CN102072954A - Research and application of electrochemiluminescent immunosensor for detecting tumor markers - Google Patents
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Abstract
The invention discloses a method for researching and applying an electrochemiluminescent immunosensor for detecting tumor markers. An electrode preparation method (shown in attached drawings as schematic diagrams) comprises the following steps of: selecting a tumor marker with higher clinical morbidity for detection; preparing a quantum dot nano-material solution; and preparing the electrochemiluminescent immunosensor by modifying an antibody onto an electrode surface by using an electrode surface modification technology. A method for detecting the tumor markers comprises the following step of connecting a modified electrode to an electrochemiluminescent instrument so as to detect a tumor marker in a sample. The electrode provided by the invention has strong specificity and high sensitivity and can reach ng level; and moreover, the time required for completing a basic detection process is short, and the cost is low. The method for detecting the tumor markers through the electrode has the advantages that: the operation is fast and simple, and both reactions and results are automatically completed and recorded by the instrument.
Description
Technical field
The present invention relates to tumor markers detection technique field, a kind of preparation that detects the electrochemiluminescimmunosensor immunosensor of tumor markers of more specifically saying so the invention still further relates to and adopts described electrochemiluminescimmunosensor immunosensor to carry out the method for diagnosing tumour mark.
Background technology
Tumour is the killer of human health.Malignant tumour also claims cancer, it is the class disease that current serious threatens human health and life, it be body be subjected to various in or external carcinogenic factor effect under, the cell of local organization has lost the normal regulation to himself growth on gene level, cause undesired hyperplasia and the neoformation that forms.Tumour be that long-term, multistage a, polygenes changes accumulation, have the complicacy of multifactor adjusting and controlled by multiple genes.The ascendant trend of tumor incidence and mortality ratio is directly threatening human health, and its early diagnosis and therapy is one of important preface field of treatment and prevention of tumour research.
Become in the growth course phenomenon that more and more has invasiveness of malignant tumour is called the evolution of tumour, its evolution process relate between the tumour cell and tumour and host between a series of complex interactions, the increment and the apoptosis that comprise tumour cell, the degraded of extracellular matrix, the change of tumour cell-cell, cell-matrix adhesion, the migration of cell, mobile, tumor-blood-vessel growth, escape host's immunosurveillance etc., these interact and mainly show the characteristics such as unlimited increment, aggressive and transfer of cell.In the growth of tumor process; because the subclone that can make tumour unusually of the sudden change of episome and coded product thereof obtains different characteristics, thereby the selection of body internal and external environment can make tumour keep in growth course should to survive, the subclone of growth, infiltration metastasis and can cause the generation of heterogeneousization of tumour.Therefore, strengthen to bring wide prospect for the treatment of tumor disease to the molecular basis of each link of tumour evolution and the understanding and the research of polygenes effect.
Immunoassay is to utilize antibody to combine with antigentic specificity and the high selectivity biochemical method set up.Detect and the peculiar tumor marker of positioning tumor cell with immunocytochemistry, more and more caused vast oncological pathology research worker's interest.There are many immunohistochemical methods to can be used for histiocytic qualitative and quantitative analysis and composition detection now.At present, main immune analysis method has electrochemical immunoanalytical, chemiluminescence immune assay, portable injection chemiluminescence immunoassay, high performance liquid chromatography chemiluminescence immune assay, capillary electrophoresis chemiluminescence immunoassay, fluidic chip chemiluminescence immunoassay etc.But be applied to the chemiluminescence immunoassay that mainly contains in the immunodiagnosis, electrochemical immunoanalytical method etc. are not enough but these detection methods exist:
1. chemiluminescence immunoassay has fast, the highly sensitive characteristics of finding speed, but the selectivity relative mistake some.Because types of organization's difference also can have one or more tumor markerses, therefore determine it is relatively difficulty of which kind of tumor disease with a kind of tumour by detected a certain specific tumors mark.
2. the electrochemical immunoanalytical method has characteristics easily and fast, and because current potential is controlled, has improved selectivity, can make diagnosis to different tumor markerses, and be applied to tumor treatment, but sensitivity is low relatively.
Based on the shortcoming of above-mentioned detection method, the present invention has set up a kind of high sensitivity, high selectivity, electrogenerated chemiluminescence immune analysis method easily and efficiently.
Summary of the invention
The technical problem to be solved in the present invention has provided that a kind of detection speed is fast, highly sensitive, selectivity is high, and reagent dosage is few, detects the preparation and the detection method of the electrochemiluminescimmunosensor immunosensor of tumor markers.
In order to solve the problems of the technologies described above, the present invention realizes by following measure: a kind of preparation method who detects the electrochemiluminescimmunosensor immunosensor of tumor markers is characterized in that may further comprise the steps:
(1) select clinical onset rate tumors of higher mark to measure;
(2) prepare the quantum dot nano material solution;
(3) utilize assembling surface modification technology etc., on the antibody modification electrode surface, make electrochemiluminescimmunosensor immunosensor.
Antibody modification of the present invention is measured to the immunosensor surface and to tumor markers and be may further comprise the steps:
(1) electrode used therein is used deionized water, acetone and ethanol ultrasonic cleaning respectively, used high-purity N afterwards
2Dry up;
(2) electrode that step (1) is obtained immerses 10-30min in the 4%NaOH solution, uses deionized water rinsing then, dry 1-2h under 120 ℃;
(3) specific antibody (Ab) is adsorbed onto the electrode surface of handling well, the not antibody of absorption is removed in washing behind 4 ℃ of incubation 12-24h, washs 2-5 time;
(4) electrode in the step (3) is added in the sample of determined antigen (Ag), 37 ℃ of incubation 1-2h wash 2-5 time;
(5) electrode in the step (4) is immersed in the specific antibody (AbE) of luminescence reagent mark, 37 ℃ of incubation 10-15min wash 2-5 time;
(6) will modify good electrode measures in conjunction with the electrogenerated chemiluminescence instrument.
The working electrode that uses of the present invention can be glassy carbon electrode, ITO electrode, gold electrode, platinum electrode.
Luminescence reagent of the present invention is trichlorine bipyridyl ruthenium and derivant, luminol and derivant thereof, acridinium ester class, lucigenin and quantum dot nano material.
Tumor markers of the present invention is alpha-fetoprotein (AFP), carcinomebryonic antigen, cancer embryo ferritin, pancreatic oncofetal antigen, cytokeratin, squama cancer associated antigen, prostate specific antigen (PSA), acid phosphatase (ACP), alkaline phosphatase (ALP), neuronspecific enolase (NSE), CEA, CA19-9, SCC, CA15-3, CA12-5, HCG, F-PSA, PAP, TMA, human chorionic gonadotrophin (hCG), Catecholamine matter.
A kind of method of fast detecting tumor markers is characterized in that comprising the steps: that the electrochemiluminescimmunosensor immunosensor that will make by above-mentioned any one method cooperates the electrogenerated chemiluminescence instrument, and tumor markers is detected.
Beneficial effect of the present invention:
1. the preparation method of tumor markers electrochemiluminescimmunosensor immunosensor, antibody modification is prepared electrochemiluminescimmunosensor immunosensor to electrode surface, adopt trichlorine bipyridyl ruthenium, luminol, acridinium ester, lucigenin and quantum dot nano material as luminescence reagent, make prepared tumor markers electrochemiluminescimmunosensor immunosensor have higher sensitivity and selectivity.
2. surface modification technology is applied in the middle of the preparation of electrochemiluminescimmunosensor immunosensor, make the preparation of tumor markers electrochemiluminescimmunosensor immunosensor of luminescence reagent synergy have controllability, improved the sensitivity and the accuracy of electrochemiluminescimmunosensor immunosensor.
3. the high specificity of electrochemiluminescimmunosensor immunosensor of the present invention since with antibody modification to electrode, and antigen carried out specific recognition, other non-specific molecules does not have influence to testing result in the sample; Highly sensitive, can reach the ng level; Reagent dosage is few, and detecting a sample only needs tens microlitre reagent; Cost is low.
4. electrochemiluminescimmunosensor immunosensor detects the method for tumor markers, highly sensitive, favorable reproducibility, electrode life is long, antijamming capability is strong, operation is simple fast, reaction and result finish and record automatically by instrument, avoided the influence of subjective factor, and guarantee that good repeatability is arranged, be convenient to on-the-spot the detection.
Description of drawings
Below in conjunction with the drawings and specific embodiments the present invention is done and to describe in further detail.
Accompanying drawing is the process synoptic diagram that antibody modification prepares electrochemiluminescimmunosensor immunosensor and tumor markers is detected to electrode surface.
Embodiment
A kind of electrochemiluminescimmunosensor immunosensor preparation method who detects tumor markers is characterized in that may further comprise the steps:
(1) select clinical onset rate tumors of higher mark to measure;
(2) prepare the quantum dot nano material solution;
(3) utilize assembling surface modification technology etc., on the antibody modification electrode surface, make electrochemiluminescimmunosensor immunosensor.
Antibody modification of the present invention is measured to the immunosensor surface and to tumor markers and be may further comprise the steps:
(1) electrode used therein is used deionized water, acetone and ethanol ultrasonic cleaning respectively, used high-purity N afterwards
2Dry up;
(2) electrode that step (1) is obtained immerses 10-30min in the 4%NaOH solution, uses deionized water rinsing then, dry 1-2h under 120 ℃;
(3) specific antibody (Ab) is adsorbed onto the electrode surface of handling well, the not antibody of absorption is removed in washing behind 4 ℃ of incubation 12-24h, washs 2-5 time;
(4) electrode in the step (3) is added in the sample of determined antigen (Ag), 37 ℃ of incubation 1-2h wash 2-5 time;
(5) electrode in the step (4) is immersed in the specific antibody (AbE) of luminescence reagent mark, 37 ℃ of incubation 10-15min wash 2-5 time;
(6) will modify good electrode measures in conjunction with the electrogenerated chemiluminescence instrument.
The working electrode that uses of the present invention can be glassy carbon electrode, ITO electrode, gold electrode, platinum electrode.
Luminescence reagent of the present invention is trichlorine bipyridyl ruthenium and derivant, luminol and derivant thereof, acridinium ester class, lucigenin and quantum dot nano material.
Tumor markers of the present invention is alpha-fetoprotein (AFP), carcinomebryonic antigen, cancer embryo ferritin, pancreatic oncofetal antigen, cytokeratin, squama cancer associated antigen, prostate specific antigen (PSA), acid phosphatase (ACP), alkaline phosphatase (ALP), neuronspecific enolase (NSE), CEA, CA19-9, SCC, CA15-3, CA12-5, HCG, F-PSA, PAP, TMA, human chorionic gonadotrophin (hCG), Catecholamine matter.
A kind of method of fast detecting tumor markers is characterized in that comprising the steps: that the electrochemiluminescimmunosensor immunosensor that will make by above-mentioned any one method cooperates the electrogenerated chemiluminescence instrument, and tumor markers is detected.
Embodiment 1 (the embryonal antigen class is as alpha-fetoprotein)
A kind of electrochemiluminescimmunosensor immunosensor preparation method who detects alpha-fetoprotein may further comprise the steps:
(1) select the higher alpha-fetoprotein of clinical onset to measure;
(2) utilize package technique, alpha-fetoprotein antibody is modified electrode surface, make electrochemiluminescimmunosensor immunosensor;
(3) working electrode is selected the ITO electrode for use, and electrode is used deionized water, acetone and ethanol ultrasonic cleaning respectively, uses high-purity N afterwards
2Dry up;
(4) electrode of handling well is immersed 10min in the 4%NaOH solution, use deionized water rinsing then, dry 2h under 120 ℃;
(5) CdTe quantum dot solution preparation: at N
2Under the protection, will now make NaHTe is the Te presoma, with CdCl
2Reaction under the condition of mercaptoacetic acid as stabilizing agent, makes water-soluble CdTe quantum dots solution;
(6) get the CdTe solution 20 μ l that prepare, ultrasonic 20min obtains homodisperse CdTe quantum dot solution;
(7) alpha-fetoprotein antibody is adsorbed onto the electrode surface of handling well, the not alpha-fetoprotein antibody of absorption is removed in washing behind 4 ℃ of incubation 24h, washs 3 times;
(8) electrode of handling well is added in the alpha-fetoprotein antigen samples, 37 ℃ of incubation 1h wash 3 times;
(9) the above-mentioned electrode of handling well is immersed in the quantum dot-labeled specificity alpha-fetoprotein antibody of CdTe, 37 ℃ of incubation 15min wash 3 times.
To make the alpha-fetoprotein electrochemiluminescimmunosensor immunosensor and cooperate the electrogenerated chemiluminescence instrument, the alpha-fetoprotein in human body, the animal blood serum sample will be detected, the results are shown in Table 1.Utilize existing method, preparation alpha-fetoprotein electrochemical immunosensor connects electrochemical workstation, and the alpha-fetoprotein in human body, the animal blood serum sample extracting solution is carried out actual detected, the results are shown in Table 1.
Table 1 alpha-fetoprotein electrochemiluminescimmunosensor immunosensor of the present invention and alpha-fetoprotein electrochemical immunosensor detect effect comparison
The result as can be seen from table 1: the alpha-fetoprotein electrochemiluminescimmunosensor immunosensor has the wideer range of linearity, higher sensitivity and lower detectability than alpha-fetoprotein electrochemical immunosensor.
Embodiment 2 (the carbohydrate mark is as CA19-9)
A kind of electrochemiluminescimmunosensor immunosensor preparation method who detects CA 19-9 may further comprise the steps:
(1) select the higher CA19-9 of clinical onset to measure;
(2) utilize package technique, the CA19-9 antibody modification to electrode surface, is made electrochemiluminescimmunosensor immunosensor;
(3) working electrode is selected the ITO electrode for use, and electrode is used deionized water, acetone and ethanol ultrasonic cleaning respectively, uses high-purity N afterwards
2Dry up;
(4) electrode of handling well is immersed 10min in the 4%NaOH solution, use deionized water rinsing then, dry 1h under 120C;
(5) CdTe quantum dot solution preparation: at N
2Under the protection, will now make NaHTe is the Te presoma, with CdCl
2Reaction under the condition of mercaptoacetic acid as stabilizing agent, makes water-soluble CdTe quantum dots solution;
(6) get the CdTe solution 20 μ l that prepare, ultrasonic 20min obtains homodisperse CdTe quantum dot solution;
(7) CA19-9 antibody is adsorbed onto the electrode surface of handling well, the not CA19-9 antibody of absorption is removed in washing behind 4 ℃ of incubation 12h, washs 3 times;
(8) electrode of handling well is added in the CA19-9 antigen samples, 37 ℃ of incubation 1h wash 3 times;
(9) the above-mentioned electrode of handling well is immersed in the quantum dot-labeled CA19-9 antibody of CdTe, 37 ℃ of incubation 15min wash 3 times.
To make the CA19-9 electrochemiluminescimmunosensor immunosensor and cooperate the electrogenerated chemiluminescence instrument, the CA19-9 in human body, the animal blood serum sample will be detected, the results are shown in Table 2.Utilize existing method, preparation CA19-9 electrochemical immunosensor connects electrochemical workstation, and the CA19-9 in human body, the animal blood serum sample extracting solution is carried out actual detected, the results are shown in Table 2.
Table 2 CA19-9 electrochemiluminescimmunosensor immunosensor of the present invention and CA19-9 electrochemical immunosensor detect effect comparison
The result as can be seen from table 2: the CA19-9 electrochemiluminescimmunosensor immunosensor has the wideer range of linearity, higher sensitivity and lower detectability than CA19-9 electrochemical immunosensor.
Embodiment 3 (protein-based) as cytokeratin
A kind of electrochemiluminescimmunosensor immunosensor preparation method who detects cytokeratin may further comprise the steps:
(1) select the higher cytokeratin of clinical onset to measure;
(2) utilize package technique, the cytokeratin antibody modification to electrode surface, is made electrochemiluminescimmunosensor immunosensor;
(3) working electrode is selected the ITO electrode for use, and electrode is used deionized water, acetone and ethanol ultrasonic cleaning respectively, uses high-purity N afterwards
2Dry up;
(4) electrode of handling well is immersed 10min in the 4%NaOH solution, use deionized water rinsing then, dry 2h under 120 ℃;
(5) CdTe quantum dot solution preparation: at N
2Under the protection, will now make NaHTe is the Te presoma, with CdCl
2Reaction under the condition of mercaptoacetic acid as stabilizing agent, makes water-soluble CdTe quantum dots solution;
(6) get the CdTe solution 20 μ l that prepare, ultrasonic 20min obtains homodisperse CdTe quantum dot solution;
(7) cytokeratin antibody is adsorbed onto the electrode surface of handling well, the not cytokeratin antibody of absorption is removed in washing behind 4 ℃ of incubation 20h, washs 4 times;
(8) electrode of handling well is added in the cytokeratin antigen samples, 37 ℃ of incubation 1h wash 4 times;
(9) the above-mentioned electrode of handling well is immersed in the quantum dot-labeled cytokeratin antibody of CdTe, 37 ℃ of incubation 15min wash 4 times.
To make the cytokeratin electrochemiluminescimmunosensor immunosensor and cooperate the electrogenerated chemiluminescence instrument, the cytokeratin in human body, the animal blood serum sample will be detected, the results are shown in Table 3.Utilize existing method, preparation cytokeratin electrochemical immunosensor connects electrochemical workstation, and the cytokeratin in human body, the animal blood serum sample extracting solution is carried out actual detected, the results are shown in Table 3.
Table 3 cytokeratin electrochemiluminescimmunosensor immunosensor of the present invention and cytokeratin electrochemical immunosensor detect effect comparison
The result as can be seen from table 3: the cytokeratin electrochemiluminescimmunosensor immunosensor has the wideer range of linearity, higher sensitivity and lower detectability than cytokeratin electrochemical immunosensor.
Embodiment 4 (enzyme is as acid phosphatase)
A kind of electrochemiluminescimmunosensor immunosensor preparation method of detection of acidic phosphatase may further comprise the steps:
(1) select the higher acid phosphatase of clinical onset to measure;
(2) utilize package technique, the acid phosphatase antibody modification to electrode surface, is made electrochemiluminescimmunosensor immunosensor;
(3) working electrode is selected the ITO electrode for use, and electrode is used deionized water, acetone and ethanol ultrasonic cleaning respectively, uses high-purity N afterwards
2Dry up;
(4) electrode of handling well is immersed 10min in the 4%NaOH solution, use deionized water rinsing then, dry 2h under 120 ℃;
(5) CdTe quantum dot solution preparation: at N
2Under the protection, will now make NaHTe is the Te presoma, with CdCl
2Reaction under the condition of mercaptoacetic acid as stabilizing agent, makes water-soluble CdTe quantum dots solution;
(6) get the CdTe solution 20 μ l that prepare, ultrasonic 20min obtains homodisperse CdTe quantum dot solution;
(7) the acid phosphatase enzyme antibody is adsorbed onto the electrode surface of handling well, the not acid phosphatase enzyme antibody of absorption is removed in washing behind 4 ℃ of incubation 18h, washs 2 times;
(8) electrode of handling well is added in the acid phosphatase antigen samples, 37 ℃ of incubation 2h wash 2 times;
(9) the above-mentioned electrode of handling well is immersed in the quantum dot-labeled acid phosphatase enzyme antibody of CdTe, 37 ℃ of incubation 15min wash 2 times.
To make the acid phosphatase electrochemiluminescimmunosensor immunosensor and cooperate the electrogenerated chemiluminescence instrument, the acid phosphatase in human body, the animal blood serum sample will be detected, the results are shown in Table 4.Utilize existing method, preparation acid phosphatase electrochemical immunosensor connects electrochemical workstation, and the acid phosphatase in human body, the animal blood serum sample extracting solution is carried out actual detected, the results are shown in Table 4.
Table 4 acid phosphatase electrochemiluminescimmunosensor immunosensor of the present invention and acid phosphatase electrochemical immunosensor detect effect comparison
The result as can be seen from table 4; The acid phosphatase electrochemiluminescimmunosensor immunosensor has the wideer range of linearity, higher sensitivity and lower detectability than acid phosphatase electrochemical immunosensor.
Embodiment 5 (steroids is as human chorionic gonadotrophin (hCG))
A kind of electrochemiluminescimmunosensor immunosensor preparation method who detects human chorionic gonadotrophin may further comprise the steps:
(1) select the higher human chorionic gonadotrophin of clinical onset to measure;
(2) utilize package technique, hCG antibodies is modified electrode surface, make electrochemiluminescimmunosensor immunosensor;
(3) working electrode selects the ITO electrode, and electrode is used deionized water, acetone and ethanol ultrasonic cleaning respectively, uses high-purity N afterwards
2Dry up;
(4) electrode of handling well is immersed 10min in the 4%NaOH solution, use deionized water rinsing then, dry 2h under 120 ℃;
(5) CdTe quantum dot solution preparation: at N
2Under the protection, will now make NaHTe is the Te presoma, with CdCl
2Reaction under the condition of mercaptoacetic acid as stabilizing agent, makes water-soluble CdTe quantum dots solution;
(6) get the CdTe solution 20 μ l that prepare, ultrasonic 20min obtains homodisperse CdTe quantum dot solution;
(7) hCG antibodies is adsorbed onto the electrode surface of handling well, the not hCG antibodies of absorption is removed in washing behind 4 ℃ of incubation 24h, washs 5 times;
(8) electrode of handling well is added in the human chorionic gonadotrophin antigen samples, 37 ℃ of incubation 2h wash 5 times;
(9) the above-mentioned electrode of handling well is immersed in the quantum dot-labeled hCG antibodies of CdTe, 37 ℃ of incubation 15min wash 5 times.
To make the human chorionic gonadotrophin electrochemiluminescimmunosensor immunosensor and cooperate the electrogenerated chemiluminescence instrument, the human chorionic gonadotrophin in human body, the animal blood serum sample will be detected, the results are shown in Table 5.Utilize existing method, preparation human chorionic gonadotrophin electrochemical immunosensor connects electrochemical workstation, and the human chorionic gonadotrophin in human body, the animal blood serum sample extracting solution is carried out actual detected, the results are shown in Table 4.
Table 5 inventor human chorionic gonadtropin electrochemiluminescimmunosensor immunosensor and human chorionic gonadotrophin electrochemical immunosensor detect effect comparison
The result as can be seen from table 5: the human chorionic gonadotrophin electrochemiluminescimmunosensor immunosensor has the wideer range of linearity, higher sensitivity and lower detectability than human chorionic gonadotrophin electrochemical immunosensor.
Claims (6)
1. preparation method who detects the electrogenerated chemiluminescence sensor of tumor markers is characterized in that may further comprise the steps:
1.1 selecting clinical onset rate tumors of higher mark measures;
1.2 the nano material of utilization is prepared nano material according to existing method;
1.3 prepare quanta point material solution;
1.4 utilize assembling surface modification technology etc., on the antibody modification electrode surface, make electrochemiluminescimmunosensor immunosensor.
2. according to the described electrode preparation method of claim 1, it is characterized in that specific antibody modified the immunosensor electrode surface and tumor markers measured may further comprise the steps:
2.1 electrode used therein is used deionized water, acetone and ethanol ultrasonic cleaning respectively, uses high-purity N afterwards
2Dry up;
2.2 the electrode that step 2.1 is obtained immerses 10-30min in the 4%NaOH solution, uses deionized water rinsing then, dry 1-2h under 120 ℃;
2.3 (Ab) is adsorbed onto the electrode surface of handling well with specific antibody, the not antibody of absorption is removed in washing behind 4 ℃ of incubation 12-24h, washs 2-5 time;
2.4 the electrode in the step 2.3 is added in the sample of determined antigen (Ag), 37 ℃ of incubation 1-2h wash 2-5 time;
2.5 the electrode in the step 2.4 is immersed in the specific antibody (AbE) of luminescence reagent mark, 37 ℃ of incubation 10-15min wash 2-5 time;
2.6 will modify good electrode measures in conjunction with the electrogenerated chemiluminescence instrument.
3. according to the described electrode preparation method of claim 1, it is characterized in that the described working electrode that uses can be glassy carbon electrode, ITO electrode, gold electrode, platinum electrode.
4. according to the described electrode preparation method of claim 1, described luminescence reagent is trichlorine bipyridyl ruthenium and derivant, luminol and derivant thereof, acridinium ester class, lucigenin and quantum dot nano material.
5. electrode preparation method according to claim 4, described tumor markers are alpha-fetoprotein (AFP), carcinomebryonic antigen, cancer embryo ferritin, pancreatic oncofetal antigen, cytokeratin, squama cancer associated antigen, prostate specific antigen (PSA), acid phosphatase (ACP), alkaline phosphatase (ALP), neuronspecific enolase (NSE), CEA, CA19-9, SCC, CA15-3, CA12-5, HCG, F-PSA, PAP, TMA, human chorionic gonadotrophin (hCG), Catecholamine matter.
6. the method for a fast detecting tumor markers is characterized in that comprising following, step: will cooperate the electrogenerated chemiluminescence instrument by the electrochemiluminescimmunosensor immunosensor that above-mentioned any one method makes, tumor markers is detected.
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