CN101339154B - Composite modified electrode test piece - Google Patents
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- CN101339154B CN101339154B CN 200710127590 CN200710127590A CN101339154B CN 101339154 B CN101339154 B CN 101339154B CN 200710127590 CN200710127590 CN 200710127590 CN 200710127590 A CN200710127590 A CN 200710127590A CN 101339154 B CN101339154 B CN 101339154B
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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 electrode test piece that amplifies the electronic signal that is used for the measurement electrochemical signals of nano-scale goldc grains sublayer and fat-soluble electron mediator layer, and contain the biology sensor of described electrode test piece.
Background technology
The biological sensing analytical technology is described as the new lover of 21st century science and technology, and biology sensor is the detection analytic system that applying biological sensing analytical technology consists of, and is to be combined by bio-identification material and various signal converter.The electrochemica biological sensor system performance is simple and easy, has simultaneously excellent sensitivity, 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 process large quantities of corpse or other object for laboratory examination and chemical testing, and wherein enzyme-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 biology sensor of enzyme immobilization can generally be divided into three developing stage, and the phase one is to utilize the dissolved oxygen that consumes in the oxidase catalyzed determinand process of general dissolved oxygen sensing electrode sensing, indirectly learns testing concentration; The another kind of product that has electrochemical activity in the enzymic catalytic reaction that detects, common such as hydrogen peroxide.The utilization of subordinate phase is mainly the adding of electronics transmitter, improve the efficient that electronics is delivered to electrode surface by the electronics transmitter, also utilize simultaneously it to have the characteristic of redox reversible, the electronics that the reception enzymic catalytic reaction produces makes electron transport substance be reduced into reduction-state, and carry out oxidation reaction successfully electronics is passed to electrode to form electric signal at electrode surface, its advantage is that the electronics transmitter has lower oxidation-reduction potential, can reduce the required current potential of whole sensing effect, the impact that other interfering material of avoiding producing because of noble potential causes.Phase III is to use some to have the enzyme of accessory factor, the resistance that electronics passes out from enzyme in the time of can adding accessory factor with reduction enzymatic oxidation or reduction reaction, modal accessory factor such as nicotinamide adenine dinucleotide (nicotinamide adenine dinucleotide; NADH), NADH can be delivered to electronics on the electrode with electronics by reversible oxidation and reduction process, many achievements in research show the method for the electronics transmission efficiency of this kind method far above the first two stage, and make sensor that higher sensitivity be arranged, but shortcoming is that the preparation process of enzyme immobilization 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, enzyme, nucleic acid, tissue or individual cells.Therefore, can select the combination of antigen, antibody and electrochemical appliance, its principle is similar to traditional solid-phase immunoassay method, but antibody or antigen are fixed on the surface of sensor, by the fixedly phase molecule mobile phase molecule combination corresponding with it, 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-electrochemistry sensor.
The immune electrochemical sensing of enzyme calibration-type is the present mode of general development the most, and heterogeneous enzyme immunoassay 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 simultaneously antigen and the determined antigen of demarcating through enzyme, (3) remove unconjugated antigen with the rinse step, insert this matrix (substrate) of demarcating enzyme 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.Immune-electrochemistry sensor is compared with traditional immunoassay, can effectively reduce a small amount of various analysis operation cost.But immune-electrochemistry sensor is often excessively low because 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 with amplification redox current signal of development, the method that it has the electric signal through strengthening thereby promotes accuracy in detection.
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.Pathogen contained in the food is a lot, need to 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 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 problem in the urgent need to address.
The 6th, 491, No. 803 United States Patent (USP)s and 1462880A number and 1462881A China's Mainland Patent Application Publication case 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, 491,803 B1 United States Patent (USP)s disclose 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 to quite strictly just can be kept the homogeneity of wire mark.1462880A number and the sequentially processing of at least three floor material such as water soluble polymer carrier, the CNT of revising and enzyme reaction layer (comprising enzyme, electronic media, stabilizing agent, damping fluid etc.) such as coating and dry carboxymethyl cellulose of 1462881A China's Mainland Patent Application Publication case, 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 easily, maybe can be embodied directly in 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 such 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 large 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 for measuring 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, the described electrode system of its partial coverage, the part that described electrode system is not covered by described electric insulation layer forms respectively terminals and the electrochemical reaction end that comprises working electrode and reference electrode;
Goldc grains sublayer with nano-scale, the electrochemical reaction end of the described working electrode of its at least part of covering; And
Fat-soluble electron mediator layer, the electrochemical reaction end of the described working electrode of its at least part of covering.
Another object of the present invention is to provide a kind of biology sensor, it comprises electrode test piece of the present invention.
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 schematic 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 schematic 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 (9) and nano-scale goldc grains sublayer (7) covering, Fig. 2 D is that the surface is through the electrochemical reaction end of 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 oxidoreducing enzyme 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 oxidoreducing enzyme (13) migrates 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 for measuring 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, the described electrode system of its partial coverage (2), the part that described electrode system (2) is not covered by described electric insulation layer form respectively terminals (4) and the electrochemical reaction end (5) that comprises working electrode (2a) and reference electrode (2b);
Goldc grains sublayer (7) with nano-scale, the electrochemical reaction end (5) of the described working electrode of its at least part of covering (2a); And
Fat-soluble electron mediator layer (9), the electrochemical reaction end (5) of the described working electrode of its at least part of covering (2a).
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 heat treatment.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 screen electrode with the screen printing metal film preferably, such as the 6th, 923,894 B2 United States Patent (USP)s disclose) or the adhesiving metal film (such as the 6th, 254,736 B1 United States Patent (USP)s disclose) 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 such as first Yin Yinmo again.Aspect an implementation according to the present invention, wherein said screen 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 approximately 0.01 to arrive 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 an implementation of the present invention, have two electric insulation layers (3) on the described electrode system (2), it is respectively across 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 (2a), is fixed on described electrochemical reaction end (5) surface by physical property absorption; Or can revise described electrochemical reaction end (5) surface (such 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 (such as antibody, part or acceptor), compound or nucleotide sequence) by follow-up chain.Be applicable to nano-scale gold particle of the present invention and be of a size of less than 100 nanometers, it is preferably approximately 5 to 50 nanometers, more preferably about 13 nanometers.According to the present invention, be applicable to nano-scale colloidal gold solution of the present invention and be chlorauride (HAuCl4) through suitable catalyzer, such as sodium citrate solution (sodium citrate), the nm of gold suspending liquid of gained after the reduction.
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 fat-soluble electronic media of the present invention and have 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 dissolve described fat-soluble electronic media includes, but is not limited to ketone, alcohols or DMSO (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 sequentially there is no successively restriction, be preferably apply first described nano-scale goldc grains sublayer after, apply again described fat-soluble electron mediator layer.
According to the present invention, so-called " at least part of covering " refers to that electrochemical reaction end (5) is covered (shown in the 6a of Fig. 1) by nano-scale goldc grains sublayer (7) and fat-soluble electron mediator layer (9) institute entirely, 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 for measuring electrochemical signals of the present invention, wherein also can comprise electrochemical reaction end (5) surface of being fixed in working electrode (2) and can with the binding member of subject matter specific binding.Be applicable to binding member of the present invention and include, but is not limited to protein (such as antibody, antigen, protein ligands or acceptor), nucleotide sequence or compound.The technician can be according to the binding characteristic of subject matter (such as antibody/antigen or ligand/receptor combination, perhaps nucleotide hybridization), select suitable binding member, and according to routine techniques (the .2001.Biomolecular Sensors such as Electra Gizeli) selected binding member is fixed in electrochemical reaction end (5) surface, for example be fixed in the electrochemical reaction end surfaces with bridging agent (8) in connection with element.Well-known such as those skilled in the art in the technical field of the invention, be applicable to subject matter of the present invention and can be medical diagnosis mark, medicine, bacterium, toxin, environmental contaminants or nucleotide.
Electrode test piece of the present invention is behind subject matter and binding member specific binding, can pass through oxidoreducing enzyme (13) and produce the electrochemical activity product with its matrix (substrate) reaction, detect again described electrochemical activity product to reach the purpose of quantitative test tested object.Be applicable to oxidoreducing enzyme of the present invention (13) and include, but is not limited to glucose oxidase, Reduction of Glucose enzyme, 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 approximately 150 to arrive 420mV.According to the present invention aspect the electrochemical measurement mode detection microbial antigen of ampere-immunosensor, the voltage of its utilization is preferably approximately 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 conjunction, or covalent bond and electrochemical reaction end (5) surface conjunction (shown in Fig. 5 (a)) by bridging agent (8).Described first antibody is through being combined in specific manner with tested antigen (11) after (shown in Fig. 5 (c)), and described tested antigen (11) again with described antigen (11) is had narrow spectrum second antibody (12)-oxidoreducing enzyme (13) complex be combined (shown in Fig. 5 (e)) form an oxidoreducing enzyme layer, carry out electrochemical measurement with hydroperoxidation again.
According to another preferably enforcement aspect of the present invention, described binding member is antigen (11), described antigen (11) is with compatibility and electrochemical reaction end (5) surface conjunction, or covalent bond and electrochemical reaction end (5) surface conjunction by bridging agent (8).Described antigen (11) is through after tested first antibody (10) is combined in specific manner, and described first antibody (10) again with can with the second antibody (12) of its combination-oxidoreducing enzyme (13) complex in conjunction with forming an oxidoreducing enzyme 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 (such as antibody, antigen, protein ligands or acceptor), nucleotide sequence or compound) of binding member.Bridging agent can be evenly and is formed dispersedly the arrangement paradigmatic structure of single or multiple lift, according to the present invention, bridging agent is the compound with difunctionality base, and the one functional group is used for the surface conjunction with electrochemical reaction end (5), and another functional group is used for being combined with binding member.Being applicable to bridging agent of the present invention is the functional group, includes, 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 (the .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 oxidoreducing enzyme layer comprises:
(a) in conjunction with first antibody (10) or bridging agent (8)-first antibody (10) complex on electrochemical reaction end (5) surface,
(b) the 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)-oxidoreducing enzyme (13) complex, and
(e) the not fixed second antibody of closing of flush away (12)-oxidoreducing enzyme (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 (11) and second antibody (12)-oxidoreducing enzyme (13) complex form antigen (11)-second antibody (12)-oxidoreducing enzyme (13) complex,
(c) in conjunction with described first antibody (10) and described antigen (11)-second antibody (12)-oxidoreducing enzyme (13) complex, or in conjunction with described bridging agent (8)-first antibody (10) complex and described antigen (11)-second antibody (12)-oxidoreducing enzyme (13) complex, and
(d) the not fixed described antigen (11) that closes of flush away-second antibody (12)-oxidoreducing enzyme (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) the 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)-oxidoreducing enzyme (13) complex, and
(e) the not fixed second antibody of closing of flush away (12)-oxidoreducing enzyme (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) in conjunction with first antibody and second antibody (12)-oxidoreducing enzyme (13) complex, form first antibody (10)-second antibody (12)-oxidoreducing enzyme (13) complex,
(c) in conjunction with described antigen and described first antibody (10)-second antibody (12)-oxidoreducing enzyme (13) complex, or in conjunction with described bridging agent (8)-antigen (11) complex and described first antibody (10)-second antibody (12)-oxidoreducing enzyme (13) complex, and
(d) the not fixed described first antibody (10) that closes of flush away-second antibody (12)-oxidoreducing enzyme (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 that forms 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 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 whereby the content of specific subject matter in the corpse or other object for laboratory examination and chemical testing.
Electrode test piece of the present invention does not need such 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 satisfies simultaneously and keep enough demands of large electronic signal.
The present invention is further illustrated for following examples, and not in order to limit the scope of the invention, the 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 working electrode (2a) and the electrode system (2) that a reference electrode (2b) is formed by each self-separation, then dry; Immediately in the same side that is printed on described electrode system (2), be coated with an electric insulation layer, and the working electrode (2a) that reserve part is exposed and reference electrode (2b) are to form terminals (4) and electrochemical reaction end (5), then an electrode test piece (such as the A of Fig. 2) of organizing in contrast A is finished in oven dry.
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 with ultrasonic vibrating device concussion it is dissolved fully, drip again at the electrode test piece of described control group A and revise described electrochemical reaction end (5), after water is removed unconjugated fat-soluble electronic media (9), finish the experimental group B electrode test piece (such 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 again the sodium citrate continuous stirring, 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), then water flush away unconjugated goldc grains sublayer and salt, carry out again the coating program of aforementioned fat-soluble electron mediator layer (9), have nano-scale goldc grains sublayer (7) electrode test piece composite modified with fat-soluble electron mediator layer (9) and finish, as experimental group C (such 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, in 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 (such as Fig. 3) of experimental group B, confirm described nano-scale goldc grains sublayer, have the characteristic of the redox electric current that amplifies the electrode of revising through fat-soluble electronic media.
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 (such 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 the experimental group E electrode test piece (such 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 diameter in the nano colloid gold particle solution of 13 nanometers, drip in described electrochemical reaction end, to form a nano-scale goldc grains sublayer (7), then unconjugated gold particle and salt are removed in washing, carry out again the coating program of aforementioned fat-soluble electron mediator layer (9), have nano-scale goldc grains sublayer (7) electrode test piece composite modified with fat-soluble electron mediator layer (9) and finish, as experimental group F (such as the F of Fig. 2).Further according to 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 (such as Fig. 4) of experimental group E, confirm described nano-scale goldc grains sublayer (7), have the characteristic of the redox electric current that amplifies the electrode of revising through fat-soluble electronic media.
Example 3.
For technology of the present invention being applied to the entity check of antigen (11), first with material and the flow process of experimental group F in the aforementioned example 2, make the electrode test piece composite modified with fat-soluble electron mediator layer (9) through nano-scale goldc grains sublayer (7).Standby monoclonal antibody with anti-E.coli O157:H7 is as first antibody (10), to 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 (such as Fig. 5) on electrode via following program:
(a) in conjunction with bridging agent (8)-first antibody (10) complex on electrode surface,
(b) the unconjugated bridging agent of flush away (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, the ampere of employing fixed voltage 300mV-immune sensing electrochemical measurement mode detection microbial antigen.Simultaneously, preparation only has the experimental group G electrode test piece (such 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 enzyme immobilization program, as the comparison of immune sensing microorganisms utilization.Confirm described fat-soluble electron mediator layer (9), have the characteristic (such as Fig. 6) of the redox electric current that amplifies again the electrode of revising through the nano-scale gold particle.
Therefore, disclosed have nano-scale goldc grains sublayer (a 7) electrode test piece composite modified with 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 to not be fixed on the electrode with covalent bonds simultaneously yet, therefore have advantages of the operation of minimizing, reduce cost.Take into account the associated electrical chemical apparatuses manufacturer of manufacturing cost, quality and the application of electrode test piece for hope, disclosed have an electrode test piece composite modified with fat-soluble electron mediator layer (9) through nano-scale goldc grains sublayer (7), really can provide effective solution.
Claims (28)
1. surperficial modified electrode test piece of be used for measuring 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, the described electrode system of its partial coverage, the part that described electrode system is not covered by described electric insulation layer forms respectively terminals and the electrochemical reaction end that comprises working electrode and reference electrode;
Nano-scale goldc grains sublayer, the electrochemical reaction end of the described working electrode of its at least part of covering; And
Fat-soluble electron mediator layer, the electrochemical reaction end of the described working electrode of its at least part of covering.
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. the described electrode test piece of arbitrary claim in 4 according to claim 1, wherein said nano-scale goldc grains sublayer covers described electrochemical reaction end fully.
6. the described electrode test piece of arbitrary claim in 4 according to claim 1, wherein said nano-scale goldc grains sublayer covers the electrochemical reaction end of described working electrode.
7. the described electrode test piece of arbitrary claim in 4 according to claim 1, wherein said nano-scale gold particle layer segment covers the electrochemical reaction end of described working electrode.
8. the described electrode test piece of arbitrary claim in 4 according to claim 1, the fat-soluble electronic media of wherein said fat-soluble electron mediator layer is selected from the group that is comprised 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 is 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. the described electrode test piece of arbitrary claim in 4 according to claim 1, wherein said fat-soluble electron mediator layer covers described electrochemical reaction end fully.
12. the described electrode test piece of arbitrary claim in 4 according to claim 1, wherein said fat-soluble electron mediator layer covers the electrochemical reaction end of described working electrode.
13. the described electrode test piece of arbitrary claim in 4 according to claim 1, the electrochemical reaction end of the described working electrode of wherein said fat-soluble electron mediator layer partial coverage.
14. the described electrode test piece of arbitrary claim in 4 according to claim 1, 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 comprised 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. the described electrode test piece of arbitrary claim in 4 according to claim 1, the electrochemical reaction end in the wherein said working electrode further comprises a binding member, and described binding member can become compound with the subject matter specific binding.
19. electrode test piece according to claim 18, wherein said binding member is selected from compound.
20. electrode test piece according to claim 18, wherein said binding member is selected from the group that protein and nucleic acid forms.
21. electrode test piece according to claim 20, wherein said protein are antibody, antigen, protein ligands or acceptor.
22. 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.
23. electrode test piece according to claim 18, wherein said binding member-subject matter compound can form the oxidoreducing enzyme layer with oxidoreducing enzyme.
24. electrode test piece according to claim 23, wherein said oxidoreducing enzyme are selected from the group that is comprised of glucose oxidase, Reduction of Glucose enzyme, Lactate Oxidase, pyruvate oxidase and hydrogen peroxidase.
25. electrode test piece according to claim 23, wherein said oxidoreducing enzyme layer comprise first antibody, antigen subject matter as described binding member, and the second antibody of being combined with described antigen-redox combined enzyme agent.
26. electrode test piece according to claim 23, wherein said oxidoreducing enzyme layer comprise the antigen as described binding member, the first antibody of being combined with described antigen, and the second antibody of being combined with described first antibody-redox combined enzyme agent.
27. a biology sensor, it comprises according to claim 1 the described electrode test piece of arbitrary claim and pick-up unit in 26.
28. biology sensor according to claim 27, wherein said pick-up unit is by voltage output device, signal receiving device, and the current sensor that forms of display device.
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