CN101339154A - Composite modified electrode test piece - Google Patents
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- CN101339154A CN101339154A CNA2007101275904A CN200710127590A CN101339154A CN 101339154 A CN101339154 A CN 101339154A CN A2007101275904 A CNA2007101275904 A CN A2007101275904A CN 200710127590 A CN200710127590 A CN 200710127590A CN 101339154 A CN101339154 A CN 101339154A
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Abstract
The invention relates to an electrode test block with modified surface which is used for measuring electrochemical signals; wherein, electronic signals are amplified through the cooperation of the layer of nanometer-sized gold particles and the layer of fat-soluble electronic media. The invention also provides a biosensor which is provided with the electrode test block.
Description
Technical field
The present invention relates to utilize the collaborative biology sensor that amplifies the electrode test piece of the electronic signal that is used to measure electrochemical signals and contain described electrode test piece of nano-scale goldc grains sublayer and fat-soluble electron mediator layer.
Background technology
The biological sensing analytical technology is described as the new lover of 21st century science and technology, and biology sensor is the check and analysis system that applying biological sensing analytical technology constitutes, and is to be combined by bio-identification material and various signal converter.The electrochemica biological sensor system performance is simple and easy, has excellent sensitivity simultaneously, therefore become an excellent sensing element transmission mechanism, add specificity (specificity) relation between biomolecule, more can solve selectivity (selectivity) problem that sensing element often faces.Because biology sensor and electrode test piece can provide the fast detecting of pin-point accuracy, therefore it can be in research with clinically in order to handle large quantities of corpse or other object for laboratory examination and chemical testing, and wherein ferment-electrochemical sensor (for example commercially available galvanochemistry blood sugar measuring system) is exactly to utilize the glucose oxidase on the electrode to carry out the concentration determination of glucose molecule.The evolution of the immobilized biology sensor of ferment can generally be divided into three developing stage, and the phase one is to utilize the dissolved oxygen that is consumed in the general dissolved oxygen sensing electrode sensing oxidation ferment catalysis determinand process, learns testing concentration indirectly; The another kind of product that has electrochemical activity in the ferment catalytic reaction that detects, common as hydrogen peroxide.The utilization of subordinate phase is mainly the adding of electron transport thing, improve the efficient of electron transport by the electron transport thing to electrode surface, also utilize it to have the characteristic of redox reversible simultaneously, the electronics that reception ferment catalytic reaction is produced makes electron transport substance be reduced into ortho states, and carry out oxidation reaction successfully electron transport is given electrode to form electric signal at electrode surface, its advantage has lower oxidation-reduction potential for the electron transport thing, can reduce the required current potential of whole sensing effect, avoid the influence that other interfering material caused that produces because of noble potential.Phase III is to use some to have the ferment of accessory factor, the resistance that electronics passes out from ferment in the time of can adding accessory factor with reduction ferment catalytic oxidation or reduction reaction, modal accessory factor such as nicotinamide adenine dinucleotide (nicotinamide adenine dinucleotide; NADH), NADH can with electronics by reversible oxidation and reduction process with electron transport to electrode, many achievements in research show the method for the electron transport efficient of this kind method far above preceding two stages, and make sensor that higher sensitivity be arranged, but shortcoming is a ferment, and immobilized preparation process is loaded down with trivial details, and its stability at room temperature is not good, the transportation that is unfavorable for commodity with store.
Utilize antibody and antigen or bifilar complementation or complementary ribonucleic or the nuclifort of part, these have high selectivity and compatibility as the biomolecule detection Design Mode, the researcher can be fixed on high selectivity and compatibility biomolecule on the various sensor, as detecting sign.Described biomolecule includes, but is not limited to antibody, various antigen, ferment, nucleic acid, tissue or individual cells.Therefore, can select the combination of antigen, antibody and electrochemical appliance for use, its principle is similar to traditional solid-phase immunoassay method, but antibody or antigen are fixed on the surface of sensor, combine by the corresponding mobile phase molecule of stationary phase molecule and its, be used for detecting the reciprocation between antibody and antigen, and enlarge detectable electric signal with the converter in the sensor and carry out quantitative test, this device is called immune electrochemical sensor.
The immune electrochemical sensing that ferment is demarcated formula is the mode of general development the most at present, and heterogeneous ferment immune analysis method wherein comprises competitive mode analysis and two kinds of sandwich style analyses.The key step of competitive mode comprises: (1) will have specific antibody to determined antigen and be fixed on electrode surface, (2) insert antigen and the determined antigen of demarcating through ferment simultaneously, (3) remove unconjugated antigen with the rinse step, insert this matrix (substrate) of demarcating ferment and carry out catalytic reaction, and generation has the product of electrochemical activity, (4) by detecting this product and then quantitative determined antigen, based on this sensing principle, gained current signal and determined antigen concentration are inverse relation in the competitive mode analysis.On the contrary, the sensing of sandwich style mechanism gained current signal and the proportional relation of determined antigen concentration.The immunity electrochemical sensor is compared with traditional immunoassay, can effectively reduce a small amount of various analysis operation cost.But immune electrochemical sensor is often low excessively because of detecting subject matter concentration when practical application, causes the signal/noise of subsequent current to compare low problem.Therefore, when the immune electrochemical measurement system of development, need a kind of biological sensing electrode test piece of development, the method that it has the electric signal through strengthening thereby promotes accuracy in detection with amplification redox current signal.
In addition, but the wide range of general immune analysis method detection of biological or biomolecule specy, and the Escherichia coli that for example often cause food poisoning and enteritis vibrios often are the research object of the immune analysis method of novelty.The pathogen that is contained in the food is a lot, need separate with selective medium with the different bacterium property that increases traditionally, and then further bacterial classification could be identified out exactly with the biochemical reaction test.This also is that traditional detection method often needs the more time and the cause of manpower now, and feels simply helpless when running into the pathogen of novel or mutation through being everlasting, and this also is the problem that presses for solution.
The 6th, 491, No. 803 United States Patent (USP)s and 1462880A number and the open case of 1462881A China's Mainland patented claim are the related application of nano-scale material in the biochemical sensing electrode.Yet described patent does not still break away from prior art, and it still needs complicated preparation process.The 6th, 491, the 803B1 United States Patent (USP) discloses a kind of test piece, but described test piece need mix all reactive materials (comprising nano metal particles) when preparing in advance, coat on the electrode by wire mark subsequently, yet its wire mark condition is wanted the quite strict homogeneity that just can keep wire mark.1462880A number and the processing of water soluble polymer carrier, the CNT of revising and ferment responding layer at least three floor materials such as (comprising ferment, electronic media, stabilizing agent, damping fluid etc.) such as coating and dry carboxymethyl cellulose in regular turn of the open case of 1462881A China's Mainland patented claim, electrode test piece manufacturing process is very complicated.Taiwan I276799 patent of invention is a kind of application of simplifying processing of CNT in the biochemical sensing electrode.
Even if but comprehensive aforementioned techniques, comprise use, compatibility biomolecule identification program, the use of nanometer material in electrochemical measurement of electronic media, repeatedly immersion, cleaning procedure in the immune identification process can not be known, maybe can be embodied directly in easily by inference, the incident low signal-to-noise ratio problem of low content immune detection subject matter can not be solved simultaneously; Therefore, concerning this industry, still existing does not need as procedure of processing loaded down with trivial details as the prior art and still can keep the demand of the electrode test piece of enough big current signal simultaneously.The present invention is exactly for solving this problem that has application.
Summary of the invention
A purpose of the present invention is to provide a kind of electrode test piece that is used to measure electrochemical signals, and its surface is through revising to increase the redox electronic signal, and it comprises:
Dull and stereotyped insulating substrate;
Electrode system with conducting film, wherein said conducting film is coated in the one side of described dull and stereotyped insulating substrate, to form working electrode and the reference electrode that separates and be not connected;
Be coated on the electric insulation layer on the described dull and stereotyped insulating substrate, its part covers described electrode system, and the part that described electrode system is not covered by described electric insulation layer forms terminals and the electrochemical reaction end that comprises working electrode and reference electrode respectively;
Goldc grains sublayer with nano-scale, it covers the electrochemical reaction end of described working electrode to small part; And
Fat-soluble electron mediator layer, it covers the electrochemical reaction end of described working electrode to small part.
Another object of the present invention is to provide a kind of biology sensor, it comprises electrode test piece of the present invention and pick-up unit.
Description of drawings
Fig. 1 (A) is that the preparation of electrode test piece and the element thereof of one embodiment of the invention launches synoptic diagram, Fig. 1 (B) three kinds of states that to be electrochemical reaction end (5) and electrode system wherein covered by nano-scale goldc grains sublayer (7) and fat-soluble electron mediator layer (9).
Fig. 2 is that the electrode of one embodiment of the invention is revised layer synoptic diagram, Fig. 2 A is the sectional view of the described electrochemical reaction end of expression, Fig. 2 B is the electrochemical reaction end that covers through fat-soluble electron mediator layer (9), Fig. 2 C is the electrochemical reaction end through fat-soluble electron mediator layer and nano-scale goldc grains sublayer (7) covering, Fig. 2 D is the electrochemical reaction end of surface through the bridging agent combination, Fig. 2 E covers and surperficial electrochemical reaction end with bridging agent through fat-soluble electron mediator layer (9), Fig. 2 F covers and surperficial electrochemical reaction end with bridging agent through fat-soluble electron mediator layer (9) and nano-scale goldc grains sublayer (7), and Fig. 2 G covers through nano-scale goldc grains sublayer (7) and surperficial electrochemical reaction end with bridging agent.
Fig. 3 is the cyclic voltammetry result of example 1 of the present invention.
Fig. 4 is the cyclic voltammetry result of example 2 of the present invention.
Fig. 5 (a) to (e) is the fixing process flow diagram of redox ferment of the embodiment of the invention 3.
Fig. 6 is the ampere-immunoassays result of example 3 of the present invention.
Embodiment
The present invention relates to a kind of electrode test piece and the biology sensor that comprises described electrode test piece, when it is characterized in that measuring the electronic signal that described redox ferment (13) migrated out, described nano-scale goldc grains sublayer (7) can collaborative electronic signal of amplifying described electrochemical measurement system with fat-soluble electron mediator layer (9).The loaded down with trivial details procedure of processing of prior art different from the past, the electrochemical reaction district of electrode test piece of the present invention only needs the job sequence of two-layer material, therefore can reduce cost of manufacture, and while amplified current signal.
A purpose of the present invention is to provide a kind of electrode test piece that is used to measure electrochemical signals, and its surface is through revising to increase the redox electronic signal, and it comprises:
Dull and stereotyped insulating substrate (1);
Have the electrode system (2) of conducting film, wherein said conducting film is coated in the one side of described dull and stereotyped insulating substrate (1), to form the working electrode (2a) and reference electrode (2b) that separates and be not connected;
Be coated on the electric insulation layer (3) on the described dull and stereotyped insulating substrate, its part covers described electrode system (2), and the part that described electrode system (2) is not covered by described electric insulation layer forms terminals (4) and the electrochemical reaction end (5) that comprises working electrode (2a) and reference electrode (2b) respectively;
Goldc grains sublayer (7) with nano-scale, it covers the electrochemical reaction end (5) of described working electrode (2a) to small part; And
Fat-soluble electron mediator layer (9), it covers the electrochemical reaction end (5) of described working electrode (2a) to small part.
According to the present invention, described dull and stereotyped insulating substrate (1) has the characteristic of flat surface and electrical isolation, and can tolerate 40 to 200 ℃ temperature capacity so that the processing of heating.The material that is suitable as dull and stereotyped insulating substrate of the present invention includes, but is not limited to Polyvinylchloride, glass fibre, polyester, phenolics plate, polyethylene terephthalate, polycarbonate, polypropylene, tygon, polyamide, polystyrene, glass or pottery.
According to the present invention, the electrode system of described conducting film (is the web plate electrode with the screen printing metal film preferably, as the 6th, 923, the 894B2 United States Patent (USP) discloses) or the adhesiving metal film (as the 6th, 254, the 736B1 United States Patent (USP) discloses) mode, be coated in the one side of described dull and stereotyped insulating substrate (1).The metal membrane material that is fit to includes, but is not limited to gold, silver, platinum or palladium, and the printer's ink that is fit to screen printing includes, but is not limited to potpourri, volatility graphite, copper China ink or the above combination of carbon ink, Jin Mo, Yin Mo, carbon ink and silver China ink, prints carbon ink again as first Yin Yinmo.Aspect of the present invention one concrete enforcement, wherein said web plate electrode comprises a silver ink layer and a carbon ink layer, and described carbon ink layer is covered on the described silver ink layer.
According to the present invention, the area of the working electrode in the described electrode system is generally greater than the area of reference electrode.
According to the present invention, described at least one electric insulation layer (3) thickness is about 0.01 to 0.6mm.The electrically insulating material of this area routine all is applicable to electric insulation layer of the present invention, and described electrically insulating material is coated on the described electrode system (2) with screen printing method.Aspect of the present invention one concrete enforcement, have two electric insulation layers (3) on the described electrode system (2), it is respectively across the centre position and the end of described electrode system (2), described electrode system (2) is separated into electrochemical reaction end (5) and terminals (4).
According to the present invention, described nano-scale goldc grains sublayer (7) is coated on the nano-scale colloidal gold solution on the electrochemical reaction end (5) of described working electrode, is fixed on described electrochemical reaction end (5) surface by physical property absorption; Or can revise described electrochemical reaction end (5) surface (as Fig. 2 F and G) with bridging agent (8) in advance, so that the nano-scale gold particle is fixed on described electrochemical reaction end (5) surface more equably, and described bridging agent (8) can close other material (for example: protein (as antibody, part or acceptor), compound or nucleotide sequence) by follow-up chain.Be applicable to that nano-scale gold particle of the present invention is of a size of less than 100 nanometers, it is preferably about 5 to 50 nanometers, more preferably about 13 nanometers.According to the present invention, be applicable to that nano-scale colloidal gold solution of the present invention is chlorauride (HAuCl
4) through suitable catalyzer, receive solution (sodium citrate), the nm of gold suspending liquid of gained after the reduction as citric acid.Bridging agent can be evenly and is formed the arrangement paradigmatic structure of single or multiple lift dispersedly, according to the present invention, bridging agent is the compound with difunctionality base, and the one functional group is used for the surface combination with electrochemical reaction end (5), and another functional group is used for combining with binding member.Be applicable to that bridging agent of the present invention is the functional group, include, but is not limited to the compound of carboxylic acid group (carboxylic acid), mercapto (thiol), alcohol radical (alcohol), amido (amine) or aldehyde radical (aldehyde); Wherein be preferably and contain aldehyde compound; And glutaraldehyde (glutaraldehyde) more preferably.
According to the present invention, described fat-soluble electron mediator layer (9) makes described fat-soluble electronic media be fixed on described electrochemical reaction end (5) surface with the absorption of physical property adhering mode by the suspending liquid of fat-soluble electronic media after with organic solvent dissolution is coated described electrochemical reaction end (5) surface.Therefore, described fat-soluble electronic media can be because of repeatedly immersion, cleaning procedure and was lost efficacy; Also removed from simultaneously with covalent bonds and be fixed in complicated procedures on the electrode system.Be applicable to that fat-soluble electronic media of the present invention has the redox characteristic that receives or supply with electronics, it includes, but is not limited to tetrathiafulvalene (tetrathiafulvalene), tetra cyanogen subculture dimethyl benzene quinone (tetracyanoquinodimethane), meldola blue (meldola blue) or ferrocene (ferrocene) or derivatives thereof; Wherein be preferably ferrocene or derivatives thereof (Joseph wang.2000.Analytical electrochemistry); More preferably 1,1 '-ferrocenedicarboxylic acid (ferrocenedicarboxylic acid).The organic solvent that is applicable to the described fat-soluble electronic media of dissolving include, but is not limited to ketone, alcohols or the inferior maple of dimethyl (Dimethyl Sulfoxide, DMSO); Wherein be preferably ethanol.
According to the present invention, the coating of described nano-scale goldc grains sublayer and described fat-soluble electron mediator layer there is no restriction successively in proper order, be preferably apply described nano-scale goldc grains sublayer earlier after, apply described fat-soluble electron mediator layer again.
According to the present invention, so-called " covering to small part " be meant electrochemical reaction end (5) entirely by nano-scale goldc grains sublayer (7) and fat-soluble electron mediator layer (9) cover (shown in the 6a of Fig. 1), or only the electrochemical reaction end (5) in the working electrode (2a) is capped (shown in the 6b of Fig. 1) fully or partly is capped (shown in the 6c of Fig. 1).
According to the surperficial modified electrode test piece that is used to measure electrochemical signals of the present invention, wherein also can comprise electrochemical reaction end (5) surface of being fixed in working electrode (2) and the binding member that can combine with the subject matter specificity.Be applicable to that binding member of the present invention includes, but is not limited to protein (as antibody, antigen, protein ligands or receiver), nucleotide sequence or compound.The technician can be according to the binding characteristic of subject matter (as antibody/antigen or ligand/receptor combination, perhaps nucleotide hybridization), select suitable binding member, and selected binding member is fixed in electrochemical reaction end (5) surface according to routine techniques (.2001.Biomolecular Sensors such as Electra Gizeli), for example binding member is fixed in the electrochemical reaction end surfaces with bridging agent (8).Well-known as those skilled in the art in the technical field of the invention, be applicable to that subject matter of the present invention can be medical diagnosis mark, medicine, bacterium, toxin, environmental contaminants or nucleotide.
Electrode test piece of the present invention at subject matter with after the binding member specificity combines, can produce the electrochemical activity product by redox ferment (13) and its matrix (substrate) reaction, detect described electrochemical activity product again to reach the purpose of quantitative test tested object.Be applicable to that redox ferment of the present invention (13) includes, but is not limited to glucose oxidase, glucose reductase, Lactate Oxidase, pyruvate oxidase or hydrogen peroxidase, wherein be preferably hydrogen peroxidase, and it can carry out electrochemical measurement with hydroperoxidation.According to the present invention, the operating voltage that utilizes the combination of hydrogen peroxidase, hydrogen peroxide and fat-soluble electronic media is about 150 to 420mV.According to the present invention aspect the electrochemical measurement mode detection microbial antigen of ampere-immunosensor, the voltage of its utilization is preferably about 300mV.
According to of the present invention one preferred enforcement aspect (as shown in Figure 5), described binding member is first antibody (10), described first antibody is with compatibility and electrochemical reaction end (5) surface combination, or covalent bond and electrochemical reaction end (5) surface combination (shown in Fig. 5 (a)) by bridging agent (8).Described first antibody through with tested antigen (11) selectivity combine (shown in Fig. 5 (c)) after, and described tested antigen (11) combines (shown in Fig. 5 (e)) formation one redox ferment layer with described antigen (11) being had narrow spectrum second antibody (12)-redox ferment (13) complex again, carries out electrochemical measurement with hydroperoxidation again.
According to another preferably enforcement aspect of the present invention, described binding member is antigen (11), and described antigen (11) is with compatibility and electrochemical reaction end (5) surface combination, or covalent bond and electrochemical reaction end (5) surface combination by bridging agent (8).Described antigen (11) through with tested first antibody (10) selectivity combine after, and described first antibody (10) again with the second antibody (12) that can combine-redox ferment (13) complex with it in conjunction with forming a redox ferment layer, carry out electrochemical measurement with hydroperoxidation again.
According to the present invention, first antibody wherein (10) can be monoclonal antibody or multi-strain antibody with second antibody (12).And the present invention not only can be used for sandwich style immunity identification binding analysis (for example shown in Figure 5), also can be used for other analysis, for example competitive mode identification binding analysis.
According to the present invention, the technician can select suitable bridging agent (8) according to the kind (for example protein (as antibody, antigen, protein ligands or receiver), nucleotide sequence or compound) of binding member.Bridging agent can be evenly and is formed the arrangement paradigmatic structure of single or multiple lift dispersedly, according to the present invention, bridging agent is the compound with difunctionality base, and the one functional group is used for the surface combination with electrochemical reaction end (5), and another functional group is used for combining with binding member.Be applicable to that bridging agent of the present invention is the functional group, include, but is not limited to the compound of carboxylic acid group (carboxylic acid), mercapto (thiol), alcohol radical (alcohol), amido (amine) or aldehyde radical (aldehyde); Wherein be preferably and contain aldehyde compound (.2001.Biomolecular Sensors such as Electra Gizeli); And glutaraldehyde (glutaraldehyde) more preferably.
According to preferred enforcement of the present invention aspect, the formation step of described redox ferment layer comprises:
(a) in conjunction with first antibody (10) or bridging agent (8)-first antibody (10) complex on electrochemical reaction end (5) surface,
(b) unconjugated first antibody of flush away (10) or bridging agent (8)-first antibody (10) complex,
(c) conjugated antigen,
(d) in conjunction with second antibody (12)-redox ferment (13) complex and
(e) the not fixed second antibody of closing of flush away (12)-redox ferment (13) complex;
Or can be in addition:
(a) in conjunction with first antibody (10) or bridging agent (8)-first antibody (10) complex on electrochemical reaction end (5) surface,
(b) conjugated antigen and second antibody (12)-redox ferment (13) complex forms antigen (11)-second antibody (12)-redox ferment (13) complex,
(c) in conjunction with described first antibody (10) and described antigen (11)-second antibody (12)-redox ferment (13) complex, or in conjunction with described bridging agent (8)-first antibody (10) complex and described antigen (11)-second antibody (12)-redox ferment (13) complex and
(d) the not fixed described antigen (11) that closes of flush away-second antibody (12)-redox ferment (13) complex together;
Or also can be:
(a) conjugated antigen (11) or bridging agent (8)-antigen (11) complex is on electrochemical reaction end (5) surface,
(b) unconjugated antigen of flush away (11) or bridging agent (8)-antigen (11) complex,
(c) in conjunction with first antibody (10),
(d) in conjunction with second antibody (12)-redox ferment (13) complex and
(e) the not fixed second antibody of closing of flush away (12)-redox ferment (13) complex;
Or can be:
(a) conjugated antigen (11) or bridging agent (8)-antigen (11) complex in electrode surface on electrochemical reaction end (5) surface,
(b), form first antibody in conjunction with first antibody and second antibody (12)-redox ferment (13) complex
(10)-second antibody (12)-redox ferment (13) complex,
(c) in conjunction with described antigen and described first antibody (10)-second antibody (12)-redox ferment (13) complex, or in conjunction with described bridging agent (8)-first antibody (10) complex and described antigen (11)-second antibody (12)-redox ferment (13) complex and
(d) the not fixed described first antibody (10) that closes of flush away-second antibody (12)-redox ferment (13) complex together.
Another object of the present invention provides a kind of biology sensor, and it comprises electrode test piece as herein described and pick-up unit.Preferably, described pick-up unit is by a voltage output device, a signal receiving device, and the current sensor formed of a display device.Described voltage output device can be exported the following voltage of 300mV to the electrochemical reaction district according to electrode test piece of the present invention, impels in a responding layer and the corpse or other object for laboratory examination and chemical testing to make the electronic media that riddles the electrochemical reaction district be oxidized into the state of oxidation by reducing condition after the specific subject matter reaction.Electric current, voltage or resistance value when described signal receiving device can change this state receive, and pass display device back, show the content of specific subject matter in the corpse or other object for laboratory examination and chemical testing whereby.
Electrode test piece of the present invention does not need as loaded down with trivial details procedure of processing as the prior art, simultaneously can the amplified current signal.In addition, electrode test piece of the present invention has the design that can reduce corpse or other object for laboratory examination and chemical testing demand, and can have a plurality of sample position (for example: electrode test piece is dropped on the electrode test piece near a corpse or other object for laboratory examination and chemical testing or with a corpse or other object for laboratory examination and chemical testing).Therefore, electrode test piece of the present invention not only prepares comparatively easy, can be more convenient and effectively from the analysans sampling, so that patient's inconvenience can be reduced to is minimum, and still can satisfy the demand that keeps enough big electronic signal simultaneously.
Following examples will the present invention is further illustrated, and not in order to limit the scope of the invention, modification that one of ordinary skill in the art can reach easily and change all are covered by in protection scope of the present invention.
[embodiment]
Example 1.
According to the 6th, 923, method shown in the embodiment one of 894B2 United States Patent (USP), the polymer resin conductive carbon paste that will contain Polyvinylchloride and polyurethane, be imprinted on half tone on the flat surface of a PVC plate substrate (1), to form the electrode system of being formed by a working electrode (2a) and a reference electrode (2b) of each self-separation (2), oven dry then; Immediately in the same side that is printed on described electrode system (2), be coated with an electric insulation layer, and the exposed working electrode (2a) of reserve part and reference electrode (2b) are dried then to form terminals (4) and electrochemical reaction end (5), finish an electrode test piece (as the A of Fig. 2) of organizing A in contrast.
Get the electrode test piece of part control group A, and at an amount of fat-soluble electronic media (1,1 '-ferrocenedicarboxylic acid (ferrocenedicarboxylic acid)) a small amount of 95% ethanolic solution of middle adding, and it is dissolved fully with ultrasonic oscillator concussion, drip again and on the electrode test piece of described control group A, revise described electrochemical reaction end (5), after water is removed unconjugated fat-soluble electronic media (9), finish experimental group B electrode test piece (as the B of Fig. 2) with fat-soluble electron mediator layer (9).Other gets the electrode test piece of part control group A, and with chlorogold solution (HAuCl
4Sigma G-4022) heats in the oil bath mode, add the sodium citrate continuous stirring again, present claret solution with formation, contain the nano colloid gold particle solution that diameter is about 13 nanometers to be ready for, dropping is at described electrochemical reaction end (5), to form a nano-scale goldc grains sublayer (7), water flush away unconjugated goldc grains sublayer and salt then, carry out the coating program of aforementioned fat-soluble electron mediator layer (9) again, have nano-scale goldc grains sublayer (7) and the composite modified electrode test piece of fat-soluble electron mediator layer (9) and finish, as experimental group C (as the C of Fig. 2).Its preferred light absorption value at 520nm of aforementioned nano colloid gold particle solution is about 0.9 to 1.2.
These three kinds of electrode test pieces, under the situation that hydrogen peroxide exists, in pH 7.2 phosphate buffer solutions, carry out cyclic voltammetric (cyclic voltammetry) analysis, the current peak of experimental group C is about 4 times (as Fig. 3) of experimental group B, confirm described nano-scale goldc grains sublayer, have the characteristic of amplification through the redox electric current of the electrode of fat-soluble electronic media modification.
Example 2.
Get the electrode test piece of part example 1 control group A, to drip as the glutaraldehyde (glutaraldehyde) of bridging agent (8) in electrochemical reaction end (5), the unconjugated bridging agent of water flush away (8) is finished an electrode test piece as experimental group D (as the D of Fig. 2) again.Get the electrode test piece of part experimental group D, and the method shown in example 1 is ready for alcohol-soluble fat-soluble electronic media (1,1 '-ferrocenedicarboxylic acid), drip on the electrode test piece of described experimental group D, revise described electrochemical reaction end (5), after water is removed unconjugated fat-soluble electronic media, finish experimental group E electrode test piece (as the E of Fig. 2) with fat-soluble electron mediator layer (9).Other gets the electrode test piece of part experimental group D, and be ready for the method shown in the example 1 and contain the nano colloid gold particle solution of diameter about 13 nanometers, drip in described electrochemical reaction end, to form a nano-scale goldc grains sublayer (7), unconjugated gold particle and salt are removed in washing then, carry out the coating program of aforementioned fat-soluble electron mediator layer (9) again, have nano-scale goldc grains sublayer (7) and the composite modified electrode test piece of fat-soluble electron mediator layer (9) and finish, as experimental group F (as the F of Fig. 2).Further according to the nano colloid gold particle solution condition and the cyclic voltammetry of example 1, measure the electrode test piece of D, E and F group, the current peak of experimental group F is about 3 times (as Fig. 4) of experimental group E, confirm described nano-scale goldc grains sublayer (8), have the characteristic of amplification through the redox electric current of the electrode of fat-soluble electronic media modification.
Example 3.
For technology of the present invention being applied to the entity check of antigen (11),, make through nano-scale goldc grains sublayer (7) and the composite modified electrode test piece of fat-soluble electron mediator layer (9) earlier with material and the flow process of experimental group F in the aforementioned example 2.Be equipped with monoclonal antibody with anti-E.coli O157:H7 as first antibody (10), will engage with hydrogen peroxidase as the anti-E.coliO157:H7 multi-strain antibody of second antibody (12) in addition, form anti-E.coli O157:H7 multi-strain antibody-hydrogen peroxide multienzyme complex.Carry out hydrogen peroxidase fixing (as Fig. 5) on electrode via following program:
(a) in conjunction with bridging agent (8)-first antibody (10) complex on electrode surface,
(b) unconjugated first antibody of flush away (10) or bridging agent (8)-first antibody (10) complex,
(c) conjugated antigen (11),
(d) in conjunction with second antibody (12)-hydrogen peroxide combined enzyme agent,
(e) the unconjugated second antibody of flush away (12)-hydrogen peroxide combined enzyme agent.
Be used as hydrogen peroxidase matrix with the hydrogen peroxide in the phosphoric acid buffer aqueous solution of pH 7.2, adopt ampere-immune sensing electrochemical measurement mode detection microbial antigen of fixed voltage 300mV.Simultaneously, preparation only has the experimental group G electrode test piece (as Fig. 2 G) of nano-scale goldc grains sublayer (7) in addition, and the electrode test piece of the experimental group D of example 2, through identical ferment immobilization program, as the comparison of immune sensing microorganism utilization.Confirm described fat-soluble electron mediator layer (9), have the characteristic (as Fig. 6) of the redox electric current that amplifies the electrode of revising through the nano-scale gold particle again.
Therefore, disclosed have nano-scale goldc grains sublayer (7) and a composite modified electrode test piece of fat-soluble electron mediator layer (9), can work in coordination with and amplify the redox current signal.Simultaneously, even if this composite modified electrode test piece experience immersion and cleaning repeatedly, being fixed in the lip-deep fat-soluble electronic media of described composite modified electrode can be because of repeatedly immersion, cleaning procedure and lost efficacy yet, need not be fixed on the electrode simultaneously yet, therefore have the advantage that reduces operation, reduces cost with covalent bonds.Take into account the associated electrical chemical apparatuses manufacturer of manufacturing cost, quality and the application of electrode test piece for hope, disclosed has through nano-scale goldc grains sublayer (7) and the composite modified electrode test piece of fat-soluble electron mediator layer (9), can provide effective solution really.
Claims (27)
1. surperficial modified electrode test piece that is used to measure electrochemical signals, it comprises:
Dull and stereotyped insulating substrate;
Electrode system with conducting film, wherein said conducting film are coated on the one side of described dull and stereotyped insulating substrate, to form working electrode and the reference electrode that separates and be not connected;
Be coated on the electric insulation layer on the described dull and stereotyped insulating substrate, its part covers described electrode system, and the part that described electrode system is not covered by described electric insulation layer forms terminals and the electrochemical reaction end that comprises working electrode and reference electrode respectively;
Nano-scale goldc grains sublayer, it covers the electrochemical reaction end of described working electrode to small part; And
Fat-soluble electron mediator layer, it covers the electrochemical reaction end of described working electrode to small part.
2. electrode test piece according to claim 1, the gold particle of wherein said nano-scale goldc grains sublayer is of a size of less than 100 nanometers.
3. electrode test piece according to claim 2, the gold particle of wherein said nano-scale goldc grains sublayer is of a size of 5 to 50 nanometers.
4. electrode test piece according to claim 3, the gold particle of wherein said nano-scale goldc grains sublayer is of a size of 13 nanometers.
5. according to the described electrode test piece of arbitrary claim in the claim 1 to 4, wherein said nano-scale goldc grains sublayer covers described electrochemical reaction end fully.
6. according to the described electrode test piece of arbitrary claim in the claim 1 to 4, wherein said nano-scale goldc grains sublayer covers the electrochemical reaction end of described working electrode.
7. according to the described electrode test piece of arbitrary claim in the claim 1 to 4, wherein said nano-scale gold particle layer segment covers the electrochemical reaction end of described working electrode.
8. according to the described electrode test piece of arbitrary claim in the claim 1 to 4, the fat-soluble electronic media of wherein said fat-soluble electron mediator layer is selected from the group that is made up of tetrathiafulvalene (tetrathiafulvalene), tetra cyanogen subculture dimethyl benzene quinone (tetracyanoquinodimethane), meldola blue (meldola blue) or ferrocene (ferrocene) or derivatives thereof.
9. electrode test piece according to claim 8, the fat-soluble electronic media of wherein said fat-soluble electron mediator layer are ferrocene (ferrocene) or ferrocene derivatives.
10. electrode test piece according to claim 9, wherein said ferrocene derivatives is 1,1 '-ferrocenedicarboxylic acid (ferrocenedicarboxylic acid).
11. according to the described electrode test piece of arbitrary claim in the claim 1 to 4, wherein said fat-soluble electron mediator layer covers described electrochemical reaction end fully.
12. according to the described electrode test piece of arbitrary claim in the claim 1 to 4, wherein said fat-soluble electron mediator layer covers the electrochemical reaction end of described working electrode.
13. according to the described electrode test piece of arbitrary claim in the claim 1 to 4, wherein said fat-soluble electronic media layer segment covers the electrochemical reaction end of described working electrode.
14. according to the described electrode test piece of arbitrary claim in the claim 1 to 4, the electrochemical reaction end of wherein said working electrode further comprises bridging agent.
15. electrode test piece according to claim 14, wherein said bridging agent contains the difunctionality base, and described functional group is selected from the group that is made up of carboxylic acid group (carboxylic acid), mercapto (thiol), alcohol radical (alcohol), amido (amine) and aldehyde radical (aldehyde).
16. electrode test piece according to claim 15, wherein said bridging agent are the compound that contains aldehyde radical.
17. electrode test piece according to claim 16, wherein said bridging agent are glutaraldehyde (glutaraldehyde).
18. according to the described electrode test piece of arbitrary claim in the claim 1 to 4, the electrochemical reaction end in the wherein said working electrode further comprises a binding member, described binding member can be combined into compound with the subject matter specificity.
19. electrode test piece according to claim 18, wherein said binding member is selected from the group that is made up of protein, nucleic acid and compound.
20. electrode test piece according to claim 19, wherein said protein are antibody, antigen, protein ligands or receiver.
21. electrode test piece according to claim 18, wherein said binding member links with the electrochemical reaction end in bridging agent and the described working electrode.
22. electrode test piece according to claim 18, wherein said binding member-subject matter compound can form redox ferment layer with the redox ferment.
23. electrode test piece according to claim 22, wherein said redox ferment is selected from the group that is made up of glucose oxidase, glucose reductase, Lactate Oxidase, pyruvate oxidase and hydrogen peroxidase.
24. second antibody-redox ferment complex that electrode test piece according to claim 22, wherein said redox ferment layer comprise first antibody, the antigen subject matter as described binding member and combine with described antigen.
25. second antibody-redox ferment complex that electrode test piece according to claim 22, wherein said redox ferment layer comprise the antigen as described binding member, the first antibody that combines with described antigen and combine with described first antibody.
26. a biology sensor, it comprises according to described electrode test piece of arbitrary claim and pick-up unit in the claim 1 to 25.
27. biology sensor according to claim 26, wherein said pick-up unit is by voltage output device, signal receiving device, and the current sensor formed of display device.
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| US5200051A (en) * | 1988-11-14 | 1993-04-06 | I-Stat Corporation | Wholly microfabricated biosensors and process for the manufacture and use thereof |
| US6241863B1 (en) * | 1998-04-27 | 2001-06-05 | Harold G. Monbouquette | Amperometric biosensors based on redox enzymes |
| TW416005B (en) * | 1999-11-11 | 2000-12-21 | Apex Biotechnology Corp | Biosensor with multiple sampling ways |
| US6491803B1 (en) * | 2001-05-18 | 2002-12-10 | Apex Biotechnology Corporation | Test strip and biosensor incorporating with nanometer metal particles |
| CN2662241Y (en) * | 2003-06-13 | 2004-12-08 | 天津南开戈德集团有限公司 | Biological enzyme electrode for biosensor |
| CN1226614C (en) * | 2003-09-03 | 2005-11-09 | 中国科学院长春应用化学研究所 | Nano immunological biosensor |
| CN1215327C (en) * | 2003-09-17 | 2005-08-17 | 中国科学院长春应用化学研究所 | Preparation method of deoxyribonucleic acid electrochemical nanometer sensor |
| CN1908665A (en) * | 2005-08-02 | 2007-02-07 | 中国科学院电子学研究所 | Blended self-assembly membrane based micro ampere immunity sensor and preparation thereof |
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