CN103048462A - Multi-parameter electrochemical immunosensor based on electrode array and preparation method thereof - Google Patents
Multi-parameter electrochemical immunosensor based on electrode array and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a multi-parameter electrochemical immunosensor based on an electrode array and a preparation method thereof, relating to the biosensor technology and the bioelectronics technology. The sensor is integrated with the electrode array, a counter electrode, an electrochemical reference electrode, a lead and an interface on a substrate. According to the sensor, the micro electro mechanical system (MEMS) technology is adopted to process and prepare the basic electrode array, silver and silver chloride (Ag/AgCl) are used for modifying the counter electrode and the electrochemical reference electrode, and electric media bodies, porous shielding films, nano materials, antibodies and the like are fixed on a detection electrode to form a multi-parameter detection system. The sensor has the characteristics of good stability and convenience for use, and can simultaneously detect a plurality of lung cancer biomarkers, so as to facilitate clinical application.
Description
Technical field
The present invention relates to biology sensor and biological electronics, relate in particular to a kind of multiparameter electrochemical immunosensor based on electrod-array and preparation method thereof.
Background technology
The incidence of disease the highest cancer in the whole world is lung cancer at present, and annual newly-increased patient's number is 1,200,000; China's patients with lung cancer total data the first in the world, our country in 2005 shows 500,000 newly-increased patients to the data of lung cancer assessment.Tumor markers (Tumor Marker) is the chemical classes material that the reflection tumour exists.They or be not present in normal adult tissue and be detected in embryonic tissue, or the content in tumor tissues substantially exceeds the content in normal structure, their existence or quantitative change can be pointed out the character of tumour, so as to understanding tissue generation, Cell Differentiation, the cell function of tumour, judge and the treatment guidance with the diagnosis, classification, the prognosis that help tumour.The clinical discovery tumor markers may exist in the pleural effusion prior to tumour cell, and pleural effusion is obtained in mainly performing the operation, so pleural effusion carries out diagnosis and postoperative intervention that tumor markers detects for lung cancer and has important directive significance in the art.Lung cancer marker has multiple, only depends on a kind ofly can't detect particularly lung cancer of cancer, and wanting to make a definite diagnosis needs the multiparameter joint-detection.Wherein carcinomebryonic antigen (CEA), neuronspecific enolase (NSE) and cytokeratin fragment (CYFRA21-1) are common lung cancer markers.CEA is 40%～50% to the diagnosis positive rate of lung cancer, and in adenocarcinoma of lung, positive rate and specificity are higher especially; NSE and small cell carcinoma of lung are in close relations, are the labels of small cell carcinoma of lung; The positive rate of CYFRA21-1 in lung squamous cancer can be up to 85.1%.These three kinds of tumor markerses are carried out quick Site Detection important reference can be provided for the early diagnosis of lung cancer.But to the detection of these marks adopt large-scale instrument detect at present more, sample need to be delivered to special laboratory, can't accomplish field quick detection.
The immunity electrochemical method combines immunoassay and Electrochemical Detection, adopts the specific binding of antigen-antibody can improve the specificity of detection, uses electrochemical method to have advantage simple to operate, highly sensitive.Adopt MEMS technique to prepare electrod-array, can form multiparameter joint inspection biology sensor, simultaneously the fast detecting Diagnostic Value of Several Serum Tumor Markers.This sensor can detect the content of a plurality of tumor markerses sensitive, fast and reliably, thus for the diagnosis of tumour clinically, by stages, observe postoperative curative effect and prognosis provides an accurately reference value.
Summary of the invention
The object of the invention is to for above-mentioned the deficiencies in the prior art, a kind of multiparameter lung cancer electrochemical immunosensor based on electrod-array and preparation method thereof is provided.
A kind of multiparameter electrochemical immunosensor based on electrod-array provided by the invention, it comprises: detecting electrode array 1, galvanochemistry contrast electrode 2, to electrode 3, lead-in wire 4, socket 6 and substrate; Wherein, described detecting electrode array 1 comprises a plurality of detecting electrodes, and described detecting electrode is immune electrochemical electrode, for detection of the various diseases mark.
The invention allows for a kind of preparation method of aforesaid multiparameter lung cancer electrochemical immunosensor based on electrod-array, the method comprises:
Step 1, with plastic substrate as substrate, etching detecting electrode array, reference electrode and to the mask pattern of electrode on its diaphragm, and thereon sputter transition bed and electrode layer;
Step 2, the mixed slurry of interval silk-screen silver and silver chloride at contrast electrode and on to electrode form integrated silver and silver chloride contrast electrode and to electrode;
Step 3, electron mediator is fixed on forms the electron mediator film on the detecting electrode in the detecting electrode array, adopt nanogold particle as adjuvant, then the filming function by the perfluorinated sulfonic acid resin is fixed in perfluorinated sulfonic acid resin nano gold composite material nafion-AuNPs film and forms the porous screened film that is mixed with nano material on the electron mediator film;
Step 4, on three detecting electrodes, fix respectively different cancer markers antibody.
Above-mentioned multiparameter lung cancer electrochemical immunosensor based on electrod-array provided by the invention and preparation method thereof can be realized the multiparameter of lung cancer marker is detected simultaneously, and this sensor performance is stable, detection speed is fast, easy to use.
Description of drawings
Fig. 1 is the multiparameter lung cancer electrochemical immunosensor structural drawing that the present invention is based on electrod-array;
Fig. 2 a is the synoptic diagram that among the present invention the substrate protection film is carved into the electrode mask pattern that needs;
Fig. 2 b be among the present invention on described electrode mask pattern the synoptic diagram of sputter conductive film;
Fig. 2 c is for take stainless steel as mask plate, forms contrast electrode and to the synoptic diagram of electrode;
Fig. 2 d is at the fixing synoptic diagram of electron mediator, porous screened film and nano material of detecting electrode;
Fig. 2 e is at the fixing synoptic diagram of glutaraldehyde and antibody of detecting electrode;
Fig. 2 f is for pasting the insulating film structure synoptic diagram for the multiparameter lung cancer electrochemical immunosensor based on electrod-array for preparing.
Embodiment
For making the purpose, technical solutions and advantages of the present invention clearer, below in conjunction with specific embodiment, and with reference to accompanying drawing, the present invention is described in more detail.
As shown in Figure 1, the present invention proposes a kind of multiparameter lung cancer electrochemical immunosensor based on electrod-array, it comprises: detecting electrode array 1, galvanochemistry contrast electrode 2 is to electrode 3, lead-in wire 4, socket 6 and substrate 5.
Wherein said detecting electrode 1 is immune electrochemical electrode, adopts the preparation of MEMS technique, identifies for detection of disease; Adopt ultrasonic or plasma method is cleaned, respectively at the fixing electron mediator of electrode surface, porous screened film, nano material, antibody etc.; Each detecting electrode all has supporting to electrode 3, galvanochemistry contrast electrode 2, described electrode is used for providing return curcuit, and described galvanochemistry contrast electrode is used for providing reference potential and keeps current potential stable; The employed material of described substrate is the organic or inorganic insulating material of good biocompatibility, preferred silicon dioxide, silicon nitride, nitrogen-oxygen-silicon, SU8, polyimide or Parylene one of them; The material of described electrode 1～3 is metal or metallic compound conductive films of good biocompatibility, preferred gold, platinum, titanium nitride or indium tin oxide wherein it
With reference to the accompanying drawings 2, the invention allows for a kind of preparation method of the multiparameter lung cancer electrochemical immunosensor based on electrod-array, details are as follows for its concrete preparation process:
1) (it is detecting electrode array, reference electrode and to the mask pattern 7 of electrode, shown in Fig. 2 a that the diaphragm of 130mm * 100mm) is carved into the electrode that needs on the plastic substrate as substrate; Sputter transition bed thereon subsequently, then sputtering electrode layer (100nm), wherein, transition bed is selected metal level such as the chromium of thickness between 10nm～30nm, described electrode layer can be selected metal or the metal oxide of thickness about 100nm, such as gold, platinum, titanium nitride or indium tin oxide, shown in Fig. 2 b; After mask 7 is removed, described plastic substrate is put into plasma etching machine, pass into O
2Clean; Wherein the operating air pressure of plasma etching machine is 3.0Pa, the O that passes into
2Flow 40ml/ minute, also pass into N
2, its flow is 120ml/ minute.
2) adopt stainless steel mask plate 81, cover the detecting electrode of residence etching, the mixed slurry of interval silk-screen silver and silver chloride at contrast electrode and on to electrode forms integrated silver and silver chloride Ag|AgCl21 contrast electrode and to electrode, shown in Fig. 2 c.
3) adopt stainless steel mask plate 81, cover contrast electrode and to electrode, utilize shitosan (CHIT) as intermediate medium electron mediator (such as the potassium ferricyanide) to be fixed on formation electron mediator film 11 on the detecting electrode; Adopt nanogold particle as adjuvant, then the filming function by perfluorinated sulfonic acid resin (nafion) is fixed in perfluorinated sulfonic acid resin nano gold composite material nafion-AuNPs film and forms the porous screened film 12 that is mixed with nano material on the electron mediator film, has represented electron mediator film 11 and the porous screened film 12 that successively forms as adopting spatial structure among Fig. 2 d on detecting electrode;
4) adopt stainless steel mask plate 83, cover contrast electrode, electrode is reached wherein two detecting electrodes, only expose a remaining detecting electrode, utilize the way of Electrostatic Absorption the polyethyleneimine (PEI) of positive electricity to be adsorbed onto the nafion-AuNPs film surface formation PEI film of electronegative detecting electrode, connect described PEI film and carcinomebryonic antigen (CEA) monoclonal antibody 14 by glutaraldehyde 13 again, form a carcinomebryonic antigen (CEA) detecting electrode, with for detection of carcinomebryonic antigen (CEA), shown in Fig. 2 e.
5) change the stainless steel mask plate, repeat previous step, respectively fixedly neuronspecific enolase (NSE) monoclonal antibody and cytokeratin fragment (CYFRA21-1) monoclonal antibody on other two detecting electrodes, finish the modification of other two detecting electrodes, be respectively applied to detect neuronspecific enolase (NSE) and cytokeratin fragment (CYFRA21-1).
6) immunosensor for preparing is placed residual glutaraldehyde reactive group on the BSA solution sealing immunosensor, to avoid non-Characteristic Adsorption; Then stick dielectric film 15 (Fig. 2 f) at sensor surface.
The invention allows for a kind of using method of described multiparameter lung cancer electrochemical immunosensor based on electrod-array.
1) preparation of reagent, sample.
Getting concentrated cleaning solution 100ul uses deionized water according to 1: 100 diluted for use; Sample to be measured (blood, pleural effusion, sputum, saliva, seminal fluid, urine, ascites) etc. is carried out pre-service, get supernatant, place equilibrium at room temperature 10-15min.
2) operation steps.
Place liquid to be measured to carry out immune response 10 minutes sensor electrode, take out; Adopt the cleansing solution after diluting repeatedly to clean 3 times; Sensor is placed PBS solution, be connected on the CHI electrochemical workstation by socket (6) and detect, obtain the testing concentration value.
Above-described specific embodiment; purpose of the present invention, technical scheme and beneficial effect are further described; institute is understood that; the above only is specific embodiments of the invention; be not limited to the present invention; within the spirit and principles in the present invention all, any modification of making, be equal to replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. multiparameter electrochemical immunosensor based on electrod-array, it comprises: detecting electrode array (1), galvanochemistry contrast electrode (2), to electrode (3), lead-in wire (4), socket (6) and substrate (5); Wherein, described detecting electrode array (1) comprises a plurality of detecting electrodes, and described detecting electrode is immune electrochemical electrode, for detection of the various diseases mark.
2. the multiparameter electrochemical immunosensor based on electrod-array according to claim 1, it is characterized in that: each electrode surface in the described detecting electrode array (1) all is fixed with electron mediator, porous screened film, nano material and antibody; Described detecting electrode adopts the preparation of MEMS technique, and adopts ultrasonic or plasma method is cleaned.
3. the multiparameter lung cancer electrochemical immunosensor based on electrod-array according to claim 1, it is characterized in that: each detecting electrode all has supporting to electrode (3) and galvanochemistry contrast electrode (2), wherein saidly be used for providing current return to electrode, described galvanochemistry contrast electrode is used for providing reference potential and keeps current potential stable.
4. the multiparameter lung cancer electrochemical immunosensor based on electrod-array according to claim 1, it is characterized in that: the employed material of described substrate is the organic or inorganic insulating material of good biocompatibility; Detecting electrode in the described detecting electrode array, galvanochemistry contrast electrode and electrode is selected metal or the metallic compound conductive film of good biocompatibility.
5. the multiparameter lung cancer electrochemical immunosensor based on electrod-array according to claim 4 is characterized in that: described substrate select silicon dioxide, silicon nitride, nitrogen-oxygen-silicon, SU8, polyimide or Parylene one of them; Described electrode select gold, platinum, titanium nitride or indium tin oxide one of them.
6. the preparation method of the multiparameter lung cancer electrochemical immunosensor based on electrod-array as claimed in claim 1, the method comprises:
Step 1, with plastic substrate as substrate, etching detecting electrode array, reference electrode and to the mask pattern of electrode on its diaphragm, and thereon sputter transition bed and electrode layer;
Step 2, the mixed slurry of interval silk-screen silver and silver chloride at contrast electrode and on to electrode form integrated silver and silver chloride contrast electrode and to electrode;
Step 3, electron mediator is fixed on forms the electron mediator film on the detecting electrode in the detecting electrode array, adopt nanogold particle as adjuvant, then the filming function by the perfluorinated sulfonic acid resin is with the perfluorinated sulfonic acid resin | and nm of gold composite material nafion-AuNPs film is fixed in the porous screened film that formation on the electron mediator film is mixed with nano material;
Step 4, on three detecting electrodes, fix respectively different disease marker antibody.
7. the preparation method of the multiparameter lung cancer electrochemical immunosensor based on electrod-array according to claim 6, it is characterized in that: described electron mediator is the potassium ferricyanide.
8. the preparation method of the multiparameter lung cancer electrochemical immunosensor based on electrod-array according to claim 6, it is characterized in that: in the described step 1, behind the complete transition bed of sputter and electrode layer, described plastic substrate is put into plasma etching machine, pass into O
2And N
2Clean, the operating air pressure of described plasma etching machine is 3.0Pa, the O that passes into
2Flow is 40ml/ minute, N
2Flow 120ml/ minute.
9. the preparation method of the multiparameter lung cancer electrochemical immunosensor based on electrod-array according to claim 6, it is characterized in that, the disease marker antibody of fixing at detecting electrode in the described step 4 is cancer markers antibody, comprise carcinomebryonic antigen, neuronspecific enolase and cytokeratin fragment, its fixation procedure is as follows:
Step 41: adopt the stainless steel mask plate, cover contrast electrode, electrode is reached wherein two detecting electrodes, only expose a remaining detecting electrode, the way of utilizing Electrostatic Absorption forms the PEI film with the perfluorinated sulfonic acid resin surface that the polyethyleneimine PEI of positive electricity is adsorbed onto electronegative detecting electrode, connect described PEI film and Cea Monoclonal Antibodies by glutaraldehyde again, form a carcinomebryonic antigen detecting electrode, with for detection of carcinomebryonic antigen;
Step 42: change the stainless steel mask plate, repeat previous step, respectively fixedly neuronspecific enolase monoclonal antibody and neuronspecific enolase monoclonal antibody on other two detecting electrodes are respectively applied to detect neuronspecific enolase and neuronspecific enolase.
10. the preparation method of the multiparameter lung cancer electrochemical immunosensor based on electrod-array according to claim 9, it is characterized in that, described on the detecting electrode respectively fixedly behind carcinomebryonic antigen, neuronspecific enolase and the cytokeratin fragment, the method also comprises:
Step 5, the described electrochemical immunosensor that will prepare place BSA solution to seal residual glutaraldehyde reactive group on it, and at its surface label upper nonconductive Film.
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Cited By (13)
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| CN103616417A (en) * | 2013-11-05 | 2014-03-05 | 刘军 | Detection device for electrochemical immune biosensor and detection and analysis method |
| CN104760922A (en) * | 2014-01-03 | 2015-07-08 | 中国科学院电子学研究所 | Ultramicro planar electrode array sensor and preparation method thereof |
| CN105116017A (en) * | 2015-09-08 | 2015-12-02 | 无锡百灵传感技术有限公司 | Chip type integrated sensor |
| CN105241930A (en) * | 2015-09-24 | 2016-01-13 | 浙江大学 | Multiparameter all-solid-state blood analysis sensor |
| CN105445339A (en) * | 2014-07-31 | 2016-03-30 | 天津大学 | Flexible differential array electrochemical glucose sensor and use method thereof |
| CN107167507A (en) * | 2017-05-16 | 2017-09-15 | 重庆石墨烯研究院有限公司 | Graphene microelectrode electrochemical test sensors with DNA molecular probe |
| CN107356651A (en) * | 2017-08-28 | 2017-11-17 | 广州商辉怡业计算机科技有限公司 | Multi parameter water quality method and system |
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| CN110146568A (en) * | 2018-11-29 | 2019-08-20 | 浙江大学山东工业技术研究院 | A kind of multi-parameter electrochemical detection electrode piece |
| 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 |
| CN113974615A (en) * | 2021-11-18 | 2022-01-28 | 中山大学 | Tissue fluid detection device and system thereof |
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| CN115950933A (en) * | 2022-11-29 | 2023-04-11 | 常州先趋医疗科技有限公司 | Self-assembled monomolecular film modified electrode and preparation method and application thereof |
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Cited By (16)
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| CN103616417A (en) * | 2013-11-05 | 2014-03-05 | 刘军 | Detection device for electrochemical immune biosensor and detection and analysis method |
| CN104760922A (en) * | 2014-01-03 | 2015-07-08 | 中国科学院电子学研究所 | Ultramicro planar electrode array sensor and preparation method thereof |
| CN104760922B (en) * | 2014-01-03 | 2016-08-24 | 中国科学院电子学研究所 | A kind of ultra micro planar electrode array sensor and preparation method thereof |
| CN105445339A (en) * | 2014-07-31 | 2016-03-30 | 天津大学 | Flexible differential array electrochemical glucose sensor and use method thereof |
| CN105116017A (en) * | 2015-09-08 | 2015-12-02 | 无锡百灵传感技术有限公司 | Chip type integrated sensor |
| CN105241930A (en) * | 2015-09-24 | 2016-01-13 | 浙江大学 | Multiparameter all-solid-state blood analysis sensor |
| CN107167507A (en) * | 2017-05-16 | 2017-09-15 | 重庆石墨烯研究院有限公司 | Graphene microelectrode electrochemical test sensors with DNA molecular probe |
| CN107356651A (en) * | 2017-08-28 | 2017-11-17 | 广州商辉怡业计算机科技有限公司 | Multi parameter water quality method and system |
| CN108204968A (en) * | 2018-02-08 | 2018-06-26 | 华南师范大学 | A kind of polynary bipolar electrode electrochemical luminescence chip of closed type and its application |
| CN108204968B (en) * | 2018-02-08 | 2019-10-08 | 华南师范大学 | A kind of application of chip in glucose and uric acid are done while being detected |
| CN110146568A (en) * | 2018-11-29 | 2019-08-20 | 浙江大学山东工业技术研究院 | A kind of multi-parameter electrochemical detection electrode piece |
| 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 |
| CN113974615A (en) * | 2021-11-18 | 2022-01-28 | 中山大学 | Tissue fluid detection device and system thereof |
| CN114441612A (en) * | 2021-12-30 | 2022-05-06 | 广州市赛特检测有限公司 | Electrochemical biosensor, detection method and application |
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