CN1614405A - Electrochemcial immunoassay for tumor marker and small size immunoassay chip - Google Patents
Electrochemcial immunoassay for tumor marker and small size immunoassay chip Download PDFInfo
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Abstract
An electrochemical method for determining immunity of tumor marker includes forming microreaction cell, placing reference and counter electrode above it and fixing antigen molecular functioning film at pool bottom, connecting enzyme labelled antibody to pool bottom at time of only proper quantity of horseradish peroxidase to label tumor marker antibody, obtaining cataltyic current on working electrode by contacting free immune matter with electronic media and having positive ratio of the current to antigen. The microvolume immune determining chip is also disclosed.
Description
Technical field
The present invention relates to the long-pending immunoassay chip of the method for immunity of tumor markers and microbody thereof, especially the chip method of immunity of the tumor markers of setting up with the electrochemical analysis technology.
Background technology
Immunoassay is to utilize antigen/antibody to react to measure a kind of high sensitivity of trace materials, the method for high selectivity, and it provides powerful measure for the clinical immunoassay of blood serum tumor markers.Usually the clinical immunoassay method of blood serum tumor markers adopts " double-antibody sandwich " method mostly, as radio immunoassay, enzyme linked immunosorbent assay, time-resolved fluorescence method, chemiluminescence and electrochemiluminescence method etc.Characteristics such as radio immunoassay disturbs and lacks because its mark is easy, highly sensitive, and range of application is wide were once becoming the most frequently used method in the clinical immunology check.But it need carry out hot operation, human body is constituted harm, limitation such as the reagent life-span is also limited, impel people constantly to explore nonradioactive labeling's immune analysis method, thereby EIA enzyme immunoassay, fluorescence and a series of nonradioactive labelings' such as time resolution immunoassay, chemiluminescence immune assay and electrochemical immunoanalytical new method is developed in succession.These methods have greatly promoted robotization, the intellectuality and networked of weeding out the old and bring forth the new of clinical immune detection project and detection means.Yet these methods need adopt spectacle case by plate (pipe), and testing process needs the incubation of long period, repeatedly washes the detection of plate and instrumentation, and sense cycle is long, complex operation step, cost height.Although occurred automatic lmunoassays analyzer, but test item is limited, instrument and reagent are quite expensive, are difficult to promote the use of, thereby are restricted in the application aspect early diagnosis, prognosis monitoring and the extensive examination of malignant tumour.How simplify the operation step, shorten analysis time, reduce cost of determination, develop new immunoassay technology, improve sensitivity, the accuracy of detection means, development minitype portable detecting instrument is problem demanding prompt solutions with the needs that adapt to extensive examination, early diagnosis and prognosis monitoring to malignant tumour.
At present, can be used for minitype portable detecting instrument and coherent detection chip thereof that blood serum tumor markers to diagnosing early malignant tumor carries out immunoassays rarely reports.Electrochemical analysis has many superiority, and is Miniaturized as test probe, not influenced by system turbidity and color, and method is highly sensitive, speed is fast, cost is low, harm is little etc.It also has the characteristics that detecting instrument is simple, be easy to microminiaturization, and the Amperometric Detection Coupled signal intensity wherein and the concentration of test substance have linear relationship, the range of linearity is wide, highly sensitive, thereby can directly detection signal be converted to the concentration value of readability directly perceived, is convenient to the layman and uses.By changing senser element and applying current potential, can realize a tractor serves several purposes easily.
Screen printing technique has been widely used in electrochemical sensor and has prepared the aspect with strong, the suitable various types of printing ink of its black bed thickness covering power, mode of printing characteristics such as restriction versatile and flexible, that be not subjected to stock size and proterties, the disposable electrode cost that is obtained is low, mutual good reproducibility, the result accurately, reliably, under 25 ℃ of drying conditions, the stability of sensor can keep 1 year.Therefore the fast detecting instrument that develops portable blood serum tumor markers immune detection with electrochemical analysis method is present available optimal path.
The solution-operated and the detected set of routine are formed in a chip, and it is not only reduced to the consumption of sample and reagent and receives or microlitre, has also improved separation efficiency and detection speed, thereby enjoys people to pay close attention in recent years.Yet this technology is mainly used in genetic analysis and expression study at present, relates to a kind of biochip test oncogene as CN03126932.X, relates to very few for immune detection.Because clinical diagnosis needs, the protein chip that is used for the tumor markers detection has caused concern, but the method for having appeared in the newspapers costs an arm and a leg, is difficult to popularize.Being used for minitype portable detecting instrument and coherent detection chip thereof that blood serum tumor markers to diagnosing early malignant tumor carries out immunoassays does not appear in the newspapers as yet.Therefore, develop new immunologic detection method, outdoorization, universalness and the family oriented of observing for the early diagnosis, curative effect and the prognosis that realize malignant tumour provide the means of necessity very meaningful.
Summary of the invention
The present invention seeks to: provide a kind of chip method of immunity and determinator----microbody that relates to tumor markers to amass detection chip, set up the chip method of immunity and the long-pending immunoassay chip of microbody of tumor markers with the electrochemical analysis technology.The object of the invention also is: utilize electrochemical analysis and screen printing technique to set up the chip immunoassay new method and the detection technique of tumor markers, and development minitype portable electro-chemistry immunity pick-up unit.
The realization of the object of the invention: the structure of the long-pending immunoassay chip of microbody of the present invention is as follows:
The long-pending immunoassay chip of microbody comprises that the annular carbon electrode that inwall is modified with electron mediator is a working electrode, the annular carbon electrode is assembled on the substrate, the inner chamber of the ring electrode that this mediator is modified is directly as micro reaction pool, Ag/AgCl contrast electrode and carbon place covering of reaction tank top to electrode, forming volume is about 40 μ L, general volume scope is controlled at little reaction chamber of 20-100 μ L, the reaction tank bottom substrate is fixed with the antigen molecule functional membrane, and this film is cut into circle and is assembled on the substrate 8, as shown in Figure 1.
The electrochemical analysis technology is set up the chip method of immunity of tumor markers:
(1) when only having an amount of HRP labelled antibody in the solution, antigen molecule generation immune response on the antigen molecule functional membrane that HRP labelled antibody and reaction tank bottom is fixed behind the incubation makes the HRP labelled antibody be connected the reaction tank bottom, this moment, immobilized HRP can't contact the electron mediator that is modified at reaction chamber wall, thereby H in can't catalytic solution
2O
2Oxidation can not get the catalytic current signal on the working electrode.
(2) when containing horseradish peroxidase (HRP) labelled antibody in the determined antigen solution, the competition immune response makes section H RP labelled antibody combine the formation immune conjugate with determined antigen and is free in the solution, and another part HRP labelled antibody is connected the reaction tank bottom.The immune conjugate that is free in the solution can touch the fixing electron mediator of reaction chamber wall, thereby can obtain catalysis H on working electrode
2O
2The catalytic current of oxidation, and this catalytic current is directly proportional with determined antigen content in the solution.
(3) can directly obtain the content of solution determined antigen by the response of catalytic current.
(4) common mediator comprises redox dye, metal complex etc.
(5) the present invention disposablely exempts to separate microbody and amasss immuno-chip by what mediator modified that annular carbon electrode inwall forms the collection incubation and be detected on one, utilize the ampere analytic approach, in conjunction with immobilization mediator technology and competitive immunoassay method, can exempt from separation determination fast to tumor markers.
Characteristics of the present invention are:
By chemical modification technology mediator is fixed on this annular carbon electrode inside surface, the inner chamber that utilizes annular carbon electrode is directly as micro reaction pool, integrate contrast electrode and to electrode in reaction tank, in conjunction with the competitive immunoassay method, utilize enzyme labelled antibody to combine the immune conjugate that generates with determined antigen and be free in the reaction tank, measure mark enzymatic oxidation H on the free immune conjugate
2O
2Catalytic current, directly obtain the concentration of determined antigen.More existing immune analysis method of this method and immunosensor have the following advantages:
(1) need not to add mediator in testing sample solution, avoided the pollution to electrode such as mediator and reaction product thereof, simplified analytical procedure, shorten analysis time, working electrode is reusable, reduces cost;
(2) incubation and detected set are formed in the long-pending chip of a microbody, the consumption of sample and reagent is few, and the incubation time is short, and need not the separating, washing step in measuring, and a step finishes, and reduces cost of determination, improves detection speed and efficient;
(3) utilize technology such as chemical modification and serigraphy to prepare immuno-chip, preparation is simple, with low cost, easy to carry, has good accuracy, repeatability and stable, not only in theory for tumor markers exempt from separate fast measuring and set up a kind of new method, and be suitable for the active demand of people to the immune sensing device, have certain market competitiveness.
The invention provides method and apparatus, overcome the shortcoming of existing immune analysis method, avoid the adding of mediator in immunoassay (stabilizing solution or nutrient solution etc.), simplify analytical procedure.Immobilization mediator technology is used to not have the exploitation of reagent biology sensor, the sensor that obtains need not to add mediator in sample solution when measuring, avoided the pollution of mediator and reaction product thereof, simplified analysis system and operation, shortened analysis time electrode surface; By incubation and detected set are formed in the long-pending chip of a microbody, not only the consumption of sample and reagent is reduced to microlitre, shorten the incubation time, reduce sample consumption, and need not the separating, washing step in measuring, measure and once finish, reduce cost of determination, improve detection speed and efficient, realize the express-analysis of clinical tumor mark.
But the present invention exempts to separate tumor markers fast measuring immuno-chip in conjunction with technical development prepared in batches such as chemical modification, little processing and serigraphy disposable, this chip cheapness, portable, be used for the detection of clinical tumor mark, to advance immunoassay technology and diagnosing early malignant tumor universalness and outdoorization and having broad application prospects aspect extensive examination and the prognosis monitoring, the development of immune science, clinical medicine, analytical chemistry and material science will be had great importance.
Description of drawings
Fig. 1 is the little reaction chamber vertical view of the present invention
Fig. 2 exempts to separate the structural representation of the long-pending immunoassay chip of microbody for the present invention is disposable
As shown in the figure: 1, inwall is modified with the annular carbon electrode of electron mediator; 2, be integrated with Ag/AgCl contrast electrode and carbon loam cake to electrode; 3, Ag/AgCl contrast electrode; 4, carbon is to electrode; 5, well; 6, electron mediator; 7, the substrate of annular carbon electrode is installed; 8, the substrate of antigen function film is installed; 9, be fixed with the functional membrane of antigen molecule; 10, immobilized antigen molecule.
Embodiment
1.1 the preparation of disposable immuno-chip
Disposablely exempt to separate the long-pending immunoassay chip of microbody and form by parts shown in Figure 1.Annular carbon electrode internal diameter 3mm, external diameter 7mm, thick 5mm, its inwall modify electron mediator 6 backs as working electrode.The functional membrane 9 that is fixed with the determined antigen molecule prepares on the PVC film, this film is cut into the circular of external diameter 2.5mm and is assembled on the substrate 8.Substrate 8 is with after substrate 7 overlaps, and annular carbon electrode is assembled in to form volume on the substrate 7 be little reaction chamber about 40 μ L.To be integrated with Ag/AgCl contrast electrode and carbon at last the loam cake 2 of electrode will be fixed on annular carbon electrode top, make this little reaction chamber bottom have nonconducting antigen function film, the top be Ag/AgCl contrast electrode and carbon to electrode, disposablely exempt to separate microbody and amass immuno-chip with mediator modifies that annular carbon electrode inwall forms the collection incubation and be detected on one.
1.2 the preparation of screen printing electrode
Utilize screen printing technique printing on the thick PVC film of 0.025mm to be integrated with Ag/AgCl contrast electrode and carbon to the loam cake (1) of electrode and substrate (2) two arrays of electrodes of the annular carbon electrode of installation.The making of electrode is by covering printing carbon-coating (carbon slurry layer width is than the silver slurry bigger 0.5-1mm of basic unit) in the silver slurry basic unit of printing, wherein loam cake comprise contrast electrode, to electrode and well.Before the silver electrode that carbon slurry is not printed on top is used in saturated KCl solution electrochemical oxidation prepare the Ag/AgCl contrast electrode; The direct conduct of electrode that is printed with the carbon slurry fully is to electrode.Silver ring that is not printed carbon slurry with the identical inside and outside footpath of annular carbon electrode is arranged at substrate top, its underpart for the band that is printed with the carbon slurry fully as lead.The electrode that prints is cut out shaping on demand.
1.3 mediator is modified the preparation of annular carbon working electrode
The spectroscopic pure carbon-point of diameter 7mm is processed into thick 5mm, and the carbocyclic ring of internal diameter 3mm, carbocyclic ring inside surface be successively with after 4,5, No. 6 homogeneous phase sand paper and the silk polishing, with the flushing of secondary water, and in secondary water ultrasonic 30 seconds.
The clean carbocyclic ring that will obtain with 502 glue is bonded at forms electrode on the substrate.This electrode in the phosphate buffered solution of 0.1mol/L pH5.0 in the scanning of+1.75V constant potential after 300 seconds, again at+0.3V to scan round between the+1.3V until obtaining stable electric current.The electrode of this electrochemical pre-treatment was soaked in the 20mmol/L glutaraldehyde solution 10-15 hour, remove the glutaraldehyde of physisorption with the flushing of secondary water after, be soaked in again in the 5mL 0.2mmol/L thionine mediator solution and obtained the mediator modified electrode in 3-8 hour.The modified electrode for preparing is soaked in set aside for use in the phosphate buffered solution of pH7.0.Except that thionine, other mediator can also be fixed in this ring electrode.
1.4 the preparation of antigen molecule functional membrane
Shitosan powder ultrasonic dissolution in 1% acetum is mixed with 1% chitosan solution.With certain density standard tumor markers to be measured (antigen) solution and this chitosan solution of 1% mixes with certain proportion and placed 12 hours down at 4 ℃.Drawing 5 these mixed solutions of μ L drips and is applied on the thick PVC film of 0.025mm at room temperature dry 5 ~ 6 hours, after the antigenic membrane that obtains is modified the PVC film and washed the antigen molecule of removing surface adsorption repeatedly through secondary water, be soaked in the phosphate buffered solution of pH7.0 stand-by in 4 ℃ of preservations.
1.5 the mensuration of tumor markers
This disposable tumor markers immuno-chip of measuring principle utilizes the ampere analytic approach, in conjunction with immobilization mediator technology and competitive immunoassay method, tumor markers is exempted from separation determination fast.When only having an amount of horseradish peroxidase marked tumor mark antibody (enzyme labelled antibody) in the solution, the antigen molecule generation immune response on the antigen molecule functional membrane that enzyme labelled antibody and reaction tank bottom is fixed behind the incubation makes it be connected the reaction tank bottom.This moment, immobilized HRP can't touch the electron mediator that is modified at reaction chamber wall, therefore can not catalytic solution in H
2O
2Oxidation can not get this catalytic current signal on working electrode; When contain in the determined antigen solution HRP labelled antibody and the time, the competition immune response makes section H RP labelled antibody combine the formation immune conjugate with determined antigen and is free in the solution, this part free immune conjugate can touch the fixing electron mediator of reaction chamber wall, thereby can obtain catalysis H on working electrode
2O
2The catalytic current of oxidation, and this catalytic current is directly proportional with determined antigen content in the solution.Response by catalytic current can directly obtain the special content of surveying antigen of solution.
The optimization of mensuration process (1) immunoassays condition comprises the incubation time, measures H in the solution
2O
2Concentration and reaction solution in the amount of enzyme labelled antibody.(2) a series of determined antigen standard solution and fixed amount enzymic-labelled antibody and the H that contain variable concentrations of preparation
2O
2Reaction solution, under optimum determining condition, determine H in the marker enzyme catalytic oxidation solution respectively
2O
2Catalytic current, obtain the bioassay standard curve of this determined antigen.(3) under optimum determining condition, measure incubation and contain determined antigen, fixed amount enzymic-labelled antibody and H
2O
2Reaction solution after H in the marker enzyme catalytic oxidation solution
2O
2Catalytic current, on the typical curve of this antigen measuring, find corresponding concentration.
Detect embodiment:
Be the application of long-pending immunoassay chip preparation of example explanation tumor markers microbody and galvanochemistry chip immunoassays new method below with carcinomebryonic antigen (CEA).The clinical meaning of change of serum C EA is that malignant tumor of digestive tract is had higher positive rate.
2.1 the preparation of CEA immunosensor
Prepare the pentanedial decoration electrode by above-mentioned steps, this electrode is soaked in 5 hours acquisition thionine modified electrodes in the 5mL 0.2mmol/L thionine solution.The thionine modified electrode for preparing is soaked in the pH7.0 phosphate buffered solution stand-by.
Shitosan powder ultrasonic dissolution in 1% acetum is mixed with 1% chitosan solution.500ng/mLCEA standard solution and this chitosan solution of 1% were with 1: 1 (V: V) mix and placed 12 hours down at 4 ℃.Draw 5 these mixed solutions of μ L and drip and be applied to the thick film modified PVC film of PVC film preparation CEA of 0.025mm, and be soaked in the phosphate buffered solution of pH7.0 stand-by 4 ℃ of preservations.
The film modified PVC film of CEA of preparation is cut into the thin rounded flakes of diameter 2.5mm and finishes the assembling of CEA immunoassay chip by above-mentioned steps.
2.2 the mensuration of CEA
(a) optimization of condition determination: change H in incubation time, the reaction solution respectively
2O
2And the amount of HRP mark CEA antibody, measure as optimum determining condition accordingly when selecting the maximum current response.
(b) mensuration of CEA: under optimal experimental conditions, respectively with series of standards CEA antigen and fixed amount HRP-CEA antibody and H
2O
2Reaction solution add in the CEA immuno-chip, measure mark HRP catalytic oxidation H
2O
2Catalytic current, obtain the typical curve that CEA measures, thus the concentration of CEA antigen in the working sample.
The use of the antigen of other tumor markers is the foregoing description roughly the same, does not also exceed the scope of the inventive method.
Claims (6)
1. the chip electrochemical method of immunity of tumor markers: it is characterized in that (1) is fixed on annular carbon electrode inside surface by chemical modification with electron transfer mediator, the inner chamber of the ring electrode that this mediator is modified is directly as micro reaction pool.Contrast electrode and electrode placed reaction tank top, the fixing antigen molecule functional membrane in reaction tank bottom; (2) when only having an amount of horseradish peroxidase marked tumor mark antibody in the solution, antigen molecule generation immune response on the antigen molecule functional membrane that enzyme labelled antibody and reaction tank bottom is fixed behind the incubation makes it be connected the reaction tank bottom, this moment, immobilized horseradish peroxidase HRP can't touch the electron mediator that is modified at reaction chamber wall, therefore can not catalytic solution in H
2O
2Oxidation can not get this catalytic current signal on working electrode; (3) when contain in the determined antigen solution HRP labelled antibody and the time, the competition immune response makes section H RP labelled antibody combine the formation immune conjugate with determined antigen and is free in the solution, this part free immune conjugate can touch the fixing electron mediator of reaction chamber wall, thereby can obtain catalysis H on working electrode
2O
2The catalytic current of oxidation, and this catalytic current is directly proportional with determined antigen content in the solution.(4) can directly obtain the content of solution determined antigen by the response of catalytic current.
2. the chip electrochemical method of immunity of tumor markers as claimed in claim 1, it is characterized in that described mediator is fixed on this annular carbon electrode inside surface, method for making is that the electrode with this electrochemical pre-treatment was soaked in the glutaraldehyde solution 10-15 hour, after removing the glutaraldehyde of physisorption with secondary water flushing, be soaked in again in the 5mL 0.2mmol/L mediator solution and obtained the mediator modified electrode in 3-8 hour.
3. the chip electrochemical method of immunity of tumor markers as claimed in claim 1, it is characterized in that the fixing antigen molecule functional membrane in described reaction tank bottom, it is after shitosan powder ultrasonic dissolution in 1% acetum is mixed with 1% chitosan solution, certain density standard determined antigen solution and this chitosan solution of 1% is mixed to drip with certain proportion be applied to that drying makes on the film.
4. the long-pending immunoassay chip of microbody: it is characterized in that the annular carbon electrode that inwall is modified with electron mediator is a working electrode, the annular carbon electrode is assembled on the substrate, the inner chamber of the ring electrode that this mediator is modified is directly as micro reaction pool, Ag/AgCl contrast electrode and carbon place covering of reaction tank top to electrode, forming volume is little reaction chamber of 20-100 μ L, the reaction tank bottom substrate is fixed with the antigen molecule functional membrane, and this film is cut into circle and is assembled on the substrate (8), that forms the collection incubation and be detected on one disposablely exempts to separate microbody and amasss immuno-chip.
5. by the long-pending immunoassay chip of the described microbody of claim 4: it is characterized in that ring electrode is carbon electrode or noble metal electrode in the structure of the long-pending immunoassay chip of microbody.
6. by the long-pending immunoassay chip of the described microbody of claim 4: the little reaction chamber volume that it is characterized in that the long-pending immunoassay chip of microbody is the little reaction chamber about 40 μ L.
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| CNB2004100411753A CN100357727C (en) | 2004-07-05 | 2004-07-05 | Electrochemcial immunoassay for tumor marker and small size immunoassay chip |
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| CNB2004100411753A CN100357727C (en) | 2004-07-05 | 2004-07-05 | Electrochemcial immunoassay for tumor marker and small size immunoassay chip |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007124669A1 (en) * | 2006-04-30 | 2007-11-08 | Nanjing University | Apparatus for electrochemical screening and early diagnosis of malignant tumor |
| CN101545902B (en) * | 2008-03-24 | 2014-01-08 | 江苏省肿瘤医院 | Automatic sampling distinguishing chemiluminescent multi-component immunological detection system and analysis method of same |
| CN101865912B (en) * | 2009-04-14 | 2014-05-14 | 南京大学 | Fast chemiluminescence immune detection system and analysis method |
| CN104805474A (en) * | 2015-03-16 | 2015-07-29 | 河北民族师范学院 | Annular carbon electrode and method for preparing Co nanowire/alumina film from same |
| CN111521652A (en) * | 2020-06-03 | 2020-08-11 | 润方(长春)生物科技有限公司 | Device and method for detecting procalcitonin in serum |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS57187643A (en) * | 1981-05-15 | 1982-11-18 | Nohmi Bosai Kogyo Co Ltd | Gas detection element and manufacture thereof |
| CN1196932C (en) * | 2003-03-26 | 2005-04-13 | 江苏省肿瘤医院 | Preparation of reagent-free ampoul immuno sensor and use thereof |
-
2004
- 2004-07-05 CN CNB2004100411753A patent/CN100357727C/en not_active Expired - Fee Related
Cited By (6)
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|---|---|---|---|---|
| WO2007124669A1 (en) * | 2006-04-30 | 2007-11-08 | Nanjing University | Apparatus for electrochemical screening and early diagnosis of malignant tumor |
| CN101545902B (en) * | 2008-03-24 | 2014-01-08 | 江苏省肿瘤医院 | Automatic sampling distinguishing chemiluminescent multi-component immunological detection system and analysis method of same |
| CN101865912B (en) * | 2009-04-14 | 2014-05-14 | 南京大学 | Fast chemiluminescence immune detection system and analysis method |
| CN104805474A (en) * | 2015-03-16 | 2015-07-29 | 河北民族师范学院 | Annular carbon electrode and method for preparing Co nanowire/alumina film from same |
| CN111521652A (en) * | 2020-06-03 | 2020-08-11 | 润方(长春)生物科技有限公司 | Device and method for detecting procalcitonin in serum |
| CN111521652B (en) * | 2020-06-03 | 2022-08-05 | 润方(长春)生物科技有限公司 | Device and method for detecting procalcitonin in serum |
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| CN100357727C (en) | 2007-12-26 |
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