CN102192938A - Homogeneous compound catalyst/enzyme structure, fabricating method thereof and application thereof - Google Patents
Homogeneous compound catalyst/enzyme structure, fabricating method thereof and application thereof Download PDFInfo
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Abstract
The invention provides a homogeneous compound catalyst/enzyme structure, a fabricating method thereof and an application thereof. According to the homogeneous compound catalyst/enzyme structure and the fabricating method thereof, an electrophoretic deposition (EPD) is employed. On the proper electrophoretic deposition condition, a plurality of catalyst particles and enzyme molecules are deposited and fixed simultaneously on a substrate, and thus a compound catalyst/enzyme film is form on the surface of the substrate. The compound catalyst/enzyme film contains a plurality of enzyme molecules, which is mixed and distributed homogeneously and used for catalyzing biomolecule reactions, as well as catalyst materials used for catalyzing electrochemical reactions. The compound structure can be used to form a working electrode of a biological recognition element of a microsensor.
Description
Technical field
The present invention is relevant with a kind of catalyst/enzymatic structure and preparation method thereof, particularly with combined type catalyst/enzymatic structure of a kind of homogeneous texture and preparation method thereof with use relevant, the method can be applied to suitable extensive fields, and it can be applied to base material immobilization of organic molecule detecting device or biology sensor (as microbiosensor) or the like.
Background technology
Please refer to Fig. 1, it illustrates a kind of synoptic diagram of basic structure of biology sensor.The basic structure of biology sensor mainly is made up of biological sensing element (Biological recognition element, Bioreceptor) 11, signal transducers (Signal transducer) 12 and signal processor (Signal processor) 13 these three part institutes.Its sensing principle is: when having after the specific biological sensing element 11 of biological chemistry combines with test substance 15 or react, physics that it produced or chemical change, can variable quantity be changed into significant electronic signal through signal transducers 12, by signal processor 13 this signal is amplified and record again, with convenient follow-up qualitative and quantitative test.
The biomaterial that can be used as the sensing element in the biology sensor generally can slightly be divided into five big classes: (a) enzyme (Enzymes), (b) antibody (Antibody) and antigen (Antigen), (c) nucleic acid (Nucleic acids), (d) receptor (Receptors), (e) tissue part (Cell organelle) or individual cells.And utilize the prepared biology sensor of different biological sensing materials is that its relative merits are separately arranged, in numerous biological sensing materials, the earliest and the most normal what be used is enzyme.Generally speaking, enzyme all has selectivity (be a kind of enzyme can only a certain particular substrate of catalysis), can reuse, easily be heated and characteristics such as soda acid influence.Enzyme can interosculate with specific determinand, and this catalysis behavior promptly can be applicable to biology sensor.
Along with global economy development promotes gradually, humanly aspect diet, also significantly change, and one of the main cause of death of its people is replaced by chronic disease gradually from acute infectious disease in life.In the middle of numerous chronic diseases, with diabetes again national high disease in vogue; According to compatriots' ten big causes of the death that 96 years Department of Health of Executive Yuan are announced, diabetes are in fourth.Though medical technology is now maked rapid progress, do not find out the methods of treatment that diabetes can be eradicated fully at present as yet.If the diabetes state of an illness is uncontrollable proper, many complication that it is followed except meeting consumes more social medical resource, also can influence sufferer itself and household's quality of the life.Monitor blood sugar concentration in the human body by the biology sensor of performance brilliance, accomplish suitable tracking and managing, then can prevent or slow down the generation of complication, and reach the purpose of early detection early treatment.And microbiosensor can provide a simple and easy measurement and measuring table easy to carry, the user can be carried so that monitor the concentration of its test substance.
Wherein, the biology sensor that can be used to monitor diabetes has many kinds of array modes, that is to say all kinds biomolecule under the signal transducer that collocation is fit to, all can be as the sensing element of analyzing glucose.Many for a long time researchs are by utilizing glucose oxidase to be fixed on the electric chemical formula glucose biological sensor, inquire into the sensing analysis of glucose, so glucose oxidase (Glucose Oxidase; GODx) be the most normal selection that is used in this research.
Glucose oxidase is that a kind of glycoprotein that contains two two similar sub-cells is formed, and the protein of this two sub-cell is linked by disulfide group, it all contains a flavin adenine dinucleotide (Flavin adenine dinucleotide in each sub-cell, FAD) be complementation group, the molecular structure of FAD is as follows.
Be subjected in the presence of the matter at electronics, GOD can be catalyzed into β-D-glucose D-glucono-δ-lactone, and the FAD on the GOD can reduce and form FADH
2Shown in 1-1:
β-D-Glucose+GOD(FAD)→GOD(FADH
2)+D-glucono-6-latone (1-1)
D-glucono-δ-lactone can with further with solution in water reaction become gluconic acid; Shown in 1-2:
D-glucono-6-lactone+H
2O→gluconic Acid (1-2)
Convolution 1-1 and formula 1-2 can obtain formula 1-3
β-D-Glucose+GOD(FAD)→GOD(FADH
2)+gluconic Acid (1-3)
Work as GOD (FADH with the oxygen in the solution
2) electronics recipient (electron acceptor), GOD (FADH-
2) electronics can be transferred to oxygen, itself be oxidized to GOD (FAD) and make it be reduced into water and make; Shown in 1-4:
GOD(FADH
2)+O
2→GOD(FAD)+H
2O
2 (1-4)
Convolution 1-4 and formula 1-3 can get formula 1-5:
β-D-Glucose+O
2→gluconic acid+H
2O
2 (1-5)
Biology sensor as shown in Figure 1 has biochemical signals is transformed into electronic signal, so that the advantage that quantize analyzing and processing and output show if use different electronic components, then can be formed the biology sensor of different kenels.In Fig. 1, signal transducer 12 is except being transformed into measurable electronic signal with the variable quantity of physics or chemistry, and under proper condition, the intensity of this electronic signal also is directly proportional with the concentration of specific chemicals.If different according to the structure of signal transducer 12 and transduction mechanism, then roughly can slightly be divided into three major types: (1) electric chemical formula biology sensor, (2) optical biologic sensor and (3) quality formula biology sensor, its grade respectively has its relative merits.
Wherein, electrochemica biological sensor is to have utilized the current potential in the electrochemical measuring method, electric current or inductance signal transducer, the biology sensor that modified electrode is formed in the cooperation, it has utilized the test substance in fixing biomolecule and sample to be tested to carry out catalytic reaction and produce product, this product carries out electrochemical redox reaction with the catalyst of electrode surface again, with the change of output current, voltage or measurement electrical conductivity, and learn testing concentration indirectly quantitatively.With the employed glucose sensor of general detection glucose is example, be fixed in the glucose oxidase molecule of electrode surface, can carry out catalytic reaction with the glucose molecule of solution to be measured and generate the hydrogen peroxide molecule, hydrogen peroxide diffuses to electrode surface and its catalyst material carries out electrochemical redox reaction, and produces current signal.Therefore, electrochemica biological sensor combines the biological sensing layer to for the selectivity (exclusive reaction) of test substance and the advantage of galvanochemistry transducer, and have need not expensive complicated instrument and equipment, signal reaction/response time fast, favorable linearity sensing range district, and advantage such as operating process is simple and easy, its simplicity helps the universalness in the following clinical practice, thereby becomes the heat subject in the research now.Characteristic according to electronic signal, the galvanochemistry transducer can be divided into three kinds again: current potential formula (Potentiometric), current type (Amperometric), conductance type (Conductometric) transducer, and wherein again with the most normal being used of current type galvanochemistry transducer.
The general method for preparing microbiosensor is catalyst to be fixed on the working electrode of microbiosensor with thick film screen printing technology (Screen Printing) earlier, finish Deng the microsensor preparation, utilize traditional enzyme immobilization method that enzyme is fixed in the surface again.Please refer to Fig. 2, it illustrates a kind of layer structure synoptic diagram of traditional microbiosensor.The shortcoming of prepared traditionally microbiosensor is: the surperficial structure of working electrode (comprising base material 21 and silver-colored line 22) of utilizing the method to make, and be the structure that belongs to catalyst/enzyme layer: its internal layer is a catalyst layer 24, skin is an enzyme layer 26.With enzyme layer 26 is that glucose oxidase is an example, and general traditional is about 60～80 μ m through immobilized glucose oxidase in the thickness of electrode surface.Layer structure as shown in Figure 2 might diffuse to catalyst layer 24 to hydrogen peroxide along with the thickness of enzyme layer 26 and cause obstruction, and make the surface by hydrogen peroxide that glucose oxidase produced, cause the matter that is spread in electrode surface to pass resistance because of this bed thickness film, and cause sensing function to descend.Particularly can cause the sensitivity (Sensitivity) of sensor under the high concentration glucose environment to descend.
Moreover, prepare microbiosensor with classic method, the operation formality of the suitable multiple tracks of needs and catalyst material and enzyme molecule are the raw material of high unit price, the formality that tradition is made microbiosensor often needs excessive catalyst material and enzyme molecule, reaching predetermined sensing function, and the production cost of microbiosensor can't be reduced.
Summary of the invention
In view of this, the purpose of this invention is to provide a kind of combined type catalyst/enzyme and preparation method thereof with homogeneous texture, it has well reproduced, stability and accuracy, and can be applicable to the various suitable extensive fields such as base material immobilization of organic molecule detecting device or biology sensor (as microbiosensor).
Propose a kind of combined type catalyst/enzymatic structure according to the present invention, it comprises a plurality of catalyst particles and a plurality of enzyme molecule of even mixing and distribution, and wherein these enzyme molecules are used for catalysis one biological molecular reaction, and these catalyst particles are used for the reaction of catalysis one electrochemical substance.Wherein, be to utilize an electrophoretic deposition and under a suitable electrophoretic deposition condition, fixing a plurality of catalyst materials of deposition and enzyme molecule on base material simultaneously, and form combined type catalyst/enzyme film in a substrate surface.This combined type catalyst enzymatic structure can be used as the working electrode in the biological sensing element of microsensor.According to the present invention, it proposes a kind of preparation method of microsensor electrode, and it comprises: a base material is provided; Provide an electrophoresis solution, comprising a plurality of catalyst particles and a plurality of enzyme molecule; And utilize an electrophoretic deposition under a suitable electrophoretic deposition condition, to deposit catalyst particle and enzyme molecule deposition simultaneously in the surface of a base material, form combined type catalyst/enzyme film with the surface in this base material, wherein this film comprises mixed uniformly catalyst particle and enzyme molecule.
Propose a kind of microbiosensor according to the present invention, it comprises a biological sensing element (Biologicalrecognition element, Bioreceptor), a signal transducer (Signal transducer) and a signal processor (Signal processor).This biological sensing element is after combining with a test substance or reacting, can produce a physical/chemical changing value, wherein the biological sensing element has a working electrode, this working electrode comprises a base material and is formed at a combined type catalyst enzyme film of substrate surface, and this film comprises a plurality of catalyst particles and a plurality of enzyme molecule of even mixing and distribution, wherein these enzyme molecules are used for catalysis one biological molecular reaction, these catalyst particles are used for the reaction of catalysis one electrochemical substance, and be catalyst material and enzyme molecule to be deposited on the base material simultaneously, with the working electrode of combined type catalyst/enzyme of forming homogeneous texture by an electrophoretic deposition.This signal transducer is that the physical/chemical changing value is changed into an electronic signal.This signal processor is the electronic signal that reception and processing signals transducer are produced.Can at room temperature have the pot-life that reaches more than 30 days through the applied microbiosensor of experiment confirm.Therefore combined type catalyst/enzyme electrode of the present invention can be applied to prepare have well reproduced, the even combined type catalyst/enzymatic structure microbiosensor of stability and accuracy.
Description of drawings
For foregoing of the present invention can be become apparent, hereinafter will elaborate to preferred embodiment of the present invention in conjunction with the accompanying drawings, wherein:
Fig. 1 illustrates a kind of synoptic diagram of basic structure of biology sensor.
Fig. 2 illustrates a kind of layer structure synoptic diagram of traditional microbiosensor.
Fig. 3 is the structural representation of working electrode that illustrates the microbiosensor of the embodiment of the invention.
Fig. 4 is the platinoiridita Nanoalloy catalyst of esca analysis Ir-mini sensor (sensor) electrode surface and the depth profiles of glucose oxidase, it is to utilize the Ya Qi From rifle (Argon Ion Gun) of 5kV to carry out etching, single etch time=100 second, etching is six times altogether, inquire into whether the composition between each element evenly distributes in the electrophoresis decorative layer, Pt and Ir represent that platinoiridita Nanoalloy catalyst, N represent that glucose oxidase, F represent that height fluoridizes (ion-exchange) resin (Nafion).
Fig. 5 is the longitudinal axis spacing of Pt and two kinds of elements of Ir in the enlarged drawing 4, with the ESCA depth profiles of clearer observation Pt and Ir.
Fig. 6 one Fig. 8 was respectively the current value that measured when applying current potential 0.3V, 0.4V and 0.5V (vs.Printed Ag/AgCl) and the result (sample number=3) of glucose sensing function, wherein collected primary current numerical value every 60 seconds.
Fig. 9 is applying under the different potentials for the EPD-PtIr+GOD-Ir-mini sensor, apply the range of linearity district of current potential 0.3V, 0.4V and 0.5V for difference, resulting oxidation current and glucose concentration curve figure, wherein working solution is PBS solution＜pH=7.4 of 0.01M 〉, collected primary current numerical value every 60 seconds.
Figure 10 A, Figure 10 B utilize electrophoretic deposition to deposit the microsensor (sample number=3) of PtIr nano metal catalyst and glucose oxidase simultaneously, in distinctly applying under current potential 0.3V and the 0.4V (vs.Printed Ag/AgCl), measure 5mM glucose solution, 5mM glucose+1.5mg/dL vitamin c solution respectively, and the current value ratio of 5mM glucose+8mg/dL uric acid solution.Wherein, Figure 10 B be among Figure 10 A each solution data compared to the relative percentage of current of 5mM glucose solution.
Figure 11 and Figure 12 are respectively and utilize the EPD-PtIr+GOD-Ir-mini sensor, applying current potential is electric current-time diagram of 0.3V and 0.4V (vs.Printed Ag/AgCl), wherein the concentration of glucose scope is 0 μ M～200 μ M, and working solution is PBS solution＜pH=7.4 of 0.01M 〉.
Figure 13 is for utilizing the EPD-PtIr+GOD-Ir-mini sensor by deciding potentiometry, applying current potential is 0.4V (vs.Printed Ag/AgCl), the resulting current time figure of measure glucose concentration scope 2mM～20mM, wherein working solution is PBS solution＜pH=7.4 of 0.01M 〉, collected primary current numerical value every 60 seconds.
Figure 14 applies current potential 0.3V (vs.Printed Ag/AgCl) for utilizing EPD-PtIr+GOD-Ir-mini sensor by deciding potentiometry, the resulting current time figure of measure glucose concentration scope 2mM～40mM wherein collected primary current numerical value every 60 seconds.
Figure 15 is drawn by the inverse that utilizes individual answering electric current and concentration to be measured among Figure 14 and is formed, it can utilize the Lineweaver-Burk equation to obtain to utilize electrophoresis by electrophoretic deposition, deposits the Kmapp and the IMax of the microsensor of PtIr nano metal catalyst and glucose oxidase.
Figure 16 is the current signal (sample number=3) of microsensor EPD-PtIr+GOD-Ir-mini sensor for the glucose solution of 5mM, and it carries out the result that current signal is measured in difference during the storage time.
Embodiment
The present invention proposes a kind of combined type catalyst enzyme membrane structure with homogeneous texture and preparation method thereof and application.Fig. 3 is the synoptic diagram that illustrates the combined type catalyst enzyme membrane structure of one embodiment of the invention.Wherein, one combined type catalyst enzyme film 35 is formed at the surface of a base material (for example being a working electrode) 33, and combined type catalyst enzyme film 35 comprises a plurality of catalyst particles 353 and the enzyme molecule 355 of even mixing and distribution, and catalyst particle 353 and enzyme molecule 355 are formed on the catalyst carrier 351.Wherein, these enzyme molecules 355 are used for catalysis one biological molecular reaction, and these catalyst particles 353 are used for the reaction of catalysis one electrochemical substance.For example in an application examples, the enzyme molecule of catalysis biological molecular reaction is to form hydrogen peroxide (H with biomolecular reaction
2O
2, Hydrogen Peroxide), the catalyst particle of catalytic electrochemical substance reaction then carries out an electrochemical redox reaction with hydrogen peroxide.Moreover, in one embodiment, catalyst carrier 351 can for example be the carbon black carrier (Carbon, XC-72R).In one embodiment, a mean grain size of catalyst particle can be between about 0.5 nanometer be to about 100 microns.Be by an electrophoretic deposition (Electrophoretic deposition in embodiments of the present invention, EPD) come simultaneously catalyst particle 353 and enzyme molecule 355 to be deposited on the surface of base material 33, when being applied to a microsensor, then can be deposited on working electrode surface simultaneously with catalyst particle 353 and enzyme molecule 355 with electrophoretic deposition.By chemical analysis electronic spectrograph (Electron Spectroscopy for Chemical Analysis, ESCA) depth analytical technology is inquired into its electrode structure, in the time of can confirming to utilize electrophoretic deposition to deposit catalyst and enzyme simultaneously, can form the combined type catalyst enzyme film of one deck homogeneous texture as shown in Figure 3.
In one embodiment, catalyst particle 353 can be a single metallic element M, a binary metal M-X, a single metal oxide MOy, a binary metal oxide MOy-XOy, a metal-metallic oxide compound substance M-MOy or comprise the optional combination of aforementioned type.Wherein y is less than 3, and M and X for example are selected from: lithium (Li), sodium (Na), magnesium (Mg), aluminium (Al), potassium (K), calcium (Ca), chromium (Cr), manganese (Mn), iron (Fe), cobalt (Co), nickel (Ni), copper (Cu), zinc (Zn), gallium (Ga), strontium (Sr), yttrium (Y), zirconium (Zr), niobium (Nb), molybdenum (Mo), ruthenium (Ru), rhodium (Rh), palladium (Pd), silver (Ag), cadmium (Cd), indium (In), tin (Sn), barium (Ba), lanthanum (La, cerium (Ce), praseodymium (Pr), neodymium (Nd), samarium (Sm), europium (Eu), gadolinium (Gd), terbium (Tb), dysprosium (Dy), holmium (Ho), erbium (Er), thulium (Tm), gold-plating (Lu), tantalum (Ta), tungsten (W), osmium (Os), iridium (Ir), platinum (Pt), gold (Au), plumbous (Pb), the group that bismuth (Bi) is formed.In one embodiment, catalyst particle 353 can for example be made up of binary metal and this binary metal oxide, and its metallic element mol ratio is less than 100% greater than 0.
Below be as the catalyst particle with platinoiridita (PtIr) nano metal catalyst, and be the related description of example as the enzyme molecule as an embodiment with glucose oxidase, it is to be used on the working electrode simultaneously fixedly platinoiridita nano metal catalyst and glucose oxidase, with the embodiment of the glucose sensor that combines with the galvanochemistry transducer.In this application examples, the enzyme molecule-glucose oxidase of catalysis biological molecular reaction-be and biomolecular reaction, and form hydrogen peroxide (H
2O
2, Hydrogen Peroxide), catalyst particle-platinoiridita nano metal catalyst-then carry out an electrochemical redox reaction with hydrogen peroxide.The content of embodiment mainly is to use electrophoretic deposition (EPD), catalyst and enzyme is fixed on the working electrode surface of miniature inductive device simultaneously, to produce the microbiosensor for preparing easy and constitutionally stable even combined type catalyst/enzymatic structure.Its content can be divided into two parts: (C1) come simultaneously platinoiridita nano metal catalyst and glucose oxidase are deposited on the electrode structure analysis of microsensor by electrophoretic deposition; (C2) simultaneously with PtIr nano metal catalyst and glucose oxidase, be deposited on the electrochemical properties analysis of microsensor.
Moreover, the present invention has same area and knows that the knowledgeable ought be as can be known, the present invention is not limited in described in following of the present invention arbitrary experimental example-uses nanometer platinoiridita (double base) metal solvent and glucose oxidase, it also can be required and make suitable selection in aforementioned catalyst molecular species that proposes and enzyme molecular species according to application conditions, and the preparation method who utilizes electrophoretic deposition reaches even combined type catalyst enzymatic structure, so the present invention also not only is confined to the application of glucose sensor.
＜C: electrophoretic deposition depositing nano metal solvent and glucose oxidase are in microsensor 〉
According to the present invention, its be utilize electrophoretic deposition simultaneously with catalyst particle and enzyme molecule deposition in microsensor.Electrophoretic deposition is by applying the driving force that electric field produces, the charged particle in the suspending liquid being deposited on the working electrode to reach the purpose of deposition.And according to a preferred embodiment of the present invention, electrophoretic deposition is simultaneously platinoiridita nano metal catalyst and glucose oxidase to be deposited on the microsensor.The compound method of electrophoresis solution has the aaerosol solution of stable phase for an amount of platinoiridita nano metal catalyst, glucose oxidase and Nafion evenly are mixed among the PBS with formation.The molecule that has electric charge in the electrophoresis solution has: the Nafion molecule dissociates hydrogen ion in water after, and make itself because of there being the inferior sulfate radical functional group to present electronegative molecule; The pI value of glucose oxidase is 4.2, and in the pH value was about 7.4 PBS, glucose oxidase can form had electronegative molecule; The carrier of platinoiridita nano metal catalyst can with Nafion ionomer (ionomer) effect, Nafion can be attached on the carrier of catalyst and make its surface have negative electricity.Therefore, be subjected under electric field drives, electronegative Nafion ionomer, glucose oxidase and platinoiridita nano metal catalyst just can move toward the anode that have positive charge, and be deposited on the working electrode surface of microsensor.
The parameter that influences electrophoretic deposition can roughly be divided into two parts: the state of (1) electrophoresis solution, the condition of (2) electrophoretic procedures.The electrophoresis solution state includes the size of suspended particles, Jie of suspended particles reaches the electric conductivity of the specific inductive capacity of current potential, solution, solution, the viscosity of solution and the factors such as degree of stability of solution.The condition of electrophoretic procedures includes sedimentation time, applies voltage, factor such as suspended particles concentration, base material electric conductivity in the solution.
In one embodiment, the step of preparation electrophoresis solution is as follows: at first, an amount of nano metal catalyst, 5%Nafion are added in the solvent, be positioned over the ultrasonic oscillation machine and made its even dispersion in 30 minutes.Then an amount of glucose oxidase is incorporated in and makes its even dispersion in the electrophoresis solution.(this step should not use the ultrasonic oscillation machine to lose activity to avoid enzyme).After preparation is finished, electrophoresis solution is stored under 4 ℃ of environment.And electrophoretic deposition step is as follows: the electrophoresis solution that will prepare be positioned over make on the stirrer particle can be dispersed in solution in.Then desire is deposited catalyst and be connected with the slot of microsensor with the microsensor of enzyme, this slot has been connected to potentiostat.Apply the electrophoretic deposition program of deciding voltage or deciding electric current.Microsensor after deposition finished is positioned over natural air drying in the air.Be positioned over afterwards in the clip chain bag with same in order to using.Yet as is known to the person skilled in the art, step that this embodiment narrated and parameter be only for reference, but not in order to limitation the present invention, uses when of the present invention its relevant administration step and parameters value, all can make appropriate change according to practical application request.
C1: deposit PtIr nano metal catalyst and glucose oxidase in microsensor simultaneously by electrophoretic deposition
The electrode structure analysis of enzyme
Be in an embodiment at aforementioned with electrophoretic deposition, the electrode that simultaneously platinoiridita nano metal catalyst and glucose oxidase is deposited on microsensor carries out structure analysis.Wherein, it is the depth ultimate analysis that utilizes chemical analysis electronic spectrograph (ESCA), inquire into the distribution scenario of different charged particle electrophoretic depositions to electrode surface, its as a result display structure can on the surface of base material, form the combined type catalyst/enzyme film of one deck homogeneous texture really as shown in Figure 3.
Below adopt the depth ultimate analysis of chemical analysis electronic spectrograph (ESCA) in the experiment of proposition embodiment, observe the distribution scenario of different charged particle electrophoretic depositions to electrode surface.
According to the binding energy (Binding Energy) of Pt, Ir, N and F and XPS depth profile (for to utilize the Ya Qi From rifle (Argon Ion Gun) of 5kV to carry out etching, etching period=300 second) resulting ultimate analysis, binding energy all corresponds to the feature binding energy position of this element as can be known, and integral area can obtain the content ratio of this element in integral body.Utilize different etching periods that the distribution of element at different depth is discussed, and observe different electrophoretic deposition particles in the composition of this composite membrane.
Fig. 4 is the platinoiridita Nanoalloy catalyst of esca analysis Ir-mini sensor (sensor) electrode surface and the depth profiles of glucose oxidase, wherein be to utilize the Ya Qi From rifle (Argon Ion Gun) of 5kV to carry out etching, single etch time=100s, etching is six times altogether, and inquire into whether the composition between each element evenly distributes in the electrophoresis decorative layer, wherein Pt and Ir represent that platinoiridita Nanoalloy catalyst, N represent that glucose oxidase, F represent Nafion.The integral body of combined type catalyst/enzyme film is formed as can be seen from Figure 4, major part is provided by the glucose oxidase that has the N atom, infer that reason should be because glucose oxidase wraps the platinoiridita nano metal catalyst of attached Nafion compared to the surface, have higher electrophoretic mobility (Electrophoretic mobility), therefore whole composition major part is all provided by glucose oxidase.And the deposition ratio that also can observe out glucose oxidase from Fig. 4 can be subjected to the influence of Nafion, because in electrophoresis solution, glucose oxidase molecule and Nafion monomer molecule surface all are to present negative charge, therefore glucose oxidase molecule and Nafion monomer molecule will take place in electrophoresis process, can compete the electrochemical activity position of electrode surface simultaneously, so when Nafion is deposited on electrode surface, can stop the deposition position of glucose oxidase molecule simultaneously.
Therefore difficult observation Pt of Fig. 4 and Ir can discuss Pt and these two kinds of elements of Ir separately by Fig. 5 in the composition of this composite membrane.Fig. 5 is the longitudinal axis spacing of Pt and two kinds of elements of Ir in the enlarged drawing 4, with the ESCA depth profiles of clearer observation Pt and Ir.Can find out among the figure that (possible cause is coated by the glucose oxidase in the electrophoresis solution by electrode surface except surface composition is more inhomogeneous, therefore outermost metal signal is than next low of internal layer), remaining etching period all presents stable composition and distributes, it demonstrates electrophoretic deposition platinoiridita nano metal catalyst, a uniform and stable structure can be provided, and the glucose oxidase that its structure will help the while electrophoretic deposition is able to held stationary in the surface.By Fig. 4 and Fig. 5 can find out electrode vertically form distribute suitable evenly, combined type catalyst/enzyme membrane structure that its proof is utilized the method can constitute really to mix is in the electrode surface of microsensor.
C2: deposit platinoiridita nano metal catalyst and glucose oxidase electrochemical properties simultaneously in microsensor
Analyze
Also carry out multiple electrochemical properties analysis in microsensor among the embodiment at depositing platinoiridita nano metal catalyst and glucose oxidase simultaneously, the linear sensing area (Linear DetectingRange) that has comprised microsensor, the lowest detection limit (Limit of detection, LOD), chaff interference test (Interference Test), repeatability of microsensor (Reproducibility) and stability test, the dynamic (dynamical) discussion of enzymic catalytic reaction, and the long-time stability of microsensor test tests such as (Long term Stability).Following series goes out part of test results for your guidance.
Also carry out related experiment for the microsensor that deposits platinoiridita nano metal catalyst and glucose oxidase simultaneously (EPD-PtIr+GOD-Ir-mini sensor) among the embodiment, it is that the preferable glucose that can detect applies potential value.When the EPD-PtIr+GOD-Ir-mini sensor under the PBS of the concentration of glucose of variable concentrations solution, measured cyclic voltammetry curve figure (cyclic voltammetry, CV), sweep limit is-0.4V to 0.4V (vs.Printed Ag/AgCl), sweep speed is 50mV/s, during the scanning number of turns 10 circles.Can find to have the oxidation current that obviously increases and rise gradually from scanning result, then have the reduction current that obviously increases and rise gradually along with hydrogen peroxide concentration about-0.25V (vs.Printed Ag/AgCl) along with hydrogen peroxide concentration about 0.2V (vs.Printed Ag/AgCl).Can find that from the result oxidation current increases thereupon along with voltage rises, it is high more that expression applies current potential, and it is high more to detect the glucose ability.But because a lot of other chaff interferences are arranged in the blood of human body, itself also be electroactive substance, if it is too high to apply current potential, chaff interference also can react together thereupon, and causes the interference of current signal.Therefore, must look for one has suitable sensing current potential, and the hydrogen peroxide that glucose oxidase is generated can have good oxidability, and can avoid interference thing and react under this current potential, will be a considerable problem.
Below experiment is to inquire into the microsensor that deposits platinoiridita nano metal catalyst and glucose oxidase by electrophoretic deposition simultaneously, apply current potential for the best of measuring variable concentrations glucose in the solution, it utilizes difference to apply current potential to carry out deciding current response method (Amperometric measurement), relatively apply the influence of current potential for the glucose sensing function.
The required linearity test district of blood sugar that generally is applied to detect the diabetic is 3～12mM.Therefore, in order to want to accord with the range of blood sugar in the actual detected blood, the glucose linearity test district of proper range is for judging whether one of key that is applicable in sensing blood sugar.In related experiment, learnt optimization measure hydrogen peroxide apply current potential be 0.3V (vs.Printed Ag/AgCl) afterwards, can change and apply potential effect, and, relatively apply current potential for current value and glucose sensing function respectively at the sensing that carries out different concentration of glucose under 0.3V, 0.4V and the 0.5V (vs.Printed Ag/AgCl).And the different results (sample number=3) that apply measured current value of current potential and glucose sensing function were respectively as Fig. 6, Fig. 7 and shown in Figure 8, wherein just collected primary current numerical value every 60 seconds.
From Fig. 6～Fig. 8, apply different potentials as can be seen and difference is all arranged for the range of linearity of glucose sensing.In Fig. 6, the glucose sense linear scope that applies 0.3V (vs.Printed Ag/AgCl) is 2mM～12mM.In Fig. 7, the glucose sense linear scope that applies 0.4V (vs.Printed Ag/AgCl) is 2mM～20mM.In Fig. 8, and the glucose sense linear scope that applies 0.5V (vs.Printed Ag/AgCl) is 4mM～20mM.Apply current potential as can be seen from the results when 0.4V (vs.Printed Ag/AgCl), have the range of linearity of higher concentration compared to 0.3V (vs.Printed Ag/AgCl).When applying current potential and increase to 0.5V (vs.PrintedAg/AgCl), it may be the influence that electrode surface produces a little subsidiary reactions, and the deviate of glucose sensing is when applying current potential and be 0.4V and 0.3V (vs.Printed Ag/AgCl) as can be seen.
Fig. 9 is that the EPD-PtIr+GOD-Ir-mini sensor is in applying under the different potentials, apply resulting oxidation current in range of linearity district and the glucose concentration curve figure of current potential 0.3V, 0.4V and 0.5V for difference, wherein working solution is PBS solution＜pH=7.4 of 0.01M 〉, collected primary current numerical value every 60 seconds.And table 1 is respectively the EPD-PtIr-Ir-mini sensor applies glucose sensing sensitivity (Sensitivity) and inspection amount line under the current potential in difference linearly dependent coefficient (liner corelation) R
2
Table 1
A. temperature: 25 ℃ (± 1 ℃)
B. electrophoretic deposition parameter: decide voltage method and apply voltage 0.5V, sedimentation time 5mins
Solution parameter (Slurry Parameters): 1mg PtIr/C nano metal catalyst+5 μ L 5%
Nafion+20mg GOD evenly mixes it and is scattered in 1mL 0.01M PBS solution
C. specimen number=3
The glucose sensing sensitivity can be along with applying the current potential rising and increasing as can be seen from Figure 9, wherein can observe out along with applying current potential and rise and the hydrogen peroxide sensing sensitivity of increase, maximum change amount (about 1.4 * 10 is arranged under 0.4V to 0.5V (vs.PrintedAg/AgCl)
-8A/mM), have higher glucose sensing sensitivity in 0.5V (vs.PrintedAg/AgCl) though apply current potential, but bigger with respect to the error that microsensor caused that applies than electronegative potential.
Therefore, from above-mentioned experimental result as can be known: will apply current potential and be located under the 0.4V (vs.Printed Ag/AgCl), and can obtain the best glucose detection range of linearity (2mM～20mM) and have lower deviate.
Embodiment afterwards will be again at applying current potential under 0.3V and 0.4V (vs.Printed Ag/AgCl), (Limiting of detection LOD) carries out related experiment and research with repeatability (Reproducibility) to disturbed test (Interference Test), detection limit.
Disturbed test
For general glucose biological sensor, vitamin C (Ascorbic Acid, AA) with uric acid (UricAcid, UA), be two kinds of electroactive interfering materials quite common in blood of human body, if to apply current potential too high, also can react together in disturbing thing thereupon, and cause the interference of current signal.Therefore, must look for one and have suitable sensing current potential,, and can avoid interference thing and under this current potential, react so that the hydrogen peroxide that glucose oxidase generates can have good oxidability.
Point out according to existing document: the vitamin C average content in the general blood of human body is about 0.4～0.6mg/dL, and the average content adult male about 3.5～7.2mg/dL of uric acid in blood of human body, and the about 2.6～6.0mg/dL of women that grows up.And in the experiment of embodiment employed vitamin C and uric acid concentration, then be set in concentration a little more than general average, its grade is respectively 1.5mg/dL and 8mg/dL, with the discussion as disturbed test.The concentration of glucose and the sensed current signal that will be in solution in the experiment add 5mM earlier, vitamin C and the uric acid that adds appropriate amount out of the ordinary in same solution thereafter, with chaff interference under the environment of observing an amount of concentration of glucose respectively for the influence of current signal.
Figure 10 A, Figure 10 B utilize electrophoretic deposition to deposit the microsensor (sample number=3) of platinoiridita nano metal catalyst and glucose oxidase simultaneously, measure 5mM glucose solution, 5mM glucose+1.5mg/dL vitamin c solution under current potential 0.3V and the 0.4V (Vs.Printed Ag/AgCl) respectively in distinctly applying, and the current value ratio of 5mM glucose+8mg/dL uric acid solution.Wherein, 10B figure be among the 10A figure each solution data compared to the relative percentage of current of 5mM glucose solution.
Can demonstrate from Figure 10 A, Figure 10 B and apply be interfered when 0.4V (vs.Printed Ag/AgCl) current affects of thing of current potential is less than applying current potential when 0.3V (vs.Printed Ag/AgCl).Generally speaking, apply the high more suffered chaff interference of current potential to influence meeting serious more, but that the result in this section experiment finds to apply current potential is higher, has selectivity preferably compared to applying electronegative potential.Infer that reason may be because lower current potential causes oxidation current less to the scarce capacity of catalysis hydrogen peroxide, therefore causes the influence of the thing that is interfered comparatively violent.Relatively, applying higher current potential can increase catalytic capability to hydrogen peroxide, thereby has increased the current response to glucose, the current response to vitamin C, uric acid is compressed, so just reduce the interference of vitamin C and uric acid.Therefore found that from the resulting disturbed test of experimental result, among the embodiment,, can obtain preferable sensor performance applying current potential under the situation of 0.4V (vs.Printed Ag/AgCl).
Microsensor detects the lowest detection limit of glucose
Also (Limit of detection LOD) carries out related experiment for utilizing electrophoretic deposition to deposit the lowest detection limit of the microsensor of platinoiridita nano metal catalyst and glucose oxidase simultaneously among the embodiment.
Figure 11 and Figure 12 are respectively and utilize the EPD-PtIr+GOD-Ir-mini sensor, applying current potential is electric current-time diagram of 0.3V and 0.4V (vs.Printed Ag/AgCl), wherein the concentration of glucose scope is 0 μ M～200 μ M, and working solution is PBS solution＜pH=7.4 of 0.01M 〉.The different concentration of glucose of each adding can calculate minimum useful signal by Figure 11 and Figure 12 respectively, then if signal/noise ratio (S/N) greater than 3, then is a useful signal; If signal/noise ratio (S/N) less than 3, then is an invalid signals.Therefore, can learn that from figure applying current potential is respectively 80 μ m and 100 μ m in the lowest detection limit of 0.3V and 0.4V (vs.Printed Ag/AgCl).Its reason may be that it is to have bigger background current (Backgroundcurrent) signal that height applies under the current potential situation, therefore resulting signal news good than too late electronegative potential under low concentration.
The repeatability of microsensor and stability
The preparation sensor is most important to be exactly to want accuracy rate height of user and the good sensing result of reappearance can be provided.Repeatability (Reproducibility) is meant different people, and different instruments under the different condition of different time or the like, all can obtain the ability of identical measured value.If have good reproducibility, expression utilizes suitable stable of the process conditions of the prepared microsensor of electrophoretic deposition, can improve the yield of microsensor and improve the confidence level of signal.Repeatability (Repeatability) is meant that sensor obtains the ability of identical measured value under identical condition, if have good repeatability, its expression utilizes the prepared microsensor of electrophoretic deposition, be to have reusable ability, the catalyst and the enzyme that utilize electrophoresis to deposit still have the structure of quite stable in by measuring process and in the process of cleaning electrode.The embodiment of the invention is also carried out related experiment at repeatability and stability (repeatability).
Figure 13 is for utilizing the EPD-PtIr+GOD-Ir-mini sensor by deciding potentiometry, in applying current potential is resulting current time figure under 0.4V (vs.Printed Ag/AgCl), the measure glucose concentration scope 2mM～20mM, wherein working solution is PBS solution＜pH=7.4 of 0.01M 〉, collected primary current numerical value every 60 seconds.Measure for three times and all use not microsensor on the same group, this experiment purpose to be to judge the height of reproducibility by measuring not microsensor on the same group.Measure all quite approaching data of acquisition as can be seen for three times from Figure 13 and table 2, its relative standard deviation (Relative Standard Deviation, RSD) be 7.14%, showing thus utilizes electrophoretic deposition on microsensor, the manufacturing process that deposits platinoiridita nano metal catalyst and glucose oxidase simultaneously is a quite stable, therefore has quite good reproducibility.
Table 2
A. temperature: 25 ℃ (± 1 ℃)
B. electrophoretic deposition parameter (EPD parameters): decide voltage method and apply voltage 0.5V, sedimentation time 5mins solution parameter (Slurry Parameters): 1mg PtIr/C nano metal catalyst+5 μ L 5%Nafion+20mgGOD evenly mix it and are scattered in 2mL 0.01M PBS solution
C. sample number=3
Table 3 be the EPD-PtIr+GOD-Ir-mini sensor by deciding potentiometry, be 0.4V (vs.Printed Ag/AgCl) in applying current potential, measure glucose concentration is resulting result under the 5mM.Three measurements all use same group of microsensor, this experiment purpose to be by same group of microsensor of duplicate measurements, to judge the height of repeatability (Repeatability).Table 3 is measured for three times as can be seen and is all obtained quite approaching data, relative standard deviation (RSD) is 7.16%, showing thus utilizes electrophoretic deposition to prepare the microsensor of platinoiridita nano metal catalyst and glucose oxidase, can have quite good repeatability, it shows by electrophoretic deposition platinoiridita nano metal catalyst and glucose oxidase is deposited on the working electrode of microsensor simultaneously, can held stationary in the structure of a good catalyst/enzyme composite bed of electrode surface formation.
Table 3
A. temperature: 25 ℃ (± 1 ℃)
B. electrophoretic deposition parameter (EPD parameters): decide voltage method and apply voltage 0.5V, sedimentation time 5mins solution parameter (Slurry Parameters): 1mg PtIr/C nano metal catalyst+5 μ L 5%Nafion+20mg GOD evenly mix it and are scattered in 2mL 0.01M PBS.
C. identical mini sensor.
Enzymic catalytic reaction dynamics
Generally can describe by the mechanism that Michaelis-Menten carried at the catalytic reaction of its enzyme; Shown in 2-2:
Wherein: the I representative detects glucose and replys electric current (A), I
MaxThe theoretical maximum of representative sensor is replied electric current (A), K
App mRepresent Michaelis constant (M), C to represent the concentration of glucose (M) of solution to be measured.
K
m AppValue (Michaelis constant) has the characteristic of judging determinand and enzyme affinity size.Low K
m AppValue then is the high affinity reaction.At testing concentration ([C]) much smaller than K
m AppThe time, reaction rate is proportional to testing concentration (first order reaction); And work as testing concentration much larger than K
m AppThe time, reacting and be zero-order reaction, its speed and testing concentration are irrelevant.If calculating K
m AppWith I
MaThe time, utilize the Lineweaver-Burk equation can obtain the K of sensor
m AppWith I
MaxShown in 2-3:
Wherein the I representative detects glucose and replys electric current (A), I
MaxThe theoretical maximum of representative sensor is replied electric current (A), K
App mRepresent Michaelis constant (M), C to represent the concentration of glucose (M) of solution to be measured.
Can utilize electric current that Figure 14 obtained in the experiment (I) and detectable concentration (C), 1/I and the 1/C in the substitution Lineweaver-Burk equation (formula 2-3) illustrates figure respectively, analyzes the correlation parameter of its biology sensor.If the sensing range of sensor is controlled at enzyme power control district, the figure of being drawn by the Lineweaver-Burk equation will present linearity, and can (be K from the slope of straight line
m App/ I
Max) with intercept (be 1/I
Max) can distinctly obtain the K of sensor
m AppWith I
Max
Figure 14 applies current potential 0.3V (vs.Printed Ag/AgCl) for utilizing EPD-PtIr+GOD-Ir-mini sensor by deciding potentiometry, measuring in the concentration of glucose scope was the resulting current time figure of 2mM～40mM, wherein collected primary current numerical value every 60 seconds.As can be seen from Figure 14 when applying current potential when 0.3V (vs.Printed Ag/AgCl), can present non-linearly in the scope of measuring high concentration glucose, represent that the rate-determing step reaction of this concentration range is reacted (Enzyme kinetic controlled reaction) for the enzyme power control.
Figure 15 is drawn by the inverse that utilizes individual answering electric current and concentration to be measured among Figure 14 and is formed, utilize Lineweaver-Burk equation (formula 2-3, linear equation) can obtain to deposit the K of the microsensor of platinoiridita nano metal catalyst and glucose oxidase by electrophoretic deposition
m AppBe 5.68mM, and I
MaxBe 6.02 * 10
-7A.Relatively can find with other document, deposit the microsensor of platinoiridita nano metal catalyst and glucose oxidase among the embodiment by electrophoretic deposition, have quite low K
m App, its representative is by the enzyme in the combined type catalyst/enzyme layer of the formed homogeneous texture of electrophoretic deposition, is to have good enzymatic activity and to the affinity of glucose.
The long-time preservation test of microsensor
For any biology sensor, the pot-life of how effectively to improve sensor is one of considerable problem.Generally by the prepared biology sensor of conventional fixed method in order to improve the pot-life of enzyme in working electrode, common store method is for being positioned over this biology sensor under 4 ℃ the environment, to prolong the activity of electrode surface enzyme molecule; But this kind store method will significantly reduce microsensor advantage easy to carry.
In view of this, in order to want to copy the custom of the biological microsensor of the actual use of general user, be to adopt the microsensor that to finish to be positioned in the clip chain bag with same and to be positioned under the room temperature to preserve in the present embodiment, to preserve test for a long time.Room temperature is the environment of about 25 ℃ (± 1 ℃).
All test in the experimentation at every turn and will measure the 1st day, the 5th day, the 10th day, the 15th day, the 20th day, the 25th day respectively, and the 30th day microsensor EPD-PtIr+GOD-Ir-mini sensor is for the current signal (sample number=3) of the glucose solution of 5mM.Figure 16 and table 4 are the detailed results that storage time and current signal are measured.
Table 4
A. temperature: 25 ℃ (± 1 ℃)
B. electrophoretic deposition parameter (EPD parameters): decide voltage method and apply voltage 0.5V, sedimentation time 5mins
Solution parameter (Slurry Parameters): 1mg PtIr/C nano metal catalyst+5 μ L 5%Nafion+20mg GOD evenly mix it and are scattered in 2mL 0.01M PBS solution
C. sample number=3
Present result shows, deposit the microsensor of platinoiridita nano metal catalyst metal catalyst and glucose oxidase simultaneously by electrophoretic deposition, still has stable sensing glucose ability being stored in for a long time under the room temperature (about 25 ℃), its reason of inference should be even compound catalyst/enzymatic structure can provide stable, three-dimensional composite structure environment, and then its enzyme stability is improved, and increase the pot-life of enzyme under room temperature.
＜comprehensive discussion 〉
According to the above embodiments, deposit the microsensor of platinoiridita nano metal catalyst and glucose oxidase simultaneously by electrophoretic deposition, the result of many related experiment that the multinomial electrochemical properties that is carried out is analyzed, comprehensive consideration electrophoresis solution parameter and electrophoretic procedures Study on Conditions, can learn: with 1mg PtIr nano metal catalyst/mL, 5 μ L 5%Nafion/mL and 20mg GOD/mL are incorporated in 0.01M PBS (pH=7.4) solution evenly to be disperseed, can access more stable electrophoresis solution, and decide voltage method in applying current potential 0.5V (vs.PrintedAg/AgCl) utilizing, under the sedimentation time 5mins, can prepare well behaved miniature glucose biology sensor.
By the depth profiles result of ESCA, as can be known electrode vertically form distribute suitable evenly, its proof utilizes the method can constitute the combined type catalyst/enzymatic structure that mixes in the electrode surface of microsensor really.Evenly combined type catalyst/enzymatic structure layer can provide a special environment, to shorten the hydrogen peroxide that generated by test substance and enzyme molecular reaction path to the catalyst surface, and make the electronics that is generated by hydrogen peroxide and catalyst surface reaction, can be directed on the base material of working electrode rapidly by the carrier of nano metal catalyst; This structure can improve answer signal and promote transducer sensitivity.
In repeatability experiment, can learn that embodiment deposits the method for platinoiridita nano metal catalyst and glucose oxidase simultaneously by electrophoretic deposition, be the processing procedure of a quite stable.Compared to the microsensor that only deposits glucose oxidase with electrophoretic deposition, optimization electrophoresis solution parameter in conjunction with the embodiments and electrophoretic procedures condition, the prepared electrophoretic deposition that forms deposits the microsensor of platinoiridita nano metal catalyst and glucose oxidase simultaneously, the glucose sensing function can be promoted to originally 1.25 times.This shows the platinoiridita nano metal catalyst of embodiment and the composite structure of glucose oxidase, can promote the glucose sensing function really.
Inquire into the result of Dynamics of Enzyme Catalysis and compare with other document, can find to deposit by electrophoretic deposition among the embodiment microsensor of platinoiridita nano metal catalyst and glucose oxidase, can have lower Michaelis constant (K in an experimental example
m App=5.68mM), this representative is by the enzyme in the combined type catalyst/enzyme layer of the formed homogeneous texture of electrophoretic deposition, has good enzymatic activity and to the affinity of glucose; The possible cause of inferring it is the combined type catalyst/enzyme layer of homogeneous texture, and a kind of special structural environment can be provided, rise and make by the enzyme stability, and the pot-life of significantly improving biology sensor.According to the experimental result test, it at least at room temperature has the pot-life that reaches more than 30 days.
Moreover, by the prepared miniature glucose biology sensor that goes out of the electrophoretic deposition conditional parameter of optimization, applying under the current potential 0.4V (vs.Printed Ag/AgCl), the sense linear scope of detected glucose is between 2mM～20mM, Michaelis constant is 5.68mM, the lowest detection limit is 0.1mM, and detection sensitivity is 2.89 μ A/mM.cm
2(R
2=0.995, R.S.D.=3.26%, N=3).
In sum, combined type catalyst/the enzyme electrode of a kind of homogeneous texture that the embodiment of the invention proposed, be to come simultaneously catalyst particle (for example being platinoiridita nano metal catalyst) and enzyme molecule (for example being glucose oxidase) to be deposited on the working electrode surface of microsensor by electrophoretic deposition, by every experiment, for example by ESCA depth analyzing electrode structure, confirmation utilizes electrophoretic deposition to deposit catalyst particle and enzyme molecule simultaneously, can form the combined type catalyst/enzyme film of one deck homogeneous texture, and it can (reach more than 30 days) for a long time and is stored in the room temperature.Experimental result shows, use preparation method of the present invention can prepare have well reproduced, the even combined type catalyst/enzymatic structure microbiosensor of stability and accuracy.
In sum, though the present invention with the embodiment exposure as above, yet it is not in order to limit the present invention.The persond having ordinary knowledge in the technical field of the present invention, without departing from the spirit and scope of the present invention, when making various changes that are equal to or replacement.Therefore, protection scope of the present invention is when looking accompanying being as the criterion that the application's claim scope defined.
Claims (12)
1. combined type catalyst enzyme membrane structure, it comprises a plurality of catalyst particles and a plurality of enzyme molecule of even mixing and distribution, and wherein these enzyme molecules are used for catalysis one biological molecular reaction, and these catalyst particles are used for the reaction of catalysis one electrochemical substance.
2. combined type catalyst enzyme membrane structure according to claim 1 is characterized in that, these enzyme molecules and these catalyst particles are to utilize an electrophoretic deposition under a suitable electrophoretic deposition condition, deposits simultaneously and is fixed in a substrate surface.
3. combined type catalyst enzyme membrane structure according to claim 1 is characterized in that, these enzyme molecules of this biomolecular reaction of catalysis are to form hydrogen peroxide with biomolecular reaction.
4. combined type catalyst enzyme membrane structure according to claim 3, it is characterized in that these enzyme molecules are to be selected from glucose oxidase, malate oxidase, the hexose oxidase, cholesterol oxidase, the aryl alcohol oxidase, the L-gulonolactone oxidase, galactose oxidase, the pyranose oxidase, L-sorbose oxidase, pyridoxol 4-oxidase, methanol oxidase, (S)-the 2-hydroxy acid oxidase, the moulting hormone oxidase, choline oxidase, the secondary alcohol oxidase, 4-Hydroxymandelate oxidase, long-chain-alcohol oxidase, glycerol-3-phosphate oxidase (glycerol-3-phosphate oxidase; EC 1.1.3.21), the Aneurine oxidase, the zinc hydroxyl stannate oxidase, N-acyl group hexosamine oxidase, the polyvinyl alcohol (PVA) oxidase, interior ester oxidase, the vanillyl alcohol oxidase, the nucleosides oxidase, D-mannitol oxidase, the xylitol oxidase, the cellobiose dehydrogenasa, hydrogenlyase, the oxidation of acetaldehyde enzyme, pyruvate oxidase, oxalate oxidase, glyoxylate oxidase, pyruvate oxidase (CoA-acetylation), the aryl aldehyde oxidase, the retinene oxidase, the ABA aldehyde oxidase, ketoglutaric dehydrogenase (succinyl group conversion); , dihydroorotate oxidase, COPRO-O, aryl-coacetylase oxidase, dihydrouracil oxidase, N-1 oxidase, tryptophane α, tryptophan side-chain alpha, PQQ synzyme, L-galactonolactone oxidase, aryl-coa dehydrogenase (acyl-CoA dehydrogenase; EC 1.3.99.3), dihydroorate dehydrogenase, the D-aspartate oxidase, the L-amino acid oxidase, the D-amino acid oxidase, amido oxidase (containing the class flavine), tremble aldehyde 5 '-phosphate synthase, amido oxidase (cupric), D-glutamate oxidase, ethanolamine oxidase, putrescine oxidase, L-glutamate oxidase, the cyclohexylamine oxidase, protein-isolated amino acid 6-oxidase, L-isolated amino acid oxidase, D-glutamate (D-aspartic acid) oxidase, the L-aspartate oxidase, glycine oxidase, L-isolated amino acid 6-oxidase, the amido dehydrogenasa, the FMN reductase, sarcosine oxidase, N-methyl-L Amino acid oxidase, N6-methyl-isolated amino acid oxidase, (S)-6-hydroxy niacin oxidase, (R)-6-hydroxy niacin oxidase, the L-pipecoline, the dimethylglycine oxidase, PAO, the DHBP oxidase, trimethylamine dehydrogenase, L-pipecoliacid dehydrogenasa, basic element of cell division dehydrogenasa, NAD (P) H oxidase, NAD (P) H dehydrogenasa (to the benzene triketone), nitrite reductase, nitroalkane oxydase, urate oxidase, 3-nitropropionic acid methyl esters oxidase, the dihydro lipoyl dehydrogenase, sulfite oxidase, thiol oxidase, glutathione oxidase (glutathione oxidase; EC 1.8.3.3), the methyl mercaptan oxidase, alkylene aminothiopropionic acid oxidase, 3-hydroxyl anthranilic acid oxidase, thunder good fortune mycin-B oxidase, the NADH peroxidase, 2-nitropropane dioxygenase, rely amino acid 2-monooxygenase, Lactate 2-monooxygenase, fluorescein 4-monooxygenase (ATP hydrolysis), phenylalanine 2-monooxygenase, the clavaminate synzyme, naphtha essence 1, the 2-dioxygenase, 4-amido ethyl benzoate 1-monooxygenase, alkanal monooxygenase, Phenylalanine 4-monooxygenase, adjacent amine Sodium Benzoate 3-monooxygenase, single phenol monooxygenase, 7-cholestenol oxidase, superoxide dismutase, the superoxides reductase, xanthine dehydrogenase, xanthine oxidase, 6-hydroxyl nicotine dehydrogenase, fragrant isobebeerine enzyme, the group that diphosphoribulose carboxylase is formed.
5. combined type catalyst enzyme membrane structure according to claim 1 is characterized in that, these catalyst particles of this electrochemical substance reaction of catalysis are to carry out an electrochemical redox reaction with hydrogen peroxide.
6. combined type catalyst enzyme membrane structure according to claim 5, it is characterized in that, redox these catalyst particles that are used for catalyzing hydrogen peroxide, comprise a single metallic element M, one binary metal M-X, one single metal oxide MOy, one binary metal oxide MOy-XOy, one metal-metallic oxide compound substance M-MOy, or be selected from the combination of aforementioned type, wherein, y be less than 3 and these catalyst particles comprise this binary metal and this binary metal oxide, and its metallic element mol ratio is less than 100% greater than 0, and a mean grain size of these catalyst particles is between about 0.5 nanometer is to about 100 microns, and M and X are selected from by lithium (Li), sodium (Na), magnesium (Mg), aluminium (Al), potassium (K), calcium (Ca), chromium (Cr), manganese (Mn), iron (Fe), cobalt (Co), nickel (Ni), copper (Cu), zinc (Zn), gallium (Ga), strontium (Sr), yttrium (Y), zirconium (Zr), niobium (Nb), molybdenum (Mo), ruthenium (Ru), rhodium (Rh), palladium (Pd), silver (Ag), cadmium (Cd), indium (In), tin (Sn), barium (Ba), lanthanum (La, cerium (Ce), praseodymium (Pr), neodymium (Nd), samarium (Sm), europium (Eu), gadolinium (Gd), terbium (Tb), dysprosium (Dy), holmium (Ho), erbium (Er), thulium (Tm), gold-plating (Lu), tantalum (Ta), tungsten (W), osmium (Os), iridium (Ir), platinum (Pt), gold (Au), plumbous (Pb), the group that bismuth (Bi) is formed.
7. combined type catalyst enzyme membrane structure according to claim 1 is characterized in that, these catalyst material particles are a plurality of platinoiridita (PtIr) nano-catalyst particles, and these enzyme molecules are a plurality of glucose oxidase molecules.
8. the preparation method of a microsensor electrode comprises:
One base material is provided;
One electrophoresis solution is provided, and it comprises a plurality of catalyst particles and a plurality of enzyme molecule; And
Utilize an electrophoretic deposition under a suitable electrophoretic deposition condition, simultaneously with these catalyst particles and these enzyme molecule depositions in the surface of this base material, form a combined type catalyst enzyme film with the surface in this base material, wherein this film comprises mixed uniformly these catalyst particles and these enzyme molecules.
9. the preparation method of microsensor electrode according to claim 8 is characterized in that, these catalyst particles are a plurality of platinoiridita (PtIr) nano metal catalyst particles, and these enzyme molecules are a plurality of glucose oxidase molecules.
10. the preparation method of microsensor electrode according to claim 9, it is characterized in that, this electrophoresis solution that is provided comprises a pH value aqueous buffer solution, these platinoiridita (PtIr) nano metal catalyst particle, these glucose oxidase molecules, and a tool water wettability ion-exchange functional group compound.
11. the preparation method of microsensor electrode according to claim 10, it is by certain voltage method (Potentiostatic Method) or certain current method (Galvanostatic Method), carries out electrophoretic deposition by different operating time and current potential/electric current.
12. a microbiosensor, it includes:
One biological sensing element, after it combines with a test substance or reacts, can produce a physical/chemical changing value, wherein this biological sensing element has a working electrode, this working electrode comprises a base material and is formed at a combined type catalyst enzyme film of this substrate surface, and this film comprises a plurality of catalyst particles and a plurality of enzyme molecule of even mixing and distribution, and wherein these enzyme molecules are used for catalysis one biological molecular reaction, and these catalyst particles are used for the reaction of catalysis one electrochemical substance;
One signal transducer, it is that this physical/chemical changing value is changed into an electronic signal; And
One signal processor, it is to receive and handle this electronic signal that this signal transducer is produced.
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| CN108031859A (en) * | 2017-11-29 | 2018-05-15 | 上海纳米技术及应用国家工程研究中心有限公司 | Method for synthesizing gold nanoclusters and products thereof and application |
| CN108624570A (en) * | 2018-05-15 | 2018-10-09 | 中南大学 | Hexavalent chrome reduction enzyme and its application in administering water body pollution of chromium |
| CN108624570B (en) * | 2018-05-15 | 2021-07-09 | 中南大学 | Hexavalent chromium reductase and application thereof in treatment of chromium pollution of water body |
| CN112547098A (en) * | 2020-12-03 | 2021-03-26 | 中国科学院海洋研究所 | Recyclable metal film and preparation thereof |
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