CN102384905A - Chemiluminescence detection method on biomarkers based on nanoparticles and application thereof - Google Patents
Chemiluminescence detection method on biomarkers based on nanoparticles and application thereof Download PDFInfo
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- CN102384905A CN102384905A CN2010102668122A CN201010266812A CN102384905A CN 102384905 A CN102384905 A CN 102384905A CN 2010102668122 A CN2010102668122 A CN 2010102668122A CN 201010266812 A CN201010266812 A CN 201010266812A CN 102384905 A CN102384905 A CN 102384905A
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Abstract
The invention belongs to the technical field of biological detection, relating to a detection method on markers in biological samples like serious illness serum, specifically to a chemiluminescence detection method on biomarkers based on nanoparticles and application thereof. The method in the invention comprises the following steps: performing covalent immobilized specific recognition on monoclonal antibodies on a polystyrene 96 pore plate, and combining them with corresponding markers, and having sandwich reaction with gold nanoparticle modified by an aptamer directing at the marker; amplifying it by NH2OH/HAuCl4, and performing high sensitivity chemiluminescence detection on the marker. The invention can identify and detect multiple disease markers. The invention can especially offer auxiliary judging basis on markers in biomarkers like high sensitivity serum for early diagnosis on serious diseases like clinical tumor. The invention can further prepare kits for early diagnosis on serious diseases like clinical tumor.
Description
Technical field
The invention belongs to technical field of biological, relate to the detection method of mark in the biological specimens such as major disease serum, be specifically related to a kind of chemical luminescence detection method and application of the biomarker based on nanoparticle.This method especially can be the early diagnosis of major diseases such as clinical tumor, and mark auxiliary judgment foundation in the biological specimens such as high sensitivity serum is provided.
Background technology
The World Health Organization's statistics has 1,000 ten thousand people to suffer from cancer in the annual world wide, die from about about 60,000 examples of number of cancer, accounts for 12% of global death toll.China's tumor incidence and the ten big malignant tumour incidence of disease sequencing display that provide according to treatment and prevention of tumour office of China Ministry of Public Health: Chinese annual cancer new cases are about 2,200,000 people; Because of the cancer mortality number is 1,600,000 people; Just had 1.3 people to die from cancer in average per 3 minutes, and the incidence of disease of cancer is rapid ascendant trend.It is the first that wherein lung cancer, breast cancer occupy the morbidity of man, women's malignant tumour respectively, and men and women's mortality of malignant tumors is the highest is lung cancer.According to Wen Hui Daily, in the past less than in the time in 20 years, China's cancer morbidity rises 69%, and mortality ratio has increased by 29.4%, and cancer has become first killer who threatens human health.The treatment early diagnosis of cancer is crucial.By present medical level, the early-stage cancer patient has approximately more than the 80%-90% and can cure, and can not only improve survival rate after the treatment, improves patient's life quality simultaneously, and therefore, early diagnosis has crucial meaning for the cancer patient.
Lung cancer is one of the most common in the middle of the cancer, that the number that causes death is maximum malignant tumour, and its incidence of disease growth rate also is in first of each malignant tumour.Prior art disclose lung cancer be divided into ED-SCLC (smallcell lung cancer, SCLC) and non-small cell lung cancer (non small cell lung cancer, NSCLC), wherein non-small cell lung cancer accounts for 80% of lung cancer sum.Because the concealment of lung cancer onset still lacks effective examination and method of early diagnosis at present.Clinical practice shows that mostly the patient is late period when symptom occurring, and prognosis is relatively poor, and 5 years total survival rates are no more than 15%, and symptom≤10% is arranged.Have research to show, in the patients with lung cancer of diagnosis, the prognosis of operative treatment obviously improves than medium and advanced lung cancer in early days, and its survival rate can reach 70%.Clinical traditional diagnostic method has methods such as rabat, bronchoscope, phlegm cytology checking, but all lacks enough specificitys and susceptibility, has been late period when most of patients with lung cancer is made a definite diagnosis.Therefore, urgent clinical needs a kind of can early diagnosis lung cancer, be fit to the supersensitive new technology generally promoted.
In recent years, aspect the auxiliary diagnosis of lung cancer, the research of tumor markers is very active.Tumor markers be meant tumour take place with breeding in, produces or secretes and be discharged in blood, cell, the body fluid by tumour cell, reflect tumour existence and a quasi-antigen substance of growing.It has advantages such as efficient, highly sensitive, convenient, that sample is prone to obtain and wound is little, therefore detects, screening and identify that tumor markers is the research emphasis of lung cancer early diagnosis research always.
The tumor markers of prior art detects general radiation or the enzyme linked immunological kit of adopting; These detection method ubiquity detection sensitivities are low; Cause that required serum consumption is big, patient's poor compliance, high, the high deficiency of maintenance cost of detecting instrument cost, thereby be unfavorable for that they are in the application clinically of basic unit's middle and small hospital.Biochip is a new and high technology that in life science, develops rapidly in recent years, has characteristics such as high flux, microminiaturization and robotization, is one of important development direction of medical science detection, but also has a segment distance from routine clinical use at present.
Summary of the invention
The objective of the invention is for overcome that detection sensitivity is low in the prior art detection of biological mark method, the serum consumption is big, detecting instrument is complicated and diagnostic fees with high deficiency; The new detection method of mark in the biological specimens such as a kind of major disease serum is provided, is specifically related to a kind of chemical luminescence detection method and application of the biomarker based on nanoparticle.
In the inventive method, Covalent Immobilization specific recognition monoclonal antibody on polystyrene 96 orifice plates combines with corresponding mark, carries out sandwich reaction with the gold nano particulate of modifying to the aptamers of this mark again; Adopt NH subsequently
2OH/HAuCl
4Amplification is carried out the high sensitivity chemiluminescence detection of mark through the gold nano particulate that immune response combines.Multiple major disease mark can be discerned, detected to the inventive method.
Particularly, the chemical luminescence detection method of the biomarker based on nanoparticle of the present invention is characterized in that it comprises step:
1) coated antibody is attached to preparation carrier antibody compound on polystyrene 96 orifice plates, seals subsequent use;
2) preparation gold nano particulate;
3) the plain gold nano particulate of modifying of preparation strepto-affinity;
4) preparation is to the gold nano particulate of target antigen aptamers dna modification;
5) NH
2OH/HAuCl
4After the amplification, add luminol, AgNO
3Substrate carries out chemical luminescent detecting.
Among the present invention, use polystyrene 96 orifice plates to combine coated antibody as carrier, after mark in the biological specimens such as standard solution or serum combines, add the gold nano particulate that aptamers is modified, formation sandwich compound is through NH
2OH/HAuCl
4After the amplification, carry out the chemiluminescence quantitative measurement;
Among the present invention, an end of said mark combines with coated antibody, and the other end combines with the gold nano particulate of modifying to the aptamers of this mark;
Among the present invention, use the gold nano particulate thing that serves as a mark, measure described major disease mark, amplify chemical luminescent detecting;
Among the present invention, with aptamers DNA identification antigen;
Among the present invention, aptamers is at first modified to gold nano particulate, carries out immune response then;
Among the present invention, the gold nano particulate that combines through immune response on 96 holes is through NH
2OH/HAuCl
4After the amplification, can significantly improve the sensitivity of detection, with horseradish peroxidase (HRP) labelling method of present use, can reach more than 10000 times;
Among the present invention, the first-selected monoclonal antibody of said antibody, and adopt covalent bond to be fixed to 96 orifice surfaces;
Among the present invention, after above-mentioned step finished, standard model and biological specimen joined on 96 orifice plates;
Among the present invention, after above-mentioned step finished, the gold nano particulate of modifying with aptamers joined in 96 orifice plates;
Among the present invention, after above-mentioned step finishes, add luminous substrate luminol and AgNO
3, carry out chemiluminescence detection; Or behind the gold nano particulate that adds the aptamers modification, add certain density NH
2OH/HAuCl
4In 96 orifice plates,, add luminous substrate luminol and AgNO subsequently with further amplification gold nano particulate
3, carry out chemiluminescence detection.
Among the present invention, described gold nano particulate amplification procedure can adopt more than 1 time.
The present invention can further prepare the kit of the early diagnosis of major diseases such as clinical tumor.
The remarkable advantage of the inventive method has:
The sample consumption is little, diagnostic fees is low, simple to operate, the suitable popularization and hypersensitivity; Can be applicable to that saliva, urine and hair etc. conveniently are easy to get, in the AT biological specimen of patient; Be the early diagnosis of major diseases such as clinical tumor, mark auxiliary judgment foundation in the biological specimens such as high sensitivity serum is provided.
For the ease of understanding, below will describe in detail the chemical luminescence detection method of the biomarker based on nanoparticle of the present invention through concrete accompanying drawing and embodiment.What need particularly point out is; Instantiation and accompanying drawing only are in order to explain; Obviously those of ordinary skill in the art can explain according to this paper, within the scope of the invention the present invention is made various corrections and change, and these corrections and change are also included in the scope of the present invention.
Description of drawings
Below in conjunction with accompanying drawing and embodiment the present invention is further specified.
Fig. 1 is a schematic diagram of the present invention.
Embodiment
Embodiment 1
1. coated antibody is attached to preparation carrier antibody compound on polystyrene 96 orifice plates
Exemplary steps is following: with certain density antibody dilution at coating buffer (0.5M Na
2HPO
4-NaH
2PO
4, pH 8.5) in, in polystyrene 96 orifice plates of surperficial carboxyl modified, add 100 μ L coating buffers, 37 ℃ are reacted 1h down, with PBS-TW washing lotion (8mM Na
2HPO
4, 2mM NaH
2PO
4, pH 7.4,0.9%NaCl, 0.05%Tween 20) wash 3 times, each 150 μ L, every hole adds PBS solution (the 8mM Na that 150 μ L contain 10%BSA
2HPO
4, 2mM NaH
2PO
4, pH 7.4,0.9%NaCl), 37 ℃ of sealing 1h, for use.
2. preparation gold nano particulate
Exemplary steps is following: 0.01% aqueous solution of chloraurate of getting 25mL after vigorous stirring and the reflux, adds 1% sodium citrate of 0.15mL rapidly in there-necked flask, treat that solution colour does not change, and 15min again refluxes.Stop heating and stirring, make solution be cooled to room temperature.Use the filtering with microporous membrane of 0.22 μ m then, obtain the collaurum of 40nm particle diameter.
3. prepare the plain gold nano particulate of modifying of strepto-affinity
Exemplary steps is following: adopt the borate buffer solution of pH 7.4, strepto-affinity element is diluted to 20 μ g/mL, add subsequently in the colloidal gold solution, under agitation colloidal gold solution and strepto-affinity cellulose solution are mixed.Behind the 10min, add 1%BSA solution, to prevent the polymerization and the deposition of the plain gold nano particulate of modifying of strepto-affinity as stabilizing agent.
4. preparation is to the gold nano particulate of target antigen aptamers modification
Exemplary steps is following: the biotin modification aptamers DNA of 80pmol is joined in the PBS solution, add the plain gold nano particulate of modifying of 40nm strepto-affinity of 48 μ L, cumulative volume 200 μ L; 37 ℃ of reaction 1h; Centrifugal 3 minutes of 12000rpm inhales the PBS-TW washing lotion washing remove to add behind the supernatant 200 μ L, and is centrifugal once more; Wash repeatedly after 3 times and to add the PBS that 800 μ L contain 1%BSA, 4 ℃ of preservations are for use.
5. chemiluminescence detection
Exemplary steps is following: (1) joins the standard and the sample (PBS dilution, 100 μ L) of different content on the 96 good orifice plates of sealing, behind 37 ℃ of reaction 1h, washes 3 times with PBS-TW; (2) add the gold nano particulate that aptamers that 100 μ L prepare is modified, 37 ℃ of reaction 1h, PBS-TW washes 3 times; (3) the 1mM NH of adding 50 μ L
2OH and 0.1mM HAuCl
4, room temperature reaction 10 minutes, PBS-TW are washed 3 times; (4) add 50 μ L 1mM luminol (0.1M NaOH) and 0.2mM AgNO
3Detect, thus the content of mark in the quantitative sample.
Claims (10)
1. based on the chemical luminescence detection method of the biomarker of nanoparticle, it is characterized in that, use polystyrene 96 orifice plates to combine coated antibody as carrier; After mark in standard solution or the biological specimen combines; Add the gold nano particulate that aptamers is modified, form the sandwich compound, after NH2OH/HAuCl4 amplifies; Carry out the chemiluminescence quantitative measurement, it comprises step:
1) coated antibody is attached to preparation carrier antibody compound on polystyrene 96 orifice plates, seals subsequent use;
2) preparation gold nano particulate;
3) the plain gold nano particulate of modifying of preparation strepto-affinity;
4) preparation is to the gold nano particulate of target antigen aptamers dna modification;
5) NH
2OH/HAuCl
4After the amplification, add luminol, AgNO
3Substrate carries out chemical luminescent detecting.
2. method according to claim 1 is characterized in that, an end of described mark combines with coated antibody, and the other end combines with the gold nano particulate of modifying to the aptamers of this mark.
3. method according to claim 1 is characterized in that, discerns antigen with aptamers DNA in the described method.
4. method according to claim 1 is characterized in that, described aptamers is at first modified to gold nano particulate, carries out immune response then.
5. method according to claim 1 is characterized in that, described antibody is monoclonal antibody, adopts covalent bond to be fixed to 96 orifice surfaces.
6. method according to claim 5 is characterized in that, after this step finished, standard model and biological specimen joined on 96 orifice plates.
7. method according to claim 6 is characterized in that, after this step finished, the gold nano particulate of modifying with aptamers joined in 96 orifice plates.
8. method according to claim 7 is characterized in that, after this step finishes, adds luminous substrate luminol and AgNO3, carries out chemiluminescence detection; Or, add certain density NH2OH/HAuCl4 and join in 96 orifice plates, further amplify gold nano particulate, add luminous substrate luminol and AgNO3 subsequently, carry out chemiluminescence detection.
9. method according to claim 8 is characterized in that, described gold nano particulate amplification procedure can adopt more than 1 time.
10. method according to claim 1 is characterized in that, described biological specimen is serum, saliva, urine or hair.
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| CN2010102668122A CN102384905A (en) | 2010-08-27 | 2010-08-27 | Chemiluminescence detection method on biomarkers based on nanoparticles and application thereof |
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| CN2010102668122A CN102384905A (en) | 2010-08-27 | 2010-08-27 | Chemiluminescence detection method on biomarkers based on nanoparticles and application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103472052A (en) * | 2013-07-02 | 2013-12-25 | 南昌大学 | Preparation method of multifunctional nanoprobes GOx/AuNPS/DNA, and applications of multifunctional nanoprobes GOx/AuNPS/DNA in kinases detection |
| CN103868914A (en) * | 2014-02-17 | 2014-06-18 | 南昌大学 | Aptamer-based method for detecting adenosine through nanogold and hydroxylamine amplification chemiluminiscence |
| CN103954611A (en) * | 2014-04-09 | 2014-07-30 | 南昌大学 | Method for gold nanoparticle chemiluminiscence amplified detection of adenosine based on aptamer technology |
| CN104964967A (en) * | 2015-05-26 | 2015-10-07 | 福建工程学院 | Method for detecting aniline in water sample by using chemiluminescence reagent |
| CN106841603B (en) * | 2017-01-23 | 2020-11-06 | 郑州轻工业学院 | Method for high-sensitivity quantitative detection of aflatoxin B1 by using glucometer |
-
2010
- 2010-08-27 CN CN2010102668122A patent/CN102384905A/en active Pending
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| 张声森: "免疫球蛋白G 的免疫纳米金催化共振散射光谱分析", 《中国优秀硕士学位论文全文数据库》 * |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103472052A (en) * | 2013-07-02 | 2013-12-25 | 南昌大学 | Preparation method of multifunctional nanoprobes GOx/AuNPS/DNA, and applications of multifunctional nanoprobes GOx/AuNPS/DNA in kinases detection |
| CN103472052B (en) * | 2013-07-02 | 2015-08-26 | 南昌大学 | The preparation method of a kind of multifunctional nano probe GOx/Au NPs/DNA and kinase assay application thereof |
| CN103868914A (en) * | 2014-02-17 | 2014-06-18 | 南昌大学 | Aptamer-based method for detecting adenosine through nanogold and hydroxylamine amplification chemiluminiscence |
| CN103954611A (en) * | 2014-04-09 | 2014-07-30 | 南昌大学 | Method for gold nanoparticle chemiluminiscence amplified detection of adenosine based on aptamer technology |
| CN104964967A (en) * | 2015-05-26 | 2015-10-07 | 福建工程学院 | Method for detecting aniline in water sample by using chemiluminescence reagent |
| CN104964967B (en) * | 2015-05-26 | 2018-02-06 | 福建工程学院 | A kind of method that aniline in water sample is detected using chemical illuminating reagent |
| CN106841603B (en) * | 2017-01-23 | 2020-11-06 | 郑州轻工业学院 | Method for high-sensitivity quantitative detection of aflatoxin B1 by using glucometer |
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Application publication date: 20120321 |