CN102192938B - Uniform compound catalyst/enzymatic structure and preparation method thereof and application - Google Patents
Uniform compound catalyst/enzymatic structure and preparation method thereof and application Download PDFInfo
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Abstract
The present invention proposes a kind of uniform compound catalyst/enzymatic structure and preparation method thereof and application, this compound catalyst/enzymatic structure and preparation method thereof utilizes an electrophoretic deposition under a suitable electrophoretic deposition condition, the fixing multiple catalyst particle of deposition and enzyme molecule simultaneously, to form a substrate surface in a compound catalyst enzyme film.This film comprises multiple enzyme molecules for catalysis biological molecular reaction of Homogeneous phase mixing and distribution, and for the catalyst material of catalytic electrochemical substance reaction.This composite structure can be used as the working electrode in the biological sensing element of microsensor.
Description
Technical field
The present invention is relevant with a kind of catalyst/enzymatic structure and preparation method thereof, particularly relevant with application with compound catalyst/enzymatic structure of a kind of homogeneous texture and preparation method thereof, the method can be applied to field quite widely, and it can be applied to base material immobilization of organic molecule detecting device or biology sensor (as microbiosensor) etc.
Background technology
Please refer to Fig. 1, it illustrates a kind of schematic diagram of basic structure of biology sensor.The basic structure of biology sensor mainly by biological sensing element (Biological recognition element, Bioreceptor) 11, signal transducers (Signal transducer) 12 and signal processor (Signal processor) 13 these three parts formed.Its sensing principle is: when having after the specific biological sensing element 11 of biological chemistry is combined with test substance 15 or reacts, its physics produced or chemical change, variable quantity can be changed into significant electronic signal through signal transducers 12, by signal processor 13, this signal is amplified and record, to facilitate follow-up Qualitative and quantitative analysis again.
The biomaterial that can be used as sensing element in biology sensor generally slightly can be divided into five large 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 biology sensor prepared by different biological sensing material to be have its respective relative merits, in numerous biological sensing materials, the earliest and the most often used enzyme.Generally speaking, enzyme all has selectivity (i.e. a kind of enzyme can only a certain particular substrate of catalysis), can reuse, is easily heated and the characteristics such as soda acid impact.Enzyme can be combined with each other with specific determinand, and namely this catalysis behavior can be applicable to biology sensor.
Along with global economic development promotes gradually, the mankind also significantly change in life with diet, and one of national main cause of death from acute infectious disease gradually by by chronic disease replace.In the middle of numerous chronic disease, be again national high disease prevailing with diabetes; According to the large cause of the death of compatriots ten that 96 years Department of Health of Executive Yuan announce, diabetes are in fourth.Although medical technology is now maked rapid progress, not yet find out methods for the treatment of diabetes can eradicated completely at present.If diabetic condition is uncontrollable proper, its adjoint many complication, except consuming more social medical resource, also can affect sufferer itself and household's quality of the life.The blood sugar concentration in human body monitored by biology sensor by performance brilliance, accomplishes suitable tracking and managing, then can prevent or slow down the generation of complication, and reach the object of early detection early treatment.And microbiosensor can provide a simple and easy measurement and measuring table easy to carry, user can be carried with 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, and that is all kinds biomolecule is being arranged in pairs or groups under applicable signal transducer, all can as the sensing element analyzing glucose.Much research is for a long time fixed on electric chemical formula glucose biological sensor by utilizing by glucose oxidase, inquires into the sensing analysis of glucose, therefore glucose oxidase (Glucose Oxidase; GODx) be the most often be used in the selection in this research.
Glucose oxidase is that a kind of glycoprotein containing two similar two sub-cells formed, and the protein of this two sub-cell linked by disulfide group, it contains a flavin adenine dinucleotide (Flavin adenine dinucleotide in each sub-cell, FAD) be complementation group, the molecular structure of FAD is as follows.
Under electronics exists by matter, β-D-glucose can be catalyzed into D-glucono-δ-lactone by GOD, and the FAD on GOD can reduce and form FADH
2; Shown in 1-1:
β-D-Glucose+GOD(FAD)→GOD(FADH
2)+D-glucono-δ-latone (1-1)
D-glucono-δ-lactone can become gluconic acid with further reacting with the water in solution; Shown in 1-2:
D-glucono-δ-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 using the oxygen in solution
2) electron acceptors (electron acceptor), GOD (FADH-
2) electrons be transferred to oxygen, and make it be reduced into hydrogen peroxide and make itself to be oxidized to GOD (FAD); Shown in 1-4:
GOD(FADH
2)+O
2→GOD(FAD)+H
2O
2(1-4)
Convolution 1-4 and formula 1-3 can obtain formula 1-5:
β-D-Glucose+O
2→gluconic acid+H
2O
2(1-5)
Biology sensor as shown in Figure 1, has and biochemical signals is transformed into electronic signal, so that the advantage of carry out quantizing analyzing and processing and output display, if apply different electronic components, then can form the biology sensor of different kenel.In FIG, signal transducer 12 is except being transformed into measurable electronic signal by the variable quantity of physics or chemistry, and under proper condition, the intensity of this electronic signal is also directly proportional to the concentration of specific chemicals.If according to the structure of signal transducer 12 and the different of transduction mechanism, then roughly slightly can 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 the current potential that make use of in electrochemical measuring method, electric current or inductance signal transducer, the biology sensor that electrode modified in cooperation forms, it makes use of and come to carry out catalytic reaction with the test substance in sample to be tested through fixing biomolecule and produce product, this product carries out electrochemical redox reaction with the catalyst of electrode surface again, with output current, voltage or measure the change of electrical conductivity, and indirect quantification learn testing concentration.Generally to detect the glucose sensor that glucose uses, be fixed on the glucose oxidase molecules of electrode surface, catalytic reaction can be carried out with the glucose molecule of solution to be measured and generate hydrogen peroxide molecule, hydrogen peroxide diffuses to electrode surface and its catalyst material carries out electrochemical redox reaction, and generation current signal.Therefore, electrochemica biological sensor combines biological sensing layer to for the selectivity (exclusive reaction) of test substance and the advantage of galvanochemistry transducer, and have without the need to quick, the good linear detection range district of instrument and equipment costly, signal reaction/response time, and the advantage such as operating process is simple and easy, its simplicity helps the universalness in future clinical application, thus becomes the heat subject in research now.According to the characteristic of electronic signal, galvanochemistry transducer can be divided into three kinds again: electric potential type (Potentiometric), current type (Amperometric), conductance type (Conductometric) transducer, be wherein the most often used with current type galvanochemistry transducer again.
The method generally preparing microbiosensor first uses thick film printing technique (Screen Printing) to be fixed on the working electrode of microbiosensor by catalyst, prepare Deng microsensor, recycled traditional enzyme immobilization method and enzyme is fixed on surface.Please refer to Fig. 2, it illustrates a kind of layer structure schematic diagram of traditional microbiosensor.The shortcoming of microbiosensor prepared is traditionally: the structure on working electrode (comprising base material 21 and the silver-colored line 22) surface utilizing the method to make, be the structure belonging to catalyst/enzyme layer: its internal layer is catalyst layer 24, skin is enzyme layer 26.For enzyme layer 26 for glucose oxidase, be generally traditionally about 60 ~ 80 μm through immobilized glucose oxidase in the thickness of electrode surface.Layer structure as shown in Figure 2 likely diffuses to catalyst layer 24 along with the thickness of enzyme layer 26 to hydrogen peroxide and causes obstruction, and make the hydrogen peroxide that surface is produced by glucose oxidase, cause because of this thickness film the matter being spread in electrode surface to pass resistance, and cause sensing function to decline.The sensitivity of sensor under high concentration glucose environment (Sensitivity) particularly can be caused to decline.
Moreover, prepare microbiosensor in the conventional way, need the operation formality of suitable multiple tracks and catalyst material and enzyme molecule are the raw material of high unit price, the formality that tradition manufactures microbiosensor often needs excessive catalyst material and enzyme molecule, to reach predetermined sensing function, and the production cost of microbiosensor cannot be reduced.
Summary of the invention
In view of this, the object of this invention is to provide a kind of compound catalyst/enzyme with homogeneous texture and preparation method thereof, it has well reproduced, stability and accuracy, and can be applicable to the various fields quite widely such as the base material immobilization of organic molecule detecting device or biology sensor (as microbiosensor).
Propose a kind of compound catalyst/enzymatic structure according to the present invention, it comprises multiple catalyst particle of Homogeneous phase mixing and distribution and multiple enzyme molecule, and wherein these enzyme molecules are used for catalysis one biomolecular reaction, and these catalyst particle are used for the reaction of catalysis one electrochemical substance.Wherein, be utilize an electrophoretic deposition and under a suitable electrophoretic deposition condition, fix multiple catalyst material and enzyme molecule in deposited on substrates simultaneously, and form one compound catalyst/enzyme film in a substrate surface.This compound 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: provide a base material; There is provided an electrophoresis solution, comprising multiple catalyst particle and multiple enzyme molecule; And utilize an electrophoretic deposition under a suitable electrophoretic deposition condition, simultaneously by depositing catalyst particle and enzyme molecule deposition in the surface of a base material, to form one compound catalyst/enzyme film in the surface of 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 being combined with a test substance or reacting, a physical/chemical changing value can be produced, wherein biological sensing element has a working electrode, this working electrode comprises a base material and is formed at a compound catalyst enzyme film of substrate surface, and this film comprises multiple catalyst particle of Homogeneous phase mixing and distribution and multiple enzyme molecule, wherein these enzyme molecules are used for catalysis one biomolecular reaction, these catalyst particle are used for the reaction of catalysis one electrochemical substance, and be by an electrophoretic deposition, catalyst material and enzyme molecule are deposited on base material simultaneously, to form the working electrode of the compound catalyst/enzyme of homogeneous texture.This signal transducer is that physical/chemical changing value is changed into an electronic signal.This signal processor is the electronic signal that reception and processing signals transducer produce.Confirm that the microbiosensor applied can at room temperature have the pot-life reaching more than 30 days through experiment.Therefore compound catalyst/enzyme electrode of the present invention can be applied to even compound catalyst/enzymatic structure microbiosensor that preparation has well reproduced, stability and accuracy.
Accompanying drawing explanation
For foregoing of the present invention can be become apparent, hereafter will elaborate to preferred embodiment of the present invention by reference to the accompanying drawings, wherein:
Fig. 1 illustrates a kind of schematic diagram of basic structure of biology sensor.
Fig. 2 illustrates a kind of layer structure schematic diagram of traditional microbiosensor.
Fig. 3 is the structural representation of the working electrode of the microbiosensor illustrating the embodiment of the present 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 utilizes the Ya Qi From rifle (Argon Ion Gun) of 5kV to etch, single etch time=100 seconds, etch six times altogether, whether the composition inquired in electrophoresis decorative layer between each element is uniformly distributed, and Pt and Ir represents platinoiridita Nanoalloy catalyst, N represents glucose oxidase, F represents high and fluoridizes (ion-exchange) resin (Nafion).
Fig. 5 is the longitudinal axis spacing of Pt and Ir two kinds of elements in enlarged drawing 4, with the ESCA depth profiles of clearer observation Pt and Ir.
Fig. 6-Fig. 8 is respectively the result (sample number=3) of current value and the glucose sensing function measured when applying current potential 0.3V, 0.4V and 0.5V (vs.Printed Ag/AgCl), wherein collects primary current numerical value every 60 seconds.
Fig. 9 is that EPD-PtIr+GOD-Ir-mini sensor is under applying different potentials, difference is applied to the range of linearity district of current potential 0.3V, 0.4V and 0.5V, the oxidation current obtained and glucose concentration curve figure, wherein working solution is the PBS solution <pH=7.4> of 0.01M, collects primary current numerical value every 60 seconds.
Figure 10 A, Figure 10 B are the microsensor (sample number=3) utilizing electrophoretic deposition simultaneously to deposit PtIr nano metal catalyst and glucose oxidase, under applying current potential 0.3V and 0.4V (vs.Printed Ag/AgCl) out of the ordinary, 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 comparatively.Wherein, Figure 10 B be in Figure 10 A each solution data compared to the Opposed Current number percent of 5mM glucose solution.
Figure 11 and Figure 12 is respectively and utilizes EPD-PtIr+GOD-Ir-mini sensor, applying current potential is the current-vs-time figure of 0.3V and 0.4V (vs.Printed Ag/AgCl), wherein glucose concentration range is 0 μM ~ 200 μMs, and working solution is the PBS solution <pH=7.4> of 0.01M.
Figure 13 is for utilizing EPD-PtIr+GOD-Ir-mini sensor by determining potentiometry, the current time figure that applying current potential obtains for 0.4V (vs.Printed Ag/AgCl), measure glucose concentration scope 2mM ~ 20mM, wherein working solution is the PBS solution <pH=7.4> of 0.01M, collects 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 determining potentiometry, the current time figure that measure glucose concentration scope 2mM ~ 40mM obtains, wherein collected primary current numerical value every 60 seconds.
Figure 15 forms by utilizing reciprocal drafting of individual answering electric current and concentration to be measured in Figure 14, it can utilize Lineweaver-Burk equation to obtain and utilize electrophoresis by electrophoretic deposition, deposit 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 of current signal measurement when the different storage time.
Embodiment
The present invention proposes a kind of compound catalyst enzyme film with homogeneous texture and preparation method thereof and application.Fig. 3 is the schematic diagram of the compound catalyst enzyme film illustrating one embodiment of the invention.Wherein, one compound catalyst enzyme film 35 is formed at the surface of a base material (being such as a working electrode) 33, and compound catalyst enzyme film 35 comprises multiple catalyst particle 353 and the enzyme molecule 355 of Homogeneous phase mixing and distribution, and catalyst particle 353 and enzyme molecule 355 are formed on catalyst carrier 351.Wherein, these enzyme molecules 355 are for catalysis one biomolecular reaction, and these catalyst particle 353 react for catalysis one electrochemical substance.Such as in an application examples, the enzyme molecule of catalysis biological molecular reaction forms 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 be such as carbon black carrier (Carbon, XC-72R).In one embodiment, a mean grain size of catalyst particle can between about 0.5 nanometer be to about 100 microns.By an electrophoretic deposition (Electrophoretic deposition in embodiments of the present invention, EPD) come catalyst particle 353 and enzyme molecule 355 to be deposited on the surface of base material 33 simultaneously, when being applied to a microsensor, then can by catalyst particle 353 with enzyme molecule 355 with electrophoretic deposition, be deposited on working electrode surface simultaneously.By chemical analysis electronic spectrograph (ElectronSpectroscopy for Chemical Analysis, ESCA) depth profile technology inquires into its electrode structure, the while of can confirming to utilize electrophoretic deposition when depositing catalyst and enzyme, the compound catalyst enzyme film of one deck homogeneous texture as shown in Figure 3 can be formed.
In an embodiment, enzyme molecule 355 can be such as be selected from glucose oxidase (Glucose Oxidase; EC1.1.3.4), malate oxidase (malate oxidase; EC 1.1.3.3), hexose oxidase (hexose oxidase; EC 1.1.3.5), cholesterol oxidase (cholesterol oxidase; EC 1.1.3.6), aryl-alcohol oxidase (aryl-alcohol oxidase; EC 1.1.3.7), ester oxidase (L-gulonolactone oxidase in L-GuA; EC 1.1.3.8), galactose oxidase (galactose oxidase; EC 1.1.3.9), pyranose oxidase (pyranoseoxidase; EC 1.1.3.10), L-sorbose oxidase (L-sorbose oxidase; EC 1.1.3.11), pyridoxol 4-oxidase (pyridoxine 4-oxidase; EC 1.1.3.12), methanol oxidase (alcohol oxidase; 1.1.3.13), (S)-2-hydroxy acid oxidase ((S)-2-hydroxy-acid oxidase; 1.1.3.15), moulting hormone oxidase (ecdysone oxidase; EC 1.1.3.16), choline oxidase (choline oxidase; EC 1.1.3.17), secondary alcohol oxidase (secondary-alcohol oxidase; EC 1.1.3.18), 4-Hydroxymandelate oxidase (4-hydroxymandelate oxidase; EC 1.1.3.19), long-chain-alcohol oxidase (long-chain-alcoholoxidase; EC 1.1.3.20), glycerol-3-phosphate oxidase (glycerol-3-phosphate oxidase; EC1.1.3.21), Aneurine oxidase (thiamine oxidase; EC 1.1.3.23), zinc hydroxyl stannate oxidase (hydroxyphytanate oxidase; EC 1.1.3.27), N-acyl group hexosamine oxidase (N-acylhexosamineoxidase; EC 1.1.3.29), polyvinyl alcohol (PVA) oxidase (polyvinyl-alcohol oxidase; EC 1.1.3.30), interior ester oxidase (D-Arabinono-1,4-lactone oxidase; EC 1.1.3.37), vanillyl alcohol oxidase (vanillyl-alcohol oxidase; EC 1.1.3.38), nucleosides oxidase (nucleoside oxidase (H2O2-forming); EC 1.1.3.39), D-MANNOSE alcohol oxidase (D-mannitol oxidase; EC1.1.3.40), xylitol oxidase (xylitol oxidase; EC 1.1.3.41), cellobiose dehydrogenase (cellobiosedehydrogenase (acceptor); EC 1.1.99.18), hydrogenlyase (formate dehydrogenase; EC 1.2.1.2), EC 1.2.3.1 (aldehyde oxidase; EC 1.2.3.1), pyruvate oxidase (pyruvateoxidase; EC 1.2.3.3), oxalate oxidase (oxalate oxidase; EC 1.2.3.4), glyoxylate oxidase (glyoxylate oxidase; EC 1.2.3.5), pyruvate oxidase (CoA-acetylation) (pyruvate oxidase (CoA-acetylating); EC 1.2.3.6), aryl aldehyde oxidase (aryl-aldehyde oxidase; EC 1.2.3.9), retinene oxidase (retinal oxidase; EC 1.2.3.11), ABA aldehyde oxidase (abscisic-aldehydeoxidase; EC 1.2.3.14), ketoglutaric dehydrogenase (succinyl group conversion) (oxoglutarate dehydrogenase (succinyl-transferring); EC 1.2.4.2), dihydroorotate oxidase (dihydroorotate oxidase; EC 1.3.3.1), COPRO-O (coproporphyrinogen oxidase; EC 1.3.3.3), aryl-CoA oxidase (acyl-CoA oxidase; EC 1.3.3.6), dihydrouracil oxidase (dihydrouraciloxidase; EC 1.3.3.7), N-1 oxidase (tetrahydroberberine oxidase; EC 1.3.3.8), tryptophane α, tryptophan side-chain alpha (tryptophan alpha, beta-oxidase; EC 1.3.3.10), PQQ synthase (pyrroloquinoline-quinone synthase; EC 1.3.3.11), ester oxidase (L-galactonolactone oxidase in L-GaA; EC 1.3.3.12), aryl-coa dehydrogenase (acyl-CoAdehydrogenase; EC 1.3.99.3), dihydroorate dehydrogenase (dihydroorotate dehydrogenase; EC 1.3.99.11), D-Asp oxidase (D-aspartate oxidase; EC 1.4.3.1), L-amino acid oxidase (L-amino-acid oxidase; EC 1.4.3.2), D-AAO (D-amino-acid oxidase; EC 1.4.3.3), amido oxidase (containing class flavine) (amine oxidase (flavin-containing); EC 1.4.3.4), tremble aldehyde 5'-phosphate synthase (pyridoxal 5'-phosphate synthase; EC 1.4.3.5), amido oxidase (cupric) (amine oxidase (copper-containing); EC 1.4.3.6), D-Glu salt oxidase (D-glutamateoxidase; EC 1.4.3.7), ethanolamine oxidase (ethanolamine oxidase; EC 1.4.3.8), putrescine oxidase (putrescine oxidase; EC 1.4.3.10), Pidolidone salt oxidase (L-glutamate oxidase; EC 1.4.3.11), cyclohexylamine oxidase (cyclohexylamine oxidase; EC 1.4.3.12), protein-isolated amino acid 6-oxidase (protein-lysine 6-oxidase; EC 1.4.3.13), L-isolated amino acid oxidase (L-lysine oxidase; EC 1.4.3.14), D-Glu salt (D-Asp) oxidase (D-glutamate (D-aspartate) oxidase; EC 1.4.3.15), L-Aspartic acid oxidase (L-aspartateoxidase; EC 1.4.3.16), glycine oxidase (glycine oxidase; EC 1.4.3.19), L-isolated amino acid 6-oxidase (L-lysine 6-oxidase; EC 1.4.3.20), amido dehydrogenasa (amine dehydrogenase; EC 1.4.99.3), FMN reductase (FMN reductase; EC 1.5.1.29), sarcosine oxidase (sarcosineoxidase; EC 1.5.3.1), N-methyl-L Amino acid oxidase (N-methyl-L-amino-acid oxidase; EC 1.5.3.2), N6-methyl-isolated amino acid oxidase (N6-methyl-lysine oxidase; EC 1.5.3.4), (S)-6-hydroxy niacin oxidase ((S)-6-hydroxynicotine oxidase; EC 1.5.3.5), (R)-6-hydroxy niacin oxidase ((R)-6-hydroxynicotine oxidase; EC 1.5.3.6), L-pipecoline (L-pipecolateoxidase; EC 1.5.3.7), dimethylglycine oxidase (dimethylglycine oxidase; EC 1.5.3.10), PAO (polyamine oxidase; EC 1.5.3.11), DHBP oxidase (Dihydrobenzophenanthridine oxidase; EC 1.5.3.12), trimethylamine dehydrogenase (trimethylaminedehydrogenase; EC 1.5.8.2), L-pipecoliacid dehydrogenasa (L-pipecolate dehydrogenase; EC 1.5.99.3), basic element of cell division dehydrogenasa (cytokinin dehydrogenase; EC 1.5.99.12), NAD (P) H oxidase (NAD (P) H oxidase; EC 1.6.3.1), NAD (P) H dehydrogenasa (to benzene triketone) (NAD (P) Hdehydrogenase (quinone); EC 1.6.5.2), nitrite reductase (nitrite reductase (NO-forming); EC 1.7.2.1), nitroalkane oxydase (nitroalkane oxidase; EC 1.7.3.1), urate oxidase (urateoxidase; EC 1.7.3.3), 3-nitropropionic acid methyl esters oxidase (3-aci-nitropropanoate oxidase; EC1.7.3.5), dihydro lipoyl dehydrogenase (dihydrolipoyl dehydrogenase; EC 1.8.1.4), sulfite oxidase (sulfite oxidase; EC 1.8.3.1), thiol oxidase (thiol oxidase; EC 1.8.3.2), glutathione oxidase (glutathione oxidase; EC 1.8.3.3), methyl mercaptan oxidase (methanethioloxidase; EC 1.8.3.4), alkylene aminothiopropionic acid oxidase (prenylcysteine oxidase; EC 1.8.3.5), 3-hydroxyl anthranilic acid oxidase (3-hydroxyanthranilate oxidase; EC 1.10.3.5), thunder good fortune mycin-B oxidase (rifamycin-B oxidase; EC 1.10.3.6), NADH peroxidase (NADH peroxidase; EC 1.11.1.1), 2-nitropropane dioxygenase (2-nitropropane dioxygenase; EC 1.13.11.32), rely amino acid 2-monooxygenase (lysine 2-monooxygenase; EC 1.13.12.2), Lactate 2-monooxygenase (lactate2-monooxygenase; EC 1.13.12.4),
fluorescein4-monooxygenase (ATP hydrolysis) (Photinus-luciferin4-monooxygenase (ATP-hydrolysing); EC 1.13.12.7), phenylalanine 2-monooxygenase (phenylalanine 2-monooxygenase; EC 1.13.12.9), clavaminate synzyme (clavaminatesynthase; EC 1.14.11.21), naphtha essence 1,2-dioxygenase (naphthalene 1,2-dioxygenase; EC 1.14.12.12), 4-aminobenzoate 1-monooxygenase (4-aminobenzoate 1-monooxygenase; EC 1.14.13.27), alkanal monooxygenase (alkanal monooxygenase (FMN-linked); EC1.14.14.3), Phenylalanine 4-monooxygenase (phenylalanine 4-monooxygenase; 1.14.16.1), adjacent amine Sodium Benzoate 3-monooxygenase (anthranilate 3-monooxygenase; EC 1.14.16.3), single phenol monooxygenase (monophenol monooxygenase; EC 1.14.18.1), 7-cholestenol oxidase (lathosteroloxidase; EC 1.14.21.6), superoxide dismutase (superoxide dismutase; EC 1.15.1.1), superoxides reductase (superoxide reductase; EC 1.15.1.2), xanthine dehydrogenase (xanthinedehydrogenase; EC 1.17.1.4), xanthine oxidase (xanthine oxidase; EC 1.17.3.2), 6-hydroxyl nicotine dehydrogenase (6-hydroxynicotinate dehydrogenase; EC 1.17.3.3), fragrant isobebeerine enzyme (reticuline oxidase; EC 1.21.3.3), diphosphoribulose carboxylase (Ribulose-bisphosphatecarboxylase; EC 4.1.1.39) group that forms.
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 is such as 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) forms.In one embodiment, catalyst particle 353 can such as be made up of binary metal and this binary metal oxide, and its metallic element mol ratio is greater than 0 to be less than 100%.
Below using platinoiridita (PtIr) nano metal catalyst as catalyst particle, and the related description using glucose oxidase as enzyme molecule as an embodiment, simultaneously it be used on working electrode to fix platinoiridita nano metal catalyst and glucose oxidase, with the embodiment of the glucose sensor be combined with 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 applies electrophoretic deposition (EPD), catalyst and enzyme are fixed on the working electrode surface of miniature inductive device simultaneously, to produce the easy and microbiosensor of constitutionally stable even compound catalyst/enzymatic structure of preparation.Its content can be divided into two parts: (C1) comes, simultaneously by platinoiridita nano metal catalyst and glucose oxidase, to be deposited on the electrode structure analysis of microsensor by electrophoretic deposition; (C2) simultaneously by PtIr nano metal catalyst and glucose oxidase, the electrochemical properties analysis of microsensor is deposited on.
Moreover, the present invention has same area, and to know that the knowledgeable works as known, the present invention to be not limited in described in following of the present invention arbitrary experimental example-to use nanometer platinoiridita (double base) metal solvent and glucose oxidase, it also can make suitable selection according to needed for application conditions in aforementioned proposed catalyst molecular species and enzyme molecular species, and utilize the preparation method of electrophoretic deposition to reach even compound catalyst enzymatic structure, therefore the present invention is also not only confined to the application of glucose sensor.
<C: electrophoretic deposition depositing nano metal catalyst and glucose oxidase are in microsensor >
According to the present invention, its be utilize electrophoretic deposition simultaneously by catalyst particle and enzyme molecule deposition in microsensor.Electrophoretic deposition is the driving force produced by applying electric field, is deposited on by the charged particle in suspending liquid on working electrode to reach the object of deposition.And according to a preferred embodiment of the present invention, electrophoretic deposition platinoiridita nano metal catalyst and glucose oxidase is deposited on microsensor simultaneously.The compound method of electrophoresis solution is by appropriate platinoiridita nano metal catalyst, glucose oxidase and Nafion Homogeneous phase mixing in PBS, to form the aaerosol solution with stable phase.Molecule with electric charge in electrophoresis solution has: after Nafion molecule dissociates hydrogen ion in water, and makes itself to present electronegative molecule because there being inferior sulfate radical functional group; The pI value of glucose oxidase is 4.2, is about in the PBS of 7.4 in pH value, and glucose oxidase can be formed has electronegative molecule; The carrier of platinoiridita nano metal catalyst can act on Nafion ionomer (ionomer), and the carrier that Nafion can be attached to catalyst makes its surface with negative electricity.Therefore, be subject under electric field drives, electronegative Nafion ionomer, glucose oxidase and platinoiridita nano metal catalyst, just can move toward the anode with positive charge, and be deposited on the working electrode surface of microsensor.
The parameter affecting electrophoretic deposition can be roughly divided into two parts: the state of (1) electrophoresis solution, the condition of (2) electrophoretic procedures.The factors such as electrophoresis solution state includes the size of suspended particles, Jie of suspended particles reaches current potential, the degree of stability of the specific inductive capacity of solution, the electric conductivity of solution, the viscosity of solution and solution.The condition of electrophoretic procedures includes sedimentation time, applies the factor such as aerosol concentration, base material electric conductivity in voltage, solution.
In one embodiment, the step preparing electrophoresis solution is as follows: first, add in solvent by appropriate nano metal catalyst, 5%Nafion, be positioned over ultrasonic oscillator and make it dispersed in 30 minutes.Then being incorporated in electrophoresis solution by appropriate glucose oxidase makes it dispersed.(this step should not use ultrasonic oscillator to lose activity to avoid enzyme).After having prepared, under electrophoresis solution being stored in 4 DEG C of environment.And electrophoretic deposition step is as follows: the electrophoresis solution prepared is positioned on stirrer and makes particle can be uniformly dispersed in solution.Then be connected for depositing catalyst with the slot of microsensor with the microsensor of enzyme, this slot is connected to potentiostat.Carry out the electrophoretic deposition program applying to determine voltage or determine electric current.Microsensor after having deposited is positioned over natural air drying in air.Be positioned over afterwards in clip chain bag with same in order to using.But as is known to the person skilled in the art, this embodiment the step that describes and parameter be only reference, and be not used to limit to the present invention, when applying of the present invention, its relevant administration step and parameters value, all can do suitable change according to practical application request.
c1: by electrophoretic deposition simultaneously in microsensor deposition PtIr nano metal catalyst and glucose oxidase
the electrode structure analysis of enzyme
Be for aforementioned with electrophoretic deposition in an embodiment, the electrode simultaneously platinoiridita nano metal catalyst and glucose oxidase being deposited on microsensor carries out structure analysis.Wherein, it is the depth ultimate analysis utilizing chemical analysis electronic spectrograph (ESCA), inquire into the distribution scenario of different band charged particle electrophoretic deposition to electrode surface, its result display structure really as shown in Figure 3, can form the compound catalyst/enzyme film of one deck homogeneous texture on the surface of base material.
Below propose the depth ultimate analysis adopting chemical analysis electronic spectrograph (ESCA) in the experiment of embodiment, observe the distribution scenario of different band charged particle electrophoretic deposition to electrode surface.
(etch for utilizing the Ya Qi From rifle (Argon Ion Gun) of 5kV according to the binding energy (Binding Energy) of Pt, Ir, N and F and XPS depth profile, etching period=300 second) ultimate analysis that obtains, known binding energy all corresponds to the feature binding energy position of this element, and integral area can obtain this element in the content ratio of entirety.Utilize different etching period to discuss the distribution of element at different depth, and observe different electrophoretic deposition particle 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 utilize the Ya Qi From rifle (Argon Ion Gun) of 5kV to etch, single etch time=100s, etch six times altogether, and whether the composition inquired in electrophoresis decorative layer between each element is uniformly distributed, wherein Pt and Ir represents platinoiridita Nanoalloy catalyst, N represents glucose oxidase, F represents Nafion.The entirety composition of compound catalyst as can be seen from Figure 4/enzyme film, major part all still provided by the glucose oxidase with atom N, infer that reason should be because glucose oxidase wraps the platinoiridita nano metal catalyst of attached Nafion compared to surface, have higher electrophoretic mobility (Electrophoretic mobility), therefore overall composition major part all provided by glucose oxidase.And the deposition ratio also can observing out glucose oxidase from Fig. 4 can be subject to the impact of Nafion, due in electrophoresis solution, glucose oxidase molecules and Nafion monomer molecule surface are all present negative charge, therefore glucose oxidase molecules and Nafion monomer molecule will be there is in electrophoresis process, the electrochemical activity position of electrode surface can be competed, so the deposition position of glucose oxidase molecules can be blocked when Nafion is deposited on electrode surface simultaneously simultaneously.
Pt and Ir is in the composition of this composite membrane in Fig. 4 more difficult observation, therefore can be discussed separately by these two kinds of elements of Pt and Ir by Fig. 5.Fig. 5 is the longitudinal axis spacing of Pt and Ir two kinds of elements in enlarged drawing 4, with the ESCA depth profiles of clearer observation Pt and Ir.Can find out in figure except surface composition more uneven (possible cause by electrode surface coated by the glucose oxidase in electrophoresis solution, therefore low next compared with internal layer of outermost metal signal), remaining etching period all presents stable composition distribution, it demonstrates electrophoretic deposition platinoiridita nano metal catalyst, can provide the structure that uniform and stable, the glucose oxidase being conducive to electrophoretic deposition is simultaneously stablized by its structure is fixed on surface.Can find out that electrode longitudinally form suitable even of distribution by Fig. 4 and Fig. 5, compound catalyst/enzyme membrane structure that its proof utilizes the method really can form to mix is in the electrode surface of microsensor.
c2: simultaneously deposit platinoiridita nano metal catalyst and glucose oxidase in the electrochemical properties of microsensor
analyze
Also for depositing platinoiridita nano metal catalyst and glucose oxidase carries out multiple electrochemical properties analysis in microsensor simultaneously in embodiment, contain the linear sense district (Linear DetectingRange) of microsensor, the lowest detection limit (Limit of detection, LOD), chaff interference test (Interference Test), the repeatability (Reproducibility) of microsensor and stability test, the discussion of simultaneous muti-component determina-tion, and the long-time stability of microsensor tests tests such as (Long term Stability).It is for reference that following series goes out part of test results.
Also carry out related experiment for the microsensor (EPD-PtIr+GOD-Ir-mini sensor) depositing platinoiridita nano metal catalyst and glucose oxidase simultaneously in embodiment, it preferably can detect glucose to apply potential value.When EPD-PtIr+GOD-Ir-mini sensor is under the PBS solution of the concentration of glucose of variable concentrations, 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, when the scanning number of turns 10 is enclosed.Can find can have about 0.2V (vs.Printed Ag/AgCl) oxidation current obviously risen gradually along with hydrogen peroxide concentration increase from scanning result, then have the reduction current obviously risen gradually along with hydrogen peroxide concentration increase about-0.25V (vs.Printed Ag/AgCl).Can find along with voltage rise from result, oxidation current increases thereupon, represents that applying current potential is higher, detects glucose ability higher.But owing to there being other chaff interference a lot of in blood of human body, itself be also electroactive substance, if it is too high to apply current potential, chaff interference also can react together, and causes the interference of current signal thereupon.Therefore, must look for one has suitable sensing current potential, and the hydrogen peroxide that glucose oxidase is generated has good oxidability, and can avoid interference thing and react under this current potential, will be a considerable problem.
Below the microsensor simultaneously depositing platinoiridita nano metal catalyst and glucose oxidase by electrophoretic deposition is inquired in experiment, current potential is applied for the best measuring different glucose in solution, it utilizes the different current potential that applies to carry out determining current response method (Amperometric measurement), compares and applies the impact of current potential for glucose sensing function.
Generally being applied to the linearity test district needed for blood sugar detecting diabetic is 3 ~ 12mM.Therefore, in order to want the range of blood sugar that can accord with in actual detection blood, the glucose linearity test district of proper range, for judging whether one of key being applicable to sensing blood sugar.After having learnt that in related experiment the applying current potential of optimization measurement hydrogen peroxide is 0.3V (vs.Printed Ag/AgCl), change can be carried out and apply potential effect, and respectively at carrying out the sensing of different concentration of glucose under 0.3V, 0.4V and 0.5V (vs.Printed Ag/AgCl), comparing and applying current potential for current value and glucose sensing function.And the current value of different applyings measured by current potential and the result (sample number=3) of glucose sensing function, be respectively as shown in Fig. 6, Fig. 7 and Fig. 8, wherein just collected primary current numerical value every 60 seconds.
Can find out that from Fig. 6 ~ Fig. 8 the range of linearity that applying different potentials senses for glucose all has difference.In figure 6, the glucose sense linear scope applying 0.3V (vs.Printed Ag/AgCl) is 2mM ~ 12mM.In the figure 7, the glucose sense linear scope applying 0.4V (vs.Printed Ag/AgCl) is 2mM ~ 20mM.In fig. 8, and apply 0.5V (vs.Printed Ag/AgCl) glucose sense linear scope be 4mM ~ 20mM.Apply current potential as can be seen from the results when 0.4V (vs.Printed Ag/AgCl), there is compared to 0.3V (vs.Printed Ag/AgCl) range of linearity of higher concentration.When applying current potential and increasing as 0.5V (vs.PrintedAg/AgCl), it may be the impact that electrode surface produces a little subsidiary reaction of ㄧ, can find out that the deviate that glucose senses is when being 0.4V and 0.3V (vs.Printed Ag/AgCl) apparently higher than applying current potential.
Fig. 9 is that EPD-PtIr+GOD-Ir-mini sensor is under applying different potentials, the oxidation current that the range of linearity district of difference applying current potential 0.3V, 0.4V and 0.5V is obtained and glucose concentration curve figure, wherein working solution is the PBS solution <pH=7.4> of 0.01M, collects primary current numerical value every 60 seconds.And table 1 is respectively the glucose sensing sensitivity (Sensitivity) of EPD-PtIr-Ir-mini sensor under difference applies current potential and linearly dependent coefficient (liner corelation) R of calibration curve
2.
Table 1
A. temperature: 25 DEG C (± 1 DEG C)
B. electrophoretic deposition parameter: determine 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 makes its Homogeneous phase mixing and is scattered in 1mL 0.01M PBS solution
C. sample number=3 are tested
Glucose sensing sensitivity can rise along with applying current potential and increase as can be seen from Figure 9, wherein can observe out and rise and the hydrogen peroxide sensing sensitivity of increase along with applying current potential, under 0.4V to 0.5V (vs.PrintedAg/AgCl), have maximum knots modification (about 1.4 × 10
-8a/mM), although apply current potential, in 0.5V (vs.PrintedAg/AgCl), there is higher glucose sensing sensitivity, but larger relative to the error applying to cause compared with the microsensor of electronegative potential.
Therefore, known from above-mentioned experimental result: applying current potential being located at 0.4V (vs.Printed Ag/AgCl), the best glucose detection range of linearity (2mM ~ 20mM) can be obtained and there is lower deviate.
Embodiment afterwards will again for applying current potential under 0.3V and 0.4V (vs.Printed Ag/AgCl), with repeatability (Reproducibility), related experiment and research are carried out to disturbed test (Interference Test), detection limit (Limiting of detection, LOD).
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 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 and have suitable sensing current potential, the hydrogen peroxide generated to enable glucose oxidase has good oxidability, and can avoid interference thing and react under this current potential.
Point out according to existing document: the vitamin C average content in 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 adult female about 2.6 ~ 6.0mg/dL.And the vitamin C used in the experiment of embodiment and uric acid concentration, be then set in the concentration a little more than general average, its etc. be respectively 1.5mg/dL and 8mg/dL, with the discussion as disturbed test.To first add the concentration of glucose of 5mM and sensed current signal in solution in experiment, distinctly add vitamin C and the uric acid of appropriate amount thereafter in the same solution, with chaff interference under the environment observing appropriate concentration of glucose respectively for the impact of current signal.
Figure 10 A, Figure 10 B are the microsensor (sample number=3) utilizing electrophoretic deposition simultaneously to deposit platinoiridita nano metal catalyst and glucose oxidase, under applying current potential 0.3V and 0.4V (vs.Printed Ag/AgCl) out of the ordinary, 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 comparatively.Wherein, 10B figure be in 10A figure each solution data compared to the Opposed Current number percent of 5mM glucose solution.
Can demonstrate from Figure 10 A, Figure 10 B apply current potential be interfered when 0.4V (vs.Printed Ag/AgCl) thing electric current impact be less than apply current potential when 0.3V (vs.Printed Ag/AgCl).Generally speaking, the impact applying the higher suffered chaff interference of current potential can be more serious, but the result in this section experiment finds that applying current potential is higher, has good selectivity 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 cause the impact of the thing that is interfered comparatively violent.Relatively, applying higher current potential can increase catalytic capability to hydrogen peroxide, thus adds the current response to glucose, makes to be compressed the current response of vitamin C, uric acid, so just reduce the interference of vitamin C and uric acid.Therefore the disturbed test obtained from experimental result found that, in embodiment, when applying current potential in 0.4V (vs.Printed Ag/AgCl), can obtain preferably sensor performance.
microsensor detects the lowest detection limit of glucose
Also related experiment is carried out for the lowest detection limit (Limit of detection, LOD) utilizing electrophoretic deposition to deposit the microsensor of platinoiridita nano metal catalyst and glucose oxidase simultaneously in embodiment.
Figure 11 and Figure 12 is respectively and utilizes EPD-PtIr+GOD-Ir-mini sensor, applying current potential is the current-vs-time figure of 0.3V and 0.4V (vs.Printed Ag/AgCl), wherein glucose concentration range is 0 μM ~ 200 μMs, and working solution is the PBS solution <pH=7.4> of 0.01M.Adding different concentration of glucose at every turn, then can calculate minimum useful signal by Figure 11 and Figure 12 respectively, if signal/noise ratio (S/N) is greater than 3, is then useful signal; If signal/noise ratio (S/N) is less than 3, then it is invalid signals.Therefore, can learn from figure that applying current potential is respectively 80 μm and 100 μm in the lowest detection limit of 0.3V and 0.4V (vs.Printed Ag/AgCl).Its reason may be that high to apply under current potential situation be have larger background current (Backgroundcurrent) signal, good than too late electronegative potential of therefore obtained at low concentrations signal news.
the repeatability of microsensor and stability
Preparing sensor most important is exactly want user's accuracy rate can be provided high and the sensing result that reappearance is good.Repeatability (Reproducibility) refers to different people, different instruments, under the different condition of different time etc., all can obtain the ability of identical measured value.If have good reproducibility, represent utilize the process conditions of the microsensor prepared by electrophoretic deposition suitable stable, the yield of microsensor can be improved and improve the confidence level of signal.Repeatability (Repeatability) refers to that sensor obtains the ability of identical measured value at identical conditions, if have good repeatability, it represents the microsensor utilized prepared by electrophoretic deposition, that there is reusable ability, the catalyst utilizing electrophoresis to deposit and enzyme, still have the structure of quite stable in by measuring process with the process at cleaning electrode.The embodiment of the present invention also carries out related experiment for repeatability and stability (repeatability).
Figure 13 is for utilizing EPD-PtIr+GOD-Ir-mini sensor by determining potentiometry, the current time figure obtained under applying current potential is for 0.4V (vs.Printed Ag/AgCl), measure glucose concentration scope 2mM ~ 20mM, wherein working solution is the PBS solution <pH=7.4> of 0.01M, collects primary current numerical value every 60 seconds.Measure for three times and all use different group microsensor, this experiment purpose is by measurement different group microsensor to judge the height of reproducibility.Can find out to measure for three times from Figure 13 and table 2 and all obtain quite close data, its relative standard deviation (Relative Standard Deviation, RSD) be 7.14%, display utilizes electrophoretic deposition on microsensor thus, the manufacturing process simultaneously depositing platinoiridita nano metal catalyst and glucose oxidase is quite stable, therefore has quite good reproducibility.
Table 2
A. temperature: 25 DEG C (± 1 DEG C)
B. electrophoretic deposition parameter (EPD parameters): determine 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 makes its Homogeneous phase mixing and is scattered in 2mL 0.01M PBS solution
C. sample number=3
Table 3 be EPD-PtIr+GOD-Ir-mini sensor by determining potentiometry, in applying current potential be 0.4V (vs.Printed Ag/AgCl), the result of measure glucose concentration for obtaining under 5mM.Three measurements all use same group of microsensor, and this experiment purpose is by duplicate measurements same group of microsensor, to judge the height of repeatability (Repeatability).Table 3 can find out that three measurements all obtain quite close data, relative standard deviation (RSD) is 7.16%, display utilizes electrophoretic deposition to prepare the microsensor of platinoiridita nano metal catalyst and glucose oxidase thus, quite good repeatability can be had, platinoiridita nano metal catalyst and glucose oxidase to be deposited on the working electrode of microsensor by its display simultaneously by electrophoretic deposition, can stablize and be fixed on the structure that electrode surface forms a good catalyst/enzyme composite bed.
Table 3
A. temperature: 25 DEG C (± 1 DEG C)
B. electrophoretic deposition parameter (EPD parameters): determine 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 makes its Homogeneous phase mixing and is scattered in 2mL 0.01M PBS.
C. identical mini sensor.
simultaneous muti-component determina-tion
The mechanism generally can carried by Michaelis-Menten for the catalytic reaction of its enzyme describes; Shown in 2-2:
Wherein: I representative detects glucose response electric current (A), I
maxtheoretical maximum response electric current (A) of representative sensor, K
app mrepresent Michaelis constant (M), concentration of glucose (M) that C represents solution to be measured.
K
m appvalue (Michaelis constant) has the characteristic judging determinand and enzyme affinity size.Low K
m appthe value then reaction in high affinity.At testing concentration ([C]) much smaller than K
m apptime, reaction rate is proportional to testing concentration (first order reaction); And when testing concentration is much larger than K
m apptime, react for zero-order reaction, its speed and testing concentration have nothing to do.If want calculating K
m appwith I
matime, utilize Lineweaver-Burk equation can obtain the K of sensor
m appwith I
max.Shown in 2-3:
Wherein I representative detects glucose response electric current (A), I
maxtheoretical maximum response electric current (A) of representative sensor, K
app mrepresent Michaelis constant (M), concentration of glucose (M) that C represents solution to be measured.
Can utilize electric current (I) and detectable concentration (C) that in experiment, Figure 14 obtains, 1/I and 1/C substituted in Lineweaver-Burk equation (formula 2-3) illustrates figure respectively, analyzes the correlation parameter of its biology sensor.If the sensing range of sensor controls in enzyme power control district, the figure drawn by Lineweaver-Burk equation will be presented linearly, and can (be K from the slope of straight line
m app/ I
max) with intercept (be 1/I
max) distinctly can 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 determining potentiometry, measuring at glucose concentration range is the current time figure that 2mM ~ 40mM obtains, and wherein collects 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-linear in the scope measuring high concentration glucose, represent that the rate-determing step reaction of this concentration range is enzyme power control reaction (Enzyme kinetic controlled reaction).
Figure 15 forms by utilizing reciprocal drafting of individual answering electric current and concentration to be measured in Figure 14, utilize Lineweaver-Burk equation (formula 2-3, linear equation) that the K of the microsensor depositing platinoiridita nano metal catalyst and glucose oxidase by electrophoretic deposition can be obtained
m appfor 5.68mM, and I
maxbe 6.02 × 10
-7a.Relatively can find with other document, in embodiment, deposit the microsensor of platinoiridita nano metal catalyst and glucose oxidase by electrophoretic deposition, there is quite low K
m app, the enzyme in the compound catalyst/enzyme layer of the homogeneous texture that its representative is formed by electrophoretic deposition has good enzymatic activity and the affinity to glucose.
the long-time preservation test of microsensor
For any one biology sensor, the pot-life of how effectively to improve sensor is one of considerable problem.General by the biology sensor prepared by traditional process for fixation in order to enzyme will be improved in the pot-life of working electrode, common store method is under this biology sensor is positioned over the environment of 4 DEG C, to extend the activity of electrode surface enzyme molecule; But this kind of store method significantly will reduce microsensor advantage easy to carry.
In view of this, in order to want the custom copying the biological microsensor of the actual use of user, be that the microsensor completed to be positioned in clip chain bag with same and to preserve under being positioned over room temperature, to preserve test for a long time by employing in the present embodiment.Room temperature is the environment of about 25 DEG C (± 1 DEG C).
All test and will measure the 1st day, the 5th day, the 10th day, the 15th day, the 20th day, the 25th day respectively, and the microsensor EPD-PtIr+GOD-Ir-mini sensor of the 30th day is for the current signal (sample number=3) of the glucose solution of 5mM in experimentation at every turn.Figure 16 and table 4 are the detailed results that storage time and current signal are measured.
Table 4
A. temperature: 25 DEG C (± 1 DEG C)
B. electrophoretic deposition parameter (EPD parameters): determine 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 makes its Homogeneous phase mixing and is scattered in 2mL 0.01M PBS solution
C. sample number=3
Current result display, deposit the microsensor of platinoiridita nano metal catalyst metal catalyst and glucose oxidase by electrophoretic deposition simultaneously, still there is stable sensing glucose ability being stored in for a long time under room temperature (about 25 DEG C), its reason of inference should be even composite 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 by electrophoretic deposition simultaneously, the result of many related experiment that the multinomial electrochemical properties carried out is analyzed, the research of comprehensive consideration electrophoresis solution parameter and electrophoretic procedures condition, can learn: by 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) dissolution homogeneity to be disperseed, more stable electrophoresis solution can be obtained, and determine voltage method in applying current potential 0.5V (vs.PrintedAg/AgCl) in utilization, under sedimentation time 5mins, miniature glucose biology sensor of good performance can be prepared.
By the depth profiles result of ESCA, known electrode is suitable even of composition distribution longitudinally, and it proves to utilize the method really in the electrode surface of microsensor, can form the compound catalyst/enzymatic structure mixed.Even compound catalyst/enzymatic structure layer can provide a special environment, to shorten the hydrogen peroxide that generated by test substance and the enzyme molecular reaction path to catalyst surface, and make the electronics that generated by hydrogen peroxide and catalyst surface reaction, can, rapidly by the carrier of nano metal catalyst, be directed on the base material of working electrode; 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 by electrophoretic deposition simultaneously, be the processing procedure of a quite stable.Compared to the microsensor only using electrophoresis deposition method glucose oxidase, optimization electrophoresis solution parameter in conjunction with the embodiments and electrophoretic procedures condition, prepared by electrophoretic deposition deposit the microsensor of platinoiridita nano metal catalyst and glucose oxidase simultaneously, glucose sensing function can be promoted to 1.25 times originally.This display platinoiridita nano metal catalyst of embodiment and composite structure of glucose oxidase, can promote glucose sensing function really.
Inquire into the result of Dynamics of Enzyme Catalysis and compare with other document, the microsensor being deposited platinoiridita nano metal catalyst and glucose oxidase in embodiment by electrophoretic deposition can be found, lower Michaelis constant (in experimental example K can be had
m app=5.68mM), the enzyme in the compound catalyst/enzyme layer of the homogeneous texture that this representative is formed by electrophoretic deposition, has good enzymatic activity and the affinity to glucose; Infer that its possible cause is the compound catalyst/enzyme layer of homogeneous texture, a kind of special structural environment can be provided, and make to be risen by enzyme stability, and significantly improve the pot-life of biology sensor.Experimentally result test, it at least at room temperature has the pot-life reaching more than 30 days.
Moreover, by the miniature glucose biology sensor prepared by the electrophoretic deposition conditional parameter of optimization, under applying 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, compound catalyst/the enzyme electrode of a kind of homogeneous texture that the embodiment of the present invention proposes, come catalyst particle (being such as platinoiridita nano metal catalyst) and enzyme molecule (being such as glucose oxidase) to be deposited on the working electrode surface of microsensor by electrophoretic deposition simultaneously, by every experiment, such as by ESCA depth profile electrode structure, confirmation utilizes electrophoretic deposition depositing catalyst particle and enzyme molecule simultaneously, compound catalyst/enzyme the film of one deck homogeneous texture can be formed, and it (can reach more than 30 days) for a long time and is stored in room temperature.Experimental result shows, and applies preparation method of the present invention and can prepare the even compound catalyst/enzymatic structure microbiosensor with well reproduced, stability and accuracy.
In sum, although the present invention with embodiment disclose as above, but itself and be not used to limit the present invention.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 equivalent change or replacement.Therefore, protection scope of the present invention is when being as the criterion of defining depending on accompanying the application's right.
Claims (10)
1. a compound catalyst enzyme film, it comprises: catalyst carrier, with multiple catalyst particle and multiple enzyme molecule of Homogeneous phase mixing and distribution, wherein aforementioned catalyst carrier is carbonaceous carrier, and these catalyst particle and these enzyme molecules are formed on aforementioned catalyst carrier, wherein said enzyme molecule and described catalyst particle are that Homogeneous phase mixing contains the electrophoresis solution of Nafion in one, Nafion makes the surface of aforementioned catalyst carrier charged, and utilize an electrophoretic deposition to make these catalyst particle, the charged aforementioned catalyst carrier of these enzyme molecules and surface deposits simultaneously and is fixed on a substrate surface, these enzyme molecules are used for catalysis one biomolecular reaction, these catalyst particle are used for the reaction of catalysis one electrochemical substance,
Wherein, these catalyst particle of this electrochemical substance of catalysis reaction are binary metal M-X, one binary metal oxide MOy-XOy, or the combination of aforementioned binary metal M-X and aforementioned binary metal oxide MOy-XOy, wherein y is less than 3, and M and X is selected from chromium (Cr), manganese (Mn), iron (Fe), cobalt (Co), nickel (Ni), copper (Cu), gallium (Ga), niobium (Nb), molybdenum (Mo), ruthenium (Ru), rhodium (Rh), palladium (Pd), silver (Ag), indium (In), tin (Sn), praseodymium (Pr), terbium (Tb), tungsten (W), iridium (Ir), platinum (Pt), gold (Au), plumbous (Pb).
2. compound catalyst enzyme film according to claim 1, is characterized in that, these enzyme Journal of Molecular Catalysis biomolecular reactions form hydrogen peroxide.
3. compound catalyst enzyme film according to claim 1, is characterized in that, these catalyst particle of this electrochemical substance of catalysis reaction carry out an electrochemical redox reaction with hydrogen peroxide.
4. compound catalyst enzyme film according to claim 3, it is characterized in that, wherein, these catalyst particle comprise this binary metal and this binary metal oxide, and the mol ratio of its metallic element M and X is greater than 0 to be less than 100%, and a mean grain size of these catalyst particle is between 0.5 nanometer to 100 micron.
5. compound catalyst enzyme film according to claim 1, is characterized in that, these catalyst particle are multiple platinoiridita (PtIr) nano-catalyst particles, and these enzyme molecules are multiple glucose oxidase molecules.
6. a preparation method for microsensor electrode, comprising:
One base material is provided;
One electrophoresis solution is provided, it comprises mixed uniformly catalyst carrier, multiple catalyst particle, multiple enzyme molecule and Nafion, and wherein, aforementioned catalyst carrier is carbonaceous carrier, these catalyst particle and these enzyme molecules are formed on aforementioned catalyst carrier, and Nafion makes the surface of aforementioned catalyst carrier charged; And
Utilize an electrophoretic deposition under a suitable electrophoretic deposition condition, aforementioned catalyst carrier charged to these catalyst particle, these enzyme molecules and surface is deposited on the surface of this base material simultaneously, to form a compound catalyst enzyme film in the surface of this base material, wherein this film comprises these catalyst particle mixed uniformly and these enzyme molecules, and these catalyst particle and these enzyme molecules are formed on aforementioned catalyst carrier
Wherein, these catalyst particle are binary metal M-X, one binary metal oxide MOy-XOy, or the combination of aforementioned binary metal M-X and aforementioned binary metal oxide MOy-XOy, wherein y is less than 3, and M and X is selected from chromium (Cr), manganese (Mn), iron (Fe), cobalt (Co), nickel (Ni), copper (Cu), gallium (Ga), niobium (Nb), molybdenum (Mo), ruthenium (Ru), rhodium (Rh), palladium (Pd), silver (Ag), indium (In), tin (Sn), praseodymium (Pr), terbium (Tb), tungsten (W), iridium (Ir), platinum (Pt), gold (Au), plumbous (Pb).
7. the preparation method of microsensor electrode according to claim 6, is characterized in that, these catalyst particle are multiple platinoiridita (PtIr) nano metal catalyst particle, and these enzyme molecules are multiple glucose oxidase molecules.
8. the preparation method of microsensor electrode according to claim 7, is characterized in that, this electrophoresis solution provided more comprises a pH value aqueous buffer solution.
9. the preparation method of microsensor electrode according to claim 8, 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.
10. a microbiosensor, it includes:
One biological sensing element, after it is combined with a test substance or reacts, a physical/chemical changing value can be produced, wherein this biological sensing element has a working electrode, this working electrode comprises a base material and is formed at a compound catalyst enzyme film of this substrate surface, and this film comprises catalyst carrier, with multiple catalyst particle and multiple enzyme molecule of Homogeneous phase mixing and distribution, aforementioned catalyst carrier is carbonaceous carrier, and these catalyst particle and these enzyme molecules are formed on aforementioned catalyst carrier, wherein said enzyme molecule and described catalyst particle are that Homogeneous phase mixing contains the electrophoresis solution of Nafion in one, Nafion makes the surface of aforementioned catalyst carrier charged, and utilizes an electrophoretic deposition to make these catalyst particle, the charged aforementioned catalyst carrier of these enzyme molecules and surface deposits simultaneously and is fixed on this substrate surface, wherein these enzyme molecules are used for catalysis one biomolecular reaction, and these catalyst particle are used for the reaction of catalysis one electrochemical substance, wherein, these catalyst particle are binary metal M-X, one binary metal oxide MOy-XOy, or the combination of aforementioned binary metal M-X and aforementioned binary metal oxide MOy-XOy, wherein y is less than 3, and M and X is selected from chromium (Cr), manganese (Mn), iron (Fe), cobalt (Co), nickel (Ni), copper (Cu), gallium (Ga), niobium (Nb), molybdenum (Mo), ruthenium (Ru), rhodium (Rh), palladium (Pd), silver (Ag), indium (In), tin (Sn), praseodymium (Pr), terbium (Tb), tungsten (W), iridium (Ir), platinum (Pt), gold (Au), plumbous (Pb),
One signal transducer, it is that this physical/chemical changing value is changed into an electronic signal; And
One signal processor, it receives and process this electronic signal that this signal transducer produces.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1057672A (en) * | 1990-06-29 | 1992-01-08 | 伊莱利利公司 | By increasing the electrophoretic deposition method for immobilizing biomolecules |
| TW344029B (en) * | 1997-05-02 | 1998-11-01 | Nat Science Council | Electrochemical sensor for measuring the concentration of hydrogen peroxide and precursor of hydrogen peroxide in liquid and method therefor |
| TW200528710A (en) * | 2004-02-26 | 2005-09-01 | Univ Tamkang | Method and chemical sensor for determining concentrations of hydrogen peroxide and its precusor in a solution |
| TW200639397A (en) * | 2005-05-10 | 2006-11-16 | Tse-Chuan Chou | Multi-purpose electrochemical type sensing system integrated with wireless telemetry |
| CN1871378A (en) * | 2003-07-10 | 2006-11-29 | 北卡罗来纳-查佩尔山大学 | Deposition method for nanostructure materials |
| TW200718939A (en) * | 2005-11-01 | 2007-05-16 | Dev Center Biotechnology | Method for detecting pesticides and bio-microsensor |
| CN101440511A (en) * | 2008-12-08 | 2009-05-27 | 华东师范大学 | Preparation of charged material horizontal gradient film |
-
2010
- 2010-03-19 CN CN201010147552.7A patent/CN102192938B/en not_active Expired - Fee Related
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1057672A (en) * | 1990-06-29 | 1992-01-08 | 伊莱利利公司 | By increasing the electrophoretic deposition method for immobilizing biomolecules |
| TW344029B (en) * | 1997-05-02 | 1998-11-01 | Nat Science Council | Electrochemical sensor for measuring the concentration of hydrogen peroxide and precursor of hydrogen peroxide in liquid and method therefor |
| CN1871378A (en) * | 2003-07-10 | 2006-11-29 | 北卡罗来纳-查佩尔山大学 | Deposition method for nanostructure materials |
| TW200528710A (en) * | 2004-02-26 | 2005-09-01 | Univ Tamkang | Method and chemical sensor for determining concentrations of hydrogen peroxide and its precusor in a solution |
| TW200639397A (en) * | 2005-05-10 | 2006-11-16 | Tse-Chuan Chou | Multi-purpose electrochemical type sensing system integrated with wireless telemetry |
| TW200718939A (en) * | 2005-11-01 | 2007-05-16 | Dev Center Biotechnology | Method for detecting pesticides and bio-microsensor |
| CN101440511A (en) * | 2008-12-08 | 2009-05-27 | 华东师范大学 | Preparation of charged material horizontal gradient film |
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