CN101059509B - Antibody/ antigen or DNA hybridization detection device based on oxidation/reduction probe, and checking method - Google Patents
Antibody/ antigen or DNA hybridization detection device based on oxidation/reduction probe, and checking method Download PDFInfo
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- CN101059509B CN101059509B CN 200710068513 CN200710068513A CN101059509B CN 101059509 B CN101059509 B CN 101059509B CN 200710068513 CN200710068513 CN 200710068513 CN 200710068513 A CN200710068513 A CN 200710068513A CN 101059509 B CN101059509 B CN 101059509B
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Abstract
The invention provides a hybridization combination check device of antibody/antigen or DNA based on oxidization/reduction probe, comprising a reference electrode, a generating electrode, a collecting electrode, and an opposite electrode, wherein the distance between the generating electrode and the collecting electrode is not longer than 100micrometer, the surfaces of the generating electrode and the collecting electrode are fixed with the antibody or single strand DNA molecule of the object, and the invention also comprises a dual-electrode constant-current potential meter for checking the electrode dynamic parameter of the collecting electrode. The invention can improve the check sensitivity. And the invention also discloses a relative check method, which measures and compares the current or electrochemical reaction resistances of the collecting electrode before and after the biological reaction to obtain the result. The invention measures the electrochemical reaction parameters on the collecting electrode, with large parameter change range and high sensitivity.
Description
Technical field
The present invention relates to a kind of antibody/antigen based on oxidation/reduction probe or DNA hybridization detection device and method.Main application fields of the present invention comprises biochemistry, environmental protection and food security etc.
Background technology
Immunoassay technology is to utilize specific reaction between antigen and the antibody to come the Fast Detection Technique that antigen or antibody are qualitatively or quantitatively determined, comprising Enzyme Immunoassay, the fluorescence immunoassay technology, immuno-gold labeling technology, immunomagnetic bead technique etc.But most immunosensor also needs the antibody of extra tape label that the combination of antigen and antibody is converted into detectable signal, has so just increased the complexity that detects, and therefore necessary exploitation need not the immune detection sensor of mark.Developed at present the immunosensor that some need not mark, they divide from principle can be divided into the quartz crystal immunosensor, surface plasma body resonant vibration immunosensor and electrochemical immunosensor etc.Among this, electrochemical immunosensor is because simple in structure, and is easy to use and caused people's very big attention.
The ultimate principle of electrochemical immunosensor that need not mark is as follows.At electrode surface, then fixing upper antibody put into solution first.Simultaneously, in detected solution, add oxidation/reduction probe, such as [Fe (CN)
6]
3-/4-As probe (here, oxidation/reduction is in fact just being equivalent to mark, and only this mark is directly to be added in the solution, and does not need to be fixed on the antibody).In solution, have measurand, for example during Escherichia coli, it will with the antibody combination of electrode surface.Antigen and antibody all are some bioelectricity media, and is non-conductive, that is to say that they are the equal of one deck megohmite insulant of electrode surface, will hinder like this oxidation/reduction to the electrochemical reaction rates at electrode surface, thereby reach testing goal.This detection principle not only can be used for the pathogenic microbes detect based on immunization, the hybridization, biotin-avidin that also can be used for DNA in conjunction with etc. detection, because these detect from the make peace reaction of antigen/antibody of principle is the same, all is the combination of dielectric biomolecule.
Traditional electrochemical immunosensor mainly by the upper electrochemical kinetics mathematic(al) parameter before and after the antigen/antibody reaction of surveying work electrode (being equivalent in the present invention produce electrode), is realized the detection of antigen such as the variation of electrochemical impedance, redox electric current etc.But also there are some problems at aspects such as Measuring Time, sensitivity,
Summary of the invention
Technical matters to be solved by this invention provide a kind of can improve sensitivity based on the antibody/antigen of oxidation/reduction probe or DNA hydridization in conjunction with pick-up unit.For this reason, the present invention is by the following technical solutions: it comprises contrast electrode, produces electrode, passive electrode and to electrode, distance between described generation electrode and the passive electrode is less than or equal to 100 microns, be fixed with the antibody of measurand or the dna molecular of strand at generation electrode and passive electrode surface, it also is provided with the bipolar electrode continuous current potentiometer that detects the electrode kinetics parameter on the passive electrode.
Owing to adopt technical scheme of the present invention, the present invention combines the dielectric property of biomaterial and the coupling effect that produces between electrode/passive electrode, with [Fe (CN)
6]
3-/4-Be example, the ferric ion of divalence is at first oxidized on the generation electrode, then arrives on the passive electrode by diffusion, is reduced at passive electrode again.Electrochemical reaction on generation electrode and passive electrode all can be subject to the impact of biological respinse, and therefore, after the biological respinse in solution, the electrochemical reaction parameters on the passive electrode has larger variation, thereby can further improve detection sensitivity.In technical scheme of the present invention, the distance that produces between electrode and the passive electrode is less, and its detection sensitivity is higher, does not contact as long as guarantee to produce between electrode and the passive electrode.Consider that technology difficulty also considers sensitivity, produce between electrode and the passive electrode distance more than or equal to 80 nanometers between less than or equal to 100 microns, be both economical and guarantee the high-precision selection of measurement result.
Another technical matters to be solved of the present invention provides a kind of highly sensitive based on the antibody/antigen of oxidation/reduction probe or the detection method of DNA hybridization detection device.For this reason, the present invention carries out respectively following operation by the following technical solutions before and after biological respinse: take contrast electrode as potential reference point, apply an oxidation voltage at the generation electrode, apply a recovery voltage on the passive electrode, then measure the electric current on the passive electrode of flowing through; Electric current after aforesaid operations finishes on the contrast passive electrode obtains measurement result;
Or before and after biological respinse, carry out respectively following operation: take contrast electrode as potential reference point, apply a recovery voltage at the generation electrode, apply an oxidation voltage on the passive electrode, then measure the electric current on the passive electrode of flowing through; Electric current after aforesaid operations finishes on the contrast passive electrode obtains measurement result.
Above-mentioned detection method can also be by the following technical solutions, take contrast electrode as potential reference point, apply an oxidation voltage at the generation electrode, apply a recovery voltage on the passive electrode, and then the less alternating voltage of an amplitude that superposes, the then electrode reaction resistance on the measurement collection electrode; Electrode reaction resistance after aforesaid operations finishes on the contrast passive electrode obtains measurement result;
Or before and after biological respinse, carry out respectively following operation: take contrast electrode as potential reference point, apply a recovery voltage at the generation electrode, apply an oxidation voltage on the passive electrode, and then the less alternating voltage of an amplitude that superposes, the then electrode reaction resistance on the measurement collection electrode; Electrode reaction resistance after aforesaid operations finishes on the contrast passive electrode obtains measurement result.
The present invention adopts the method for the electrochemical reaction parameters on the measurement collection electrode to measure, and parameter variation range is large, can obtain higher detection sensitivity.
Description of drawings
Fig. 1 is the schematic top plan view of structure of the present invention.
Fig. 2 is the schematic side view of structure of the present invention.
Fig. 3 is for adopting the structure schematic top plan view of the present invention of interdigital electrode embodiment.
Embodiment
With reference to attached Fig. 1 and 2.The present invention includes contrast electrode 1, produce electrode 2, passive electrode 3 and to electrode 4, the distance between described generation electrode and the passive electrode is less than or equal to 100 microns, and the bipolar electrode continuous current potentiometer 8 that detects the electrode kinetics parameter on the passive electrode.Accompanying drawing 3 adopts the enforcement synoptic diagram of interdigital electrode form for the present invention, in the drawings, contrast electrode, identical with Fig. 1 and 2 to the drawing reference numeral of electrode, the drawing reference numeral that produces electrode is 6, the drawing reference numeral of passive electrode is 7.
According to the difference of detected object, if antigen or pathogenic microbes detect are producing electrode and passive electrode surface and be fixed with the antibody of measurand.
Here, the effect of contrast electrode mainly is to be used to provide voltage reference, and electrode is used to provide the electrode bypass, so that electric current is from flowing through to electrode rather than from contrast electrode, to avoid electric current on the impact of the voltage of contrast electrode.Producing electrode and passive electrode is the electrode that plays a major role among the present invention.
Present embodiment is used for colibacillary detection.In the present embodiment, produce the structure (as shown in Figure 3) that electrode 6 and passive electrode 7 adopt interdigital electrode, its purpose mainly is the working area that increases simultaneously two electrodes under the prerequisite that keeps generation electrode and passive electrode fine pitch.Contrast electrode, generation electrode, passive electrode and electrode all adopted the integrated circuit fabrication technique of standard, the method by sputter or evaporation forms in substrate of glass, and wherein the material that adopts of contrast electrode is silver, and the generation electrode, passive electrode and the material that electrode is adopted are gold.The interdigital width that produces electrode and passive electrode is 10 microns, and interdigital distance also is 10 microns.At the Escherichia coli antibody of electrode surface by biochemical method affixed merchandise commonly used.In reality detects, at first will install to immerse and contain [Fe (CN)
6]
3-/4-Solution in, general control [Fe (CN)
6]
3-/4-Concentration is less than 100mM, present embodiment [Fe (CN)
6]
3-/4-Concentration be 10mM, take contrast electrode as benchmark, apply 0.4V voltage at the generation electrode, and apply at passive electrode-0.2V voltage, test adds and contains the electric current of flowing through on the passive electrode of colibacillary detected solution front and back respectively, realizes colibacillary detection.Electric current on the passive electrode before and after the biological respinse is compared, and when not having Escherichia coli in the solution, recording electric current at passive electrode is 800 microamperes, and colibacillary concentration is 10 in solution
7During cfu/ml, recording electric current at passive electrode is 300 microamperes.And colibacillary concentration is 10 in solution
10During cfu/ml, recording electric current at passive electrode is 50 microamperes.
Above-mentioned detection method also can be: take contrast electrode as potential reference point, apply a recovery voltage at the generation electrode respectively before biological respinse, apply an oxidation voltage on the passive electrode, then measure the electric current on the passive electrode of flowing through; After finishing, biological respinse repeats again aforesaid operations; Electric current after aforesaid operations finishes on the contrast passive electrode obtains measurement result.
The pick-up unit that present embodiment provides is identical with embodiment 1.In testing process, at first will install to immerse and contain 10mM[Fe (CN)
6]
3-/4-Solution in, take contrast electrode as benchmark, apply 0.4V voltage at the generation electrode, and apply at passive electrode-the 0.2V DC voltage less alternating voltage of an amplitude that superposes, generally, alternating voltage is less than 10mV, and the proportion of present embodiment is the sinusoidal voltage of 10mV to the 1MHz amplitude from 1Hz, adopt respectively the test of standard Electrode with Electrochemical Impedance Spectroscopy to add and contain electrode reaction resistance on the passive electrode of colibacillary detected solution front and back, contrast realizes colibacillary detection.Impedance on the passive electrode before and after the biological respinse is compared, and when not having Escherichia coli in the solution, recording electrode impedance at passive electrode is 400 ohm, and colibacillary concentration is 10 in solution
7During cfu/ml, recording electrode impedance at passive electrode is 900 ohm of peaces.And colibacillary concentration is 10 in solution
10During cfu/ml, recording electrode impedance at passive electrode is 2000 ohm.
Above-mentioned detection method also can be: take contrast electrode as potential reference point, before biological respinse, apply-recovery voltage at the generation electrode respectively, apply an oxidation voltage on the passive electrode, and then the less alternating voltage of an amplitude that superposes, the then electrode reaction resistance on the measurement collection electrode; After finishing, biological respinse repeats again aforesaid operations; Electrode reaction resistance after aforesaid operations finishes on the contrast passive electrode obtains measurement result.
The detection of embodiment 3DNA hydridization
The structure of the detecting device of present embodiment and embodiment one are basic identical, just at electrode surface fixedly single stranded DNA rather than Escherichia coli antibody.In reality detects, at first will install to immerse and contain 10mM[Fe (CN)
6]
3-/4-Solution in, take reference electrode as benchmark, apply 0.4V voltage at the generation electrode, and apply at passive electrode-the 0.2V DC voltage, test adds electrode reaction resistance on the detected solution front and back passive electrode that contains the corresponding DNA strand respectively, impedance on the passive electrode before and after the reaction is compared, realize the detection of DNA hydridization.
Claims (2)
1. one kind based on the antibody/antigen of oxidation/reduction probe or the detection method of DNA hybridization detection device, it is characterized in that: described pick-up unit comprises contrast electrode, produces electrode, passive electrode and to electrode, distance between described generation electrode and the passive electrode more than or equal to 80 nanometers less than or equal to 100 microns, be fixed with the antibody of detected object or the dna molecular of strand at generation electrode and passive electrode surface, it also is provided with the bipolar electrode continuous current potentiometer that detects the electrode kinetics parameter on the passive electrode; Produce electrode and be applied in oxidation voltage, passive electrode is applied in recovery voltage; Described detection method is carried out respectively following operation before and after biological respinse: take contrast electrode as potential reference point, apply an oxidation voltage at the generation electrode, apply a recovery voltage on the passive electrode, then measure the electric current on the passive electrode of flowing through; The measurement result that electric current after aforesaid operations finishes on the contrast passive electrode obtains.
2. one kind based on the antibody/antigen of oxidation/reduction probe or the detection method of DNA hybridization detection device, it is characterized in that: described pick-up unit comprises contrast electrode, produces electrode, passive electrode and to electrode, distance between described generation electrode and the passive electrode more than or equal to 80 nanometers less than or equal to 100 microns, be fixed with the antibody of detected object or the dna molecular of strand at generation electrode and passive electrode surface, it also is provided with the bipolar electrode continuous current potentiometer that detects the electrode kinetics parameter on the passive electrode; Produce electrode and be applied in oxidation voltage, passive electrode is applied in recovery voltage; Described detection method is carried out respectively following operation before and after biological respinse: take contrast electrode as potential reference point, apply an oxidation voltage at the generation electrode, apply a recovery voltage on the passive electrode, then superpose one less than the less alternating voltage of the amplitude of recovery voltage, then the electrode reaction resistance on the measurement collection voltage; Electrode reaction resistance after aforesaid operations finishes on the contrast passive electrode obtains measurement result.
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| CN1699929A (en) * | 2005-05-11 | 2005-11-23 | 浙江大学 | Electrochemical micro flow measurement method and electrochemical micro flow sensor |
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| CN1699929A (en) * | 2005-05-11 | 2005-11-23 | 浙江大学 | Electrochemical micro flow measurement method and electrochemical micro flow sensor |
Non-Patent Citations (6)
| Title |
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| Determination of bacterial cell concentrations by electrical measurements;Thomas G Wheeler et al;《Journal of clinical microbiology》;19750131;第1卷(第1期);全文 * |
| Thomas G Wheeler et al.Determination of bacterial cell concentrations by electrical measurements.《Journal of clinical microbiology》.1975,第1卷(第1期),全文. |
| 基于纳米金胶标记DNA探针的电化学DNA传感器研究;蔡宏等;《高等学校化学学报》;20030831;第24卷(第8期);全文 * |
| 张灯.检测大肠杆菌的电化学阻抗谱免疫生物传感器研究.《中国优秀硕士学位论文全文数据库》.2005,(第5期),全文. |
| 检测大肠杆菌的电化学阻抗谱免疫生物传感器研究;张灯;《中国优秀硕士学位论文全文数据库》;20050915(第5期);全文 * |
| 蔡宏等.基于纳米金胶标记DNA探针的电化学DNA传感器研究.《高等学校化学学报》.2003,第24卷(第8期),全文. |
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