CN200982972Y - Malignancy electrochemical screening and early diagnostic apparatus - Google Patents
Malignancy electrochemical screening and early diagnostic apparatus Download PDFInfo
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- CN200982972Y CN200982972Y CN 200620073068 CN200620073068U CN200982972Y CN 200982972 Y CN200982972 Y CN 200982972Y CN 200620073068 CN200620073068 CN 200620073068 CN 200620073068 U CN200620073068 U CN 200620073068U CN 200982972 Y CN200982972 Y CN 200982972Y
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Abstract
A malignant tumor electrochemistry screening and early-stage diagnosis meter is characterized in that an immunoassay chip(1) is connected with a time-resolved multi-channel potentiostat (2) through an interface, the time-resolved multi-channel potentiostat(2) is connected with a data processing and display system(3) through the interface, wherein the immunoassay chip(1) comprises eight working electrode which fixedly have different antigen-molecule functional film, an Ag wire(a), an Ag/AgC1 reference electrode(b), a carbon pair electrode(c) and an insulating film(d). We can use the competition of getting an enzyme labeled antibody which is in the incubated solution between the antigen molecule which is fixedly arranged on the surface of the electrode and the antigen molecule which is in the sample to make some enzyme labeled antibodies connect with the surface of the electrode, thereby achieving a catalyst electric current and indirectly achieving the contents of the eight antigens which are in the solution and ready to be tested. Through the software processing, the testing result will be displayed by pictures and colors which are easy to observe and read. The diagnosis meter has the advantages of low cost, intelligence and quick testing, and has a broad application prospect on the aspects of monitoring and screening the tumor.
Description
One, technical field
The utility model relates to a kind of medical diagnostic equipment, specifically relates to a kind of immunoassays and fast electrochemical examination and early diagnosing instrument of tumor markers.
Two, background technology
Immunoassay is to utilize the reaction of high specific between the antigen/antibody realize antagonist, antigen or the related substances a kind of high sensitivity that detects, 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.Because the radiological hazard and the limitation in reagent life-span of radio immunoassay, nonradioactive labeling's immune analysis 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, the number that can detect mark simultaneously is limited, and instrument and reagent are also quite expensive, is difficult to promote the use of, thereby is restricted in the application aspect early diagnosis, prognosis monitoring and the extensive examination of malignant tumour.
In recent years, along with the development of genetic chip and protein-chip, immuno-chip obtains people's attention.Existing company releases the instrument that utilizes chemiluminescence and CCD method binding energy to detect 13 tumor markerses simultaneously, but this instrument price is very expensive, also has limitation aspect detection by quantitative, thereby is difficult to promote.How simplify the operation step, shorten analysis time, reduce cost of determination, develop new immunoassay technology, improve sensitivity, the accuracy of detection means, developing high-throughout multi-tumor marker detector is problem demanding prompt solutions with the needs that adapt to extensive examination, early diagnosis and prognosis monitoring to malignant tumour.
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 at high flux multi-tumor marker context of detection potentialization, the report that can measure the electrochemical immunoanalytical method of 2 kinds of tumor markerses was simultaneously arranged in 2005, and these authors further proposed many marks method of electrochemical immunoanalytical simultaneously again in 2006.These methods have certain practicality, but because of not providing concentration value intuitively, can not be provided at general practical image result in the chip analysis, more do not break through the specific defective of tumor associated antigen, develop detecting instrument, and promote the use of.
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, the consumption of sample and reagent is few, mutual good reproducibility, the result accurately, reliably, under 25 ℃ of drying conditions, sensor can obtain well stability.Utilize this technology and electrochemical analysis method, the present invention proposes the multi-channel electrochemical detection chip of fast measuring kinds of tumors mark simultaneously, and according to the needs of malignant tumour examination and early diagnosis, by experiment and clinical control, threshold value is set, image result is combined with uniting diagnosis, further develop malignant tumour electrochemical screening and early diagnosing instrument with this chip.This instrument can be the intuitive image result with the Amperometric Detection Coupled signal transition, so that the layman uses.Simultaneously, can report the concentration value of the kinds of tumors mark relevant intuitively, so that carry out course of disease monitoring with the course of disease.
This invention comprises multi-channel electrochemical detection chip and multi-channel electrochemical detecting instrument, it is directly perceived to report the result, detect with low cost, the automaticity height, combine and overcome the specific defective of tumor associated antigen with uniting diagnosis, can carry out early detection, early diagnosis and the course of disease monitoring of malignant tumour, realize the universalness and the family oriented that detect, to improve the prevention and control of cancer efficiency of resource, also very meaningful to the development of China's treatment and prevention of tumour cause.
Three, summary of the invention
1. the purpose of this utility model is: provide a kind of, malignant tumour electrochemical screening and the early diagnosing instrument that can measure the kinds of tumors mark simultaneously.
2. technical scheme
1) malignant tumour electrochemical screening and early diagnosing instrument:
Malignant tumour electrochemical screening and early diagnosing instrument as shown in Figure 1, by eight passage immunoassay chips (1), time resolution hyperchannel potentiostat (2) and data processing and display system (3) are formed; Eight passage immunoassay chips (1) are connected with time resolution hyperchannel potentiostat (2) by interface, and data processing and display system (3) also is connected with time resolution hyperchannel very potentiometer (2) by interface;
2) eight passage immunoassay chips, comprise that eight working electrodes (M1, M2, M3, M4, M5, M6, M7, M8), Ag/AgCl contrast electrode (b) and carbon that the functional membrane of fixing different tumor associated antigens respectively and these functional membrane modify are to electrode (c), silver lead (a), dielectric film (d), as shown in Figure 2.This chip (1) is directly inserted on the time resolution hyperchannel potentiostat (2) by interface and is measured, and shows the qualitative detection result by data processing and display system (3) with color of image.
3) the Electrochemical Detection principle of malignant tumour electrochemical screening and early diagnosing instrument: based on the competitive reaction pattern, with the mixed liquor of the plurality of enzymes labeling antibody solution of sample (antigen) and specific concentrations as incubation liquid, utilize the enzyme labelled antibody of corresponding horseradish peroxidase-labeled in not synantigen fixing on the immuno-chip and the sample antigenic competition reaction incubation liquid, thereby make horseradish peroxidase be fixed on electrode surface, catalysis H
2O
2With the reaction of electron transfer mediator, the content of the big or small indirect detection determined antigen by catalytic current.
4) this process realizes by the time resolution hyperchannel potentiostat (the 2nd parts among Fig. 1) of development.Common potentiostat single job can only detect 1 sample, and commercial hyperchannel potentiostat comprises a plurality of constant potential system, and cost is higher, and the number of detected object directly depends on constant potential system number simultaneously, is unfavorable for improving practicality.The time resolution hyperchannel potentiostat of the present invention's design, use single potentiostat system, by the design control system, change the order of working electrode on the detection chip within a short period of time, obtain the information that " simultaneously " obtains a plurality of objects (kinds of tumors mark) content, and show with the peak-shaped curve image its corresponding threshold value of peak-shaped curve of the various tumor markers content in the detected sample is compared, show qualitative and quantitative result by last form after the software processes, as shown in Figure 3 with image.
Common potentiostat demonstrates peak shape result among Fig. 3, by automatic processing, can provide peak point current, utilizes working curve, and the tester can obtain the concentration of detected object.
5) data processing and display system: the present invention is according to the needs of malignant tumour examination and early diagnosis, by experiment and clinical control, threshold value is set, and the electro-chemistry immunity Amperometric Detection Coupled signal that the multi-channel electrochemical detection chip is obtained carries out image and shows.The sample that peak-to-peak signal surpasses threshold value 15% is set to redness (●), and expression is positive; That near peak-to-peak signal sample of 15% threshold value is set to is orange (
), represent suspicious; The sample that peak-to-peak signal is lower than threshold value 15% is set to blueness (zero), and expression is negative, and shows qualitative and quantitative result (see figure 3) with the form of image.
6) further will unite the diagnosis notion and introduce result's demonstration, analyze the testing result of kinds of tumors mark automatically, propose the examination result, carry out early diagnosis.Utilize quantitative result to carry out course of disease monitoring simultaneously.The malignant tumour electrochemical screening and the early diagnosing instrument that propose have overcome the specific defective of tumor associated antigen, and it is low to detect cost, can be convenient to the layman and use.
3. beneficial effect
1) the present invention utilizes the enzyme labelled antibody of the horseradish peroxidase-labeled in antigen fixing on the immuno-chip and the sample antigenic competition reaction incubation liquid, thereby makes horseradish peroxidase be reacted to electrode surface in conjunction with the competitive immunoassay method, thus catalysis H
2O
2With the reaction of electron transfer mediator, the content of the big or small indirect detection determined antigen by catalytic current.This method need not instrumentation and detects, and sense cycle is short, and operation steps is simple, is easy to robotization, intellectuality, is convenient to the layman and uses.
2) but the present invention in conjunction with technical developments such as chemical modification, little processing and serigraphys the disposable multi-tumor marker immuno-chip of fast measuring simultaneously of prepared in batches, not only the consumption of sample and reagent is reduced to microlitre, shorten the incubation time, reduce sample consumption, and can once finish the mensuration of kinds of tumors mark, reduce cost of determination, improve detection speed and efficient.
3) the present invention proposes the time-resolved notion of Electrochemical Detection, develop time resolution hyperchannel potentiostat hyperchannel potentiostat, pass through sequence detection, the signal of sample on the different detecting electrodes obtains the information that " simultaneously " obtains a plurality of objects (kinds of tumors mark) content on the acquisition chip.
4) image of electrochemical results shows.By clinical control, obtain corresponding threshold value, utilize threshold value and signal contrast, with the qualitative and quantitative result of form demonstration of image.
5) the present invention will unite diagnosis notion introducing result and show that the testing result that the multi-channel electrochemical detection chip obtains is carried out analyzing and processing, and proposition examination result carries out early diagnosis, breaks through the specific defective of tumor associated antigen.
This diagnostic equipment is with low cost, and is Miniaturized, and intellectuality to advance immunoassay technology and diagnosing early malignant tumor universalness and outdoorization, is having broad application prospects aspect extensive examination and the prognosis monitoring.
Four, description of drawings
Fig. 1. screening of malignant tumour galvanochemistry and early diagnosing instrument structural representation
(1)-eight passage immunoassay chip, (2)-time resolution hyperchannel potentiostat, (3)-data processing and display system
Fig. 2. the structural representation of eight passage immunoassay chips
(a)-and the Ag lead, (b)-the Ag/AgCl contrast electrode, (c) carbon is to electrode, (d)-and dielectric film, M1, M2, M3, M4, M5, M6, M7, M8 represent to be fixed with eight working electrodes of different antigen function films respectively.
Fig. 3. the analyte detection process synoptic diagram of screening of malignant tumour galvanochemistry and early diagnosing instrument
Red ●-positive (warning), orange
-suspicious, blue zero feminine gender (eliminating); C1, C2, C3, C4, C5, C6, C7, C8 represent the concentration of eight tumor markerses recording respectively.
Five, embodiment
1. the preparation of eight passage immunoassay chips
Utilize screen printing technique the PVC film on printing be integrated with Ag/AgCl contrast electrode, carbon substrate to electrode and a plurality of (8) carbon working electrode, the making of electrode is elder generation's printed silver conductive layer on the PVC film, printing Ag/AgCl slurry formation Ag/AgCl contrast electrode (b) on the position of contrast electrode then, print the carbon slurry at working electrode and carbon on the position to electrode and form 8 carbon working electrode M1, M2, M3, M4, M5, M6, M7, M8 and carbon to electrode (c), green oil dielectric film (d) is gone up in printing at last.Green oil dielectric film (d) both can have been protected silver conductive layer (a), also can control the volume of reaction.
2. 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 ℃, draw 1 this mixed solution of μ L and drip and be applied to working electrode surface, obtain to comprise fixing eight film modified working electrodes of different tumor associated antigen molecular functionizations.
3. the mensuration of tumor markers
3.1 measuring principle
This malignant tumour electrochemical screening and early diagnosing instrument utilize the ampere analytic approach, in conjunction with the competitive immunoassay method, tumor markers are exempted from separation determination fast.When only having an amount of horseradish peroxidase marked tumor mark antibody (enzyme labelled antibody) in the incubation liquid, the antigen molecule generation immune response on the antigen molecule functional membrane that enzyme labelled antibody and electrode surface are fixed behind the incubation makes it be connected to electrode surface.H in the at this moment immobilized HRP energy catalytic solution
2O
2With the electron transfer mediator reaction, thereby can on working electrode, obtain catalysis H
2O
2The catalytic current of oxidation, and determined antigen content is inversely proportional in this catalytic current and the solution.Can obtain the content of determined antigen in the solution indirectly by the response of catalytic current.
3.2 mensuration process
(1) cross interference reaction experiment.In this full-automatic malignant tumour electrochemical screening and early diagnosing instrument, used the immune detection chip of eight passages.In order to prevent contingent cross interference reaction between each electrode, the different immune detection chip of design a series of activities interelectrode distance is selected the minimum electrode spacing that does not produce cross interference in detection.
The optimization of (2) immunoassays condition comprises incubation time, heated culture temperature, pH value, measures H in the solution
2O
2Amount with enzyme labelled antibody in the concentration of electron transfer mediator and the reaction solution.
(3) a series of determined antigen standard solution of variable concentrations and the incubation liquid of fixed amount enzymic-labelled antibody of containing of preparation under optimum determining condition, determine marker enzyme catalysis H respectively
2O
2With the catalytic current of the reaction of electron transfer mediator, obtain the bioassay standard curve of this determined antigen.
(4) under optimum determining condition, incubation contains determined antigen and fixed amount enzymic-labelled antibody, measures marker enzyme catalysis H
2O
2With the catalytic current of electron transfer mediator reaction, on the typical curve of this antigen measuring, find corresponding concentration.
(5) its corresponding threshold of the concentration of the tumor markers in the actual sample that records, designing treatment software, the sample that peak-to-peak signal surpasses threshold value 15% is set to redness (●), and expression is positive; That near peak-to-peak signal sample of 15% threshold value is set to is orange (
), represent suspicious; The sample that peak-to-peak signal is lower than threshold value 15% is set to blueness (zero), and expression is negative, with the qualitative and quantitative result of form demonstration of image.
(6) with catalytic current signal and threshold signal contrast, show the qualitative detection result with color of image.
(7) will unite the diagnosis notion and introduce result's demonstration, the testing result that the multi-channel electrochemical detection chip obtains will be carried out analyzing and processing, propose the examination result.
3.3 specific embodiment:
With carcinomebryonic antigen (CEA), alpha-fetoprotein (AFP), oophoroma related antigen (CA125), breast cancer correlation antigen (CA153), pancreas human primary gastrointestinal cancers mark (CA199), cancer markers (CA50), cancer in digestive system mark (CA242), prostate cancer specific antigen (PSA) is the application of example explanation malignant tumour electrochemical screening and early diagnosing instrument.
1) preparation of the detection chip of malignant tumour electrochemical screening and early diagnosing instrument
Shitosan powder ultrasonic dissolution in 1% acetum is mixed with 1% chitosan solution.CEA, AFP, CA125, CA153, CA199, CA50, CA242, PSA standard solution respectively with this chitosan solution of 1% with 1: 1 (V: V) mix and placed 12 hours down at 4 ℃.Above-mentioned mixed solution is respectively got 1 μ L and is dripped respectively and be applied to eight working electrode surfaces, under the room temperature dry 5-6 hour.Preserve stand-by under 4 ℃ of dry situation.
2) mensuration of CEA, AFP, CA125, CA153, CA199, CA50, CA242, PSA
(1) optimization of condition determination; Change incubation time, heated culture temperature, pH value respectively, measure H in the solution
2O
2With the amount of various enzyme labelled antibodies in the concentration of thionine and the reaction solution, measure as optimum determining condition accordingly when selecting the maximum current response.
(2) mensuration of CEA, AFP, CA125, CA153, CA199, CA50, CA242, PSA: under optimal experimental conditions, respectively with the incubation liquid of series of standards CEA, AFP, CA125, CA153, CA199, CA50, CA242, PSA antigen and fixed amount enzyme labelled antibody, add in the immune detection chip of full-automatic malignant tumour electrochemical screening and early diagnosing instrument and carry out incubation, mark HRP catalytic oxidation H is measured in the intact back of incubation
2O
2Catalytic current, obtain the typical curve that CEA, AFP, CA125, CA153, CA199, CA50, CA242, PSA measure respectively, thus the concentration of CEA, AFP, CA125, CA153, CA199, CA50, CA242, the various antigens of PSA in the working sample.
(3) concentration of CEA, AFP in the sample of measuring, CA125, CA153, CA199, CA50, CA242, PSA antigen is compared with their threshold values separately respectively,, directly detection signal is converted to concentration value and examination result and shows by software processes.
(4) concentration of the different tumor markerses of analysis is utilized and is united diagnosis proposition examination result.
Claims (1)
1. malignant tumour electrochemical screening and early diagnosing instrument is characterized in that it by eight passage immunoassay chips (1), and time resolution hyperchannel potentiostat (2) and data processing and display system (3) are formed; Eight passage immunoassay chips (1) are connected with time resolution hyperchannel potentiostat (2) by interface, and time resolution hyperchannel potentiostat (2) is connected with data processing and display system (3) by interface; Eight passage immunoassay chips (1), eight working electrodes (M1, M2, M3, M4, M5, M6, M7, M8), Ag/AgCl contrast electrode (b) and the carbon that comprise the functional membrane modification of fixing different tumor associated antigens respectively are to electrode (c), silver lead (a), dielectric film (d), this chip (1) directly inserts on the time resolution hyperchannel potentiostat (2) by interface and measures, and shows the qualitative detection result by data processing and display system (3) with color of image.
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| CN 200620073068 CN200982972Y (en) | 2006-04-30 | 2006-04-30 | Malignancy electrochemical screening and early diagnostic apparatus |
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| CN 200620073068 CN200982972Y (en) | 2006-04-30 | 2006-04-30 | Malignancy electrochemical screening and early diagnostic apparatus |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1866018B (en) * | 2006-04-30 | 2012-03-28 | 南京大学 | Electrochemical screening and early diagnosing instrument for malignant tumor |
| CN103411981A (en) * | 2013-07-25 | 2013-11-27 | 宁波大学 | Method for identifying stomach cancer glandular epithelium cell by using dielectric spectral characteristics |
-
2006
- 2006-04-30 CN CN 200620073068 patent/CN200982972Y/en not_active Expired - Lifetime
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1866018B (en) * | 2006-04-30 | 2012-03-28 | 南京大学 | Electrochemical screening and early diagnosing instrument for malignant tumor |
| CN103411981A (en) * | 2013-07-25 | 2013-11-27 | 宁波大学 | Method for identifying stomach cancer glandular epithelium cell by using dielectric spectral characteristics |
| CN103411981B (en) * | 2013-07-25 | 2016-06-22 | 宁波大学 | One utilizes dielectric and magnetic characteristic to differentiate the epithelioglandular method of gastric cancer |
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