claim 1ly it is characterized in that based on the immunohistochemical analysis detection method on the quantum dot basis in the step 1, the pH of described borate buffer solution is 9.18.
3, according to claim 1ly it is characterized in that based on the immunohistochemical analysis detection method on the quantum dot basis in the step 1, the pH of described phosphate buffer solution is 7.4.
4, according to claim 1ly it is characterized in that in the step 1, described surface has carboxyl or amino water-soluble quantum dot is InP based on the immunohistochemical analysis detection method on the quantum dot basis, InAs, InCaAs, InAs/Cdse, InAs/ZnSe, InAs/InP, ZnS, CdS, HgS, ZnSe, CdSe, HgSe, PbSe, HgTe, CaAs, CdS/ZnS, CdS/Pbs, ZnS/CdS, ZnS/CdS, CdSe/CdS, CdSe/Cuse, CdSe/HgTe, CdSe/ZnSe, CdSe/HgSe, CdTe, MgS, Mgse, MgTe, CaS, the combination of one or more among Case or the CaTe.
5, according to claim 1ly it is characterized in that based on the immunohistochemical analysis detection method on the quantum dot basis that in the step 1, described surface has carboxyl or amino water-soluble quantum dot, emission wavelength is 520nm～680nm.
6, according to claim 1ly it is characterized in that in the step 2, described one anti-freeze-dried powder one for phosphate buffer solution dilution in 1: 500 is anti-based on the immunohistochemical analysis detection method on the quantum dot basis.
7, according to claim 1ly it is characterized in that based on the immunohistochemical analysis detection method on the quantum dot basis in the step 2, described two quantum dot-labeled anti-compounds are specially, 300 times of the two quantum dot-labeled anti-compounds dilutions that step 1 is obtained.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102072959A (en) * | 2010-11-16 | 2011-05-25 | 武汉大学 | Method for simultaneously labelling collagen type IV, macrophage and neovascularisation of tumors |
| CN102169090A (en) * | 2011-01-13 | 2011-08-31 | 武汉大学 | Method for preparing quantum dot marked catalase fluorescent probe |
| CN102305780A (en) * | 2011-07-05 | 2012-01-04 | 武汉大学 | Preparation method of ZnSe quantum dot mark hydrogen peroxidase fluorescent probe |
| CN102081040B (en) * | 2009-11-27 | 2012-05-30 | 中国科学院理化技术研究所 | Method for detecting enzymatic activity by using quantum dot fluorescence |
| CN102620979A (en) * | 2011-01-31 | 2012-08-01 | 上海舜百生物科技有限公司 | Bone tissue immune-histochemical antigen retrieval method and related kit |
| CN110244041A (en) * | 2019-06-26 | 2019-09-17 | 吉林大学 | It is a kind of detect albumen kit and its application |
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2009
- 2009-05-07 CN CNA2009100507481A patent/CN101551387A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102081040B (en) * | 2009-11-27 | 2012-05-30 | 中国科学院理化技术研究所 | Method for detecting enzymatic activity by using quantum dot fluorescence |
| CN102072959A (en) * | 2010-11-16 | 2011-05-25 | 武汉大学 | Method for simultaneously labelling collagen type IV, macrophage and neovascularisation of tumors |
| CN102072959B (en) * | 2010-11-16 | 2013-08-14 | 武汉大学 | Method for simultaneously labelling collagen type IV, macrophage and neovascularisation of tumors |
| CN102169090A (en) * | 2011-01-13 | 2011-08-31 | 武汉大学 | Method for preparing quantum dot marked catalase fluorescent probe |
| CN102620979A (en) * | 2011-01-31 | 2012-08-01 | 上海舜百生物科技有限公司 | Bone tissue immune-histochemical antigen retrieval method and related kit |
| CN102620979B (en) * | 2011-01-31 | 2014-06-18 | 上海舜百生物科技有限公司 | Bone tissue immune-histochemical antigen retrieval method and related kit |
| CN102305780A (en) * | 2011-07-05 | 2012-01-04 | 武汉大学 | Preparation method of ZnSe quantum dot mark hydrogen peroxidase fluorescent probe |
| CN110244041A (en) * | 2019-06-26 | 2019-09-17 | 吉林大学 | It is a kind of detect albumen kit and its application |
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Application publication date: 20091007 |