CN106957887A

CN106957887A - Improved barrier film for enzyme vivo sensing device

Info

Publication number: CN106957887A
Application number: CN201710256511.3A
Authority: CN
Inventors: A.施泰布; M.蒂勒; K-H.克尔克; E.里格; A.利希特
Original assignee: F Hoffmann La Roche AG
Current assignee: F Hoffmann La Roche AG
Priority date: 2012-03-27
Filing date: 2013-03-27
Publication date: 2017-07-18
Also published as: CA2867766A1; CN104334740B; RU2014142913A; CA2867766C; JP6374860B2; CN104334740A; RU2611038C2; WO2013144255A1; JP2015515305A

Abstract

The present invention relates to the electrode system of the analyte concentration under the conditions of for measuring in vivo, it includes the electrode of enzyme molecule with fixation and improved diffusion barrier, and the diffusion barrier controls diffusion of the analyte from the body fluid around the electrode system to the enzyme molecule.

Description

Improved barrier film for enzyme vivo sensing device

The application is the divisional application of following application：The applying date：On March 27th, 2013；Application number： 201380027747.X(PCT/EP2013/056619)；Denomination of invention：Ibid.

Invention field

The present invention relates to for measure in vivo under the conditions of analyte concentration electrode system, it include with fixation enzyme The electrode of molecule, and the improved diffusion barrier for controlling the analyte to be spread from the body fluid around the electrode system to enzyme molecule.

In addition, the present invention relates to for measure in vivo under the conditions of analyte concentration electrode system, it include have consolidate The electrode of fixed enzyme molecule, the diffusion barrier for optionally controlling the analyte to be spread from the outside of electrode system to enzyme molecule, and Form the improved spacer film of at least a portion outer layer of electrode system.

Background technology

Sensor with implantable or pluggable electrode system is conducive to measuring physiologically meaningful in patient's body Analyte, such as lactic acid or glucose.The conductive enzyme layer of working electrode of this kind of system, is combined with enzyme wherein Molecule, it discharges charge carrier by catalyzed conversion analyte molecule.In this process, electric current, institute are produced as measurement signal The amplitude for stating signal is relevant with analyte concentration.

Such electrode system is for example known from WO 2007/147475 and WO 2010/028708, and its content is led to Cross and be incorporated herein by reference.

The working electrode of the electrode system is provided with diffusion barrier, and it controls analyte to be determined from around the electrode The body fluid of system organizes diffusion to the enzyme molecule being fixed in enzyme layer.According to WO 2010/028708, the electrode system Diffusion barrier is the solid solution of at least two different polymer, the solid solution of preferred acrylate.The polymer can be altogether The copolymer or butyl methacrylate and methacrylic acid of polymers, such as methyl methacrylate and hydroxyethyl methacrylate The copolymer of hydroxyl ethyl ester.

WO 2007/147475 discloses the diffusion barrier being made up of the polymer with amphion structure.It is such poly- One example of compound is poly- (2- methylacryoyloxyethyl phosphocholine -co-s n-BMA).The amphion Polymer can be mixed with another polymer (such as polyurethane).

But, the shortcoming using polymer or the mixture of copolymer is that the preparation of the mixture is applied to sensing with it Device is numerous and diverse and may be problematic.Generally, polymer to be mixed is dissolved respectively, then by the solution formed to The ratio mixing of prestige.But, this may result in one of described component precipitation, so as to cause processability problems, such as in spraying During.When the mixture includes the polymer with ion characteristic, i.e. there is the moon when one of polymer to be mixed is included During the monomer of ion or cation group, bigger difficulty can be produced.But, the presence of such charged group has to dissolubility There is strong influence, making it difficult to find suitable for electropolymer and without the solvent of both electric polymers.

WO 2006/058779 discloses a kind of sensor based on enzyme, and it has that combines to include at least one polymer The diffusion of material and enzyme layer, and particle carries enzyme, the wherein particle is dispersed in the polymeric material.The polymer can be included Hydrophily and hydrophobic polymer chains sequence, such as polymer can be high or low water imbibition polyether-polyurethane copolymers. Do not disclose and use the block copolymer with least one hydrophilic block and at least one hydrophobic block as diffusion layer.

EP-A-2163190 describes a kind of electrode system for being used to measure analyte concentration in vivo, and it, which is included, has conductance The counterelectrode (counterelectrode) of body, and with the electric conductor for being disposed with the enzyme layer comprising fixed enzyme molecule thereon Working electrode.Diffusion barrier controls diffusion of the analyte from ambient body fluid to enzyme molecule.The diffusion barrier can include hydrophiling Polyurethane, it can (it can be polyethylene glycol by 4,4'- methylene-bis- (cyclohexyl isocyanates) and diol mixture And polypropylene glycol) polycondensation obtain.Hydrophilic polyurethane layer can covered with spacer, such as butyl methacrylate with The copolymer of 2- methylacryoyloxyethyls-phosphorylcholine.Do not disclose using with least one hydrophilic block and extremely A kind of few block copolymer of hydrophobic block is used as diffusion layer.Do not disclose using comprising more than 50 mol-% hydrophily lists yet The hydrophilic copolymers of (methyl) acrylic monomers of body.

Summary of the invention

It is an object of the invention to provide diffusion barrier on the electrode system of enzyme (enzymatic) vivo sensing device, it is carried Desired physicochemical property has been supplied, and it may be easily manufactured.

This purpose is by providing by single with least one hydrophilic block and at least one hydrophobic block Diffusion barrier that block copolymer is constituted reaches.The hydrophily and hydrophobic block are covalently attached each other.It is preferred that should Block is (methyl) acrylate polymer block.

The diffusion barrier based on block copolymer provides following excellent physicochemical property：

(i) diffusion barrier for analyte to be determined permeability,

(ii) Penetration Signature of the diffusion barrier, its acts and efforts for expediency (wetability) for being suitable to the electrode and long-term action (sensor Drift),

(iii) mechanical flexibility of the diffusion barrier, it allows multielectrode vivo sensing device of the manufacture with extension；

(iv) effective introducing of the ionic group into the diffusion layer, i.e. can effectively adjust the cation or the moon in polymer The density of ionic charge, this is with the repulsion or attraction of charged analyte and/or to cell adherence (such as to coming from body around The monocyte of liquid or tissue) control it is relevant.

Subject of the present invention be for measure in vivo under the conditions of analyte concentration electrode system, it include have fixation Enzyme molecule electrode, and diffusion barrier from the electrode system to enzyme molecule that spread outside of control analyte is characterised by this Diffusion barrier includes the block copolymer with least one hydrophilic block and at least one hydrophobic block.

Preferably, the diffusion barrier is comprising single, i.e. only a kind of to have at least one hydrophilic block and at least one Plant the block copolymer of hydrophobic block, i.e. in the absence of other polymer or copolymer.It is highly preferred that the diffusion barrier by Single block copolymer with least one hydrophilic block and at least one hydrophobic block is constituted.

The electrode system of the present invention is adapted for insertion into or implanted, such as in mammalian body such as human body.The electrode system System in desired analyte, such as extracellular space (small―gap suture), in blood or drenches suitable for measurement body fluid and/or body tissue Desired analyte in hand shaft or in intercellular space.

The electrode system being inserted or implanted into is suitable to short application use, and such as 3-14 days, or prolonged application, such as 6-12 was individual Month.During during this is inserted or implanted into, desired analyte can be determined by continuously or discontinuously measuring.

The electrode system of the present invention is preferably the part of enzyme nonfluid (ENF) sensor, wherein the enzyme for determining analyte turns Change.It is preferred that the sensor includes working electrode, it has the enzyme molecule of the fixation for transformation assay thing, and the conversion causes to produce Electric signal.The enzyme may reside in the layer for covering the electrode.Furthermore, it is possible to there is redox mediator and/or electro-catalysis Agent and conductive particle and pore former.Such electrode description is in such as WO 2007/147475, and its content is by drawing With being incorporated herein.

The region of working electrode is the sensitizing range of sensor.The sensitizing range is provided with diffusion barrier, and it is controlled point Analysis thing from it is outside for example around the electrode system body fluid and/or organize diffusion to enzyme molecule.The diffusion barrier can be such as It is the coating for covering enzyme layer, i.e. enzyme-free layer.But, it is also feasible that diffusion control particle is incorporated into the enzyme layer Serve as diffusion barrier.The hole of such as enzyme layer can be filled with the polymer of control analyte molecule diffusion.The diffusion barrier Typically about 2-20 μm of thickness, e.g., from about 2-15 μm, or about 5-20 μm, particularly from about 5-10 μm or about 10-15 μm (is being dried shape Under state).

The diffusion barrier of the electrode system of the present invention includes block copolymer, preferably with least one hydrophilic block and The single block copolymer of at least one hydrophobic block.The block copolymer can include the block of alternate sequence, i.e. hydrophilic Property block is connected on hydrophobic block.The hydrophily and hydrophobic block are covalently attached each other in polymer molecule.This gathers The mean molecule quantity (by weight) of compound is typically 20-70kD, particularly 25-60kD, and more particularly 30-50kD.This is embedding The mol ratio typically about 75% (hydrophily) of hydrophily and hydrophobic parts in section copolymer：25% (hydrophobicity)-about 25% (parent It is aqueous)：75% (hydrophobicity), about 65% (hydrophily)：35% (hydrophobicity)-about 35% (hydrophily)：65% (hydrophobicity) or about 60% (hydrophily)：40% (hydrophobicity)-about 40% (hydrophily)：In the range of 60% (hydrophobicity).

The hydrophilic block of the block copolymer is by least 90%, at least 95% and particularly completely by hydrophilic monomer unit Composition.Its length is typically 50-400, for example 50-200, or 150-300, particularly 100-150, or 200-250 monomer Molecule.The hydrophobic block of the copolymer is by least 90%, more particularly at least 95% and even more particularly completely by hydrophobicity Monomeric unit is constituted.Its length is typically 50-300, such as 50-200 or 150-250, particularly 80-150 or 170-200 Individual monomeric unit.

The hydrophilic block and/or hydrophobic block are preferably made up of the unit based on (methyl) acrylic acid.More preferably should Both hydrophilic block and hydrophobic block are all made up of the monomeric unit based on (methyl) acrylic acid.

The hydrophilic monomer unit of the hydrophilic block is preferably selected from hydrophily (methyl) acrylate, i.e. in the alcohol of ester There is polar group (that is, OH, OCH in part₃Or OC₂H₅) ester, with acid amides (NH₂) or N- alkyl-or N, N- dialkyl group acyl Hydrophily (methyl) acrylamide of amine groups, the wherein alkyl include 1-3 C atom and optional hydrophilic radical, such as OH、OCH₃Or OC₂H₅, and suitably have charged group, such as anion or cation group, (methyl) acrylic acid units, Such as acrylic acid (acrylate) or methacrylic acid (methacrylate).In addition it is possible to use the combination of monomeric unit.

Instantiation for the preferred monomeric unit of the hydrophilic block is selected from：

Acrylic acid 2- hydroxyl ethyl esters,

HEMA (HEMA),

Acrylic acid 2- methoxy acrylates,

Methacrylic acid 2- methoxy acrylates,

Acrylic acid 2- ethoxy ethyl esters,

Methacrylic acid 2- ethoxy ethyl esters,

Acrylic acid 2- or 3- hydroxypropyl acrylate,

Methacrylic acid 2- or 3- hydroxypropyl acrylate (2- or 3-HPMA),

Acrylic acid 2- or 3- methoxyl group propyl ester,

Methacrylic acid 2- or 3- methoxyl group propyl ester,

Acrylic acid 2- or 3- ethyoxyl propyl ester,

Methacrylic acid 2- or 3- ethyoxyl propyl ester,

Acrylic acid 1- or 2- glyceride,

Methacrylic acid 1- or 2- glyceride,

Acrylamide,

Methacrylamide,

N- alkyl-or N, N- dialkylacrylamides, and

N- alkyl-or N, N- dialkyl methyl acid amides, wherein alkyl include 1-3 C atom, such as methyl, ethyl or propyl group,

Acrylic acid (acrylate),

Methacrylic acid (methacrylate) and combinations thereof.

It is preferred that hydrophilic monomer be HEMA (HEMA) and/or methacrylic acid 2- or 3- hydroxypropyl acrylate (2- or 3-HPMA).It is highly preferred that the hydrophilic block is made up of at least two different hydrophilic monomer units.For example it can To be the random copolymer of at least two different hydrophilic monomer units (such as HEMA and 2-HPMA).

In order to which ionic group is introduced into monomer, can charged monomeric units, such as acrylic acid (acrylate) and/or Methacrylic acid (methacrylate), is incorporated in the hydrophilic block.Therefore, in one embodiment of the invention In, the hydrophilic block can by least one non-ionic hydrophilic monomeric unit (for example, as described above) and at least one from Sub- hydrophilic monomer unit is made, and wherein the ionic monomer units exist with preferred 1-20mol% mole.It is embedding in hydrophily In the case that section is comprising ionic monomer units (such as acrylic or methacrylic acid), and selected from (methyl) acrylamide, especially It is N, the copolymerization of the hydrophilic monomer of N- dialkylacrylamides or N, N- dialkyl methacrylamides is preferred.

The hydrophobic monomer units of the hydrophobic block be preferably selected from hydrophobic acrylic acid and/or methacrylic acid unit, The monomeric unit of styrene-based or its combination.Preferably, the hydrophobic monomer units are selected from hydrophobicity (methyl) acrylate, Ester for example with the alcohol part containing 1-3 C atom but without hydrophilic radical.Monomeric unit for the hydrophobic block Instantiation is selected from：

Methyl acrylate,

Methyl methacrylate (MMA),

Ethyl acrylate,

EMA (EMA),

Acrylic acid just or isopropyl ester,

Methacrylic acid just or isopropyl ester,

N-butyl acrylate,

N-BMA (BUMA),

Acrylic acid peopentyl ester,

Methacrylic acid peopentyl ester and combinations thereof.

The hydrophobic block preferably comprises at least two kinds of different hydrophobic monomer units, and it is for example as random copolymer And exist.In a preferred embodiment, the hydrophobic block includes methyl methacrylate (MMA) and methacrylic acid N-butyl (BUMA).In an especially preferred embodiment, the hydrophobic block is MMA and BUMA random copolymer. Mol ratio between MMA and BUMA is preferably about 60% (MMA)：40% (BUMA)-about 40% (MMA)：60% (BUMA), e.g., from about 50%(MMA)：50%(BUMA).The glass transition temperature of the hydrophobic block is preferably 100 DEG C or lower, 90 DEG C or lower or 80 DEG C or it is lower, e.g., from about 40-80 DEG C.In an alternative embodiment, the hydrophobic block can be by styrene units Composition, for example, be made up of the polystyrene that glass transition temperature is about 95 DEG C.

The block copolymer used in the present invention can manufacture (B ker et al., Macromolecules according to known method 34 (2001), 7477-7488).

The block copolymer can be applied on electrode system by routine techniques, such as by providing the block copolymer Solution in suitable solvent or solvent mixture (such as organic solvent, such as ether), applies the solution to prefabricated electricity Electrode systems are simultaneously dried thereon.

When the block copolymer is contacted with water, it is in 37 DEG C of temperature and 7.4 pH (water-bearing phosphate salt buffer 10mM KH₂PO₄, 10mM NaH₂PO₄With 147mM NaCI) under show preferably from about 15 weight %-30 weight % (be based on polymer dry weight) Water absorption rate (water uptake).

In addition to the block copolymer, the diffusion barrier can also include other component, particularly non-polymeric component, It can disperse and/or be dissolved in the polymer.These other compounds include the increasing of plasticizer, particularly bio-compatible Mould agent, such as tri trimellitate-(2- ethylhexyls) ester and/or glycerine.

The diffusion barrier of the present invention has the high effective diffusion cofficient D to glucose_eff, in 37 DEG C of temperature and 7.4 pH Under, it is preferably >=10^-10cm²/ s, more preferably >=510^-10cm²/ s, and even more preferably >=10^-9 cm²/ s, such as up to 10^-7 Or 10^-8 cm²/s.Effective diffusion cofficient is preferably determined according to below equation as described in Example 4：

D_eff=SE_m/F·L_m·5182∙10^-8

Wherein SE_mIt is the sensitivity of working electrode, F is the area of working electrode, and L_mIt is the thickness of diffusion barrier.SE_mAnd L_mCan Determined with as described embodiments.

The electrode system of the present invention measures the dense of analyte under the conditions of being suitable in vivo (that is, when being inserted or implanted into internal) Degree.The analyte can be any molecule or ion being present in tissue or body fluid, for example oxygen, carbon dioxide, salt (sun from Son and/or anion), fat or fatty ingredient, carbohydrate or carbohydrate ingredient, albumen or protein component or its The biomolecule of its type.Particularly preferably determine the analysis that can be effectively shifted between body fluid, such as blood, and tissue Thing, such as oxygen, carbon dioxide, sodium cation, cl anion, glucose, urea, glycerine, Lactic acid and Pyruvic acid.

The electrode system includes the enzyme being fixed on electrode.The enzyme is suitable to determine desired analyte.It is preferred that the enzyme can The electric signal that catalyzed conversion analyte and thus generation can be detected by the electric conductor of working electrode.Enzyme for measuring analyte Preferably oxidizing ferment, such as glucose oxidase or LO or dehydrogenase, such as glucose dehydrogenase or lactic dehydrogenase Enzyme.In addition to enzymes, the enzyme layer can also include elctro-catalyst or redox mediator, and it is conducive to electronics to working electrode The transfer of conductive component (such as graphite particle).Suitable elctro-catalyst is metal oxide (such as manganese dioxide) or organic gold Belong to compound (such as Cobalt Phthalocyanine).In a preferred embodiment, the redox mediator can degrade hydrogen peroxide, by This offsets the oxygen consumption around working electrode.In a different embodiment, redox mediator can be covalently bound to On enzyme, and it is achieved in the Direct electron transfer to working electrode.Suitable redox mediator for Direct electron transfer It is prothetic group (prosthetic group), such as PQQ (PQQ), flavin adenine dinucleotide (FAD) (FAD) or other Known prothetic group.The enzyme being fixed on electrode is for example described in WO 2007/147475, and its content is incorporated herein by reference.

One preferred embodiment of the electrode system include with electric conductor counterelectrode and with electric conductor (thereon Enzyme layer and diffusion barrier are set) working electrode.The enzyme layer is preferably designed for the form of multiple pieces (field), and it is separated by Certain distance, at least for example, at least 0.3 mm or 0.5 mm, is arranged on the conductor of working electrode.Each piece of the working electrode can To form each a series of working electrode.Between these blocks, the conductor of the working electrode can be covered by insulating barrier.Pass through On the top that the block of enzyme layer is arranged on to the opening of electric insulation layer, signal to noise ratio can be improved.Such setting is disclosed in WO In 2010/028708, its content is incorporated herein by reference.

The electrode system of the present invention can be additionally comprised can supply the reference electrode of reference potential for working electrode, for example Ag/Ag-Cl reference electrodes.In addition, the electrode system of the present invention can have other counterelectrode and/or working electrode.

The electrode system can be the part of sensor, such as by being connected to voltage-stablizer and for amplifying the electrode system Measurement signal amplifier.The sensor is preferably enzyme nonfluid (ENF) sensor, more preferably electrochemistry ENF sensors.Should The electrode of electrode system can be arranged in the substrate for carrying voltage-stablizer or be attached on the circuit board for carrying voltage-stablizer.

Another theme of the present invention is related to the block with least one hydrophilic block and at least one hydrophobic block Copolymer as the diffusion barrier of enzyme electrode purposes.The block copolymer preferably as described above, such as single block copolymer. The diffusion barrier and enzyme electrode are it is also preferred that as described above.

Brief description of the drawings

The other details and advantage of the present invention is based on exemplary and is explained with reference to the accompanying drawings.

Fig. 1 shows an exemplary of the electrode system of the present invention.

Fig. 2 shows Fig. 1 partial enlarged view.

Fig. 3 shows Fig. 1 another partial enlarged view.

Fig. 4 is shown along Fig. 2 section line CC section.

Fig. 5 shows four kinds of glucose sensors for being provided with different block polymers (C, F, D, B) as barrier layer The sensitivity (and standard deviation) of (in 10mM glucose).

Fig. 6 shows four kinds of glucose sensors for being provided with different block polymers (A, C, D, F) as barrier layer Sensor drift.

Fig. 7 shows block copolymer A curve (2 experiment) of the electrical conductivity dependent on the time.

Fig. 8 shows block copolymer F curve (3 experiment) of the electrical conductivity dependent on the time.

Fig. 9 is shown for the thickness respectively for 2.77 μm or 4.43 μm, when block copolymer H electrical conductivity is depended on Between curve.

Figure 10 shows the fibrinogen relative to uncoated plate (blank) in vitro to different isolation membrane polymers The adhesion of (Adapt and Eudragit E100).

Figure 11 show with barrier film (Adapt and Lipidure CM5206) coating or it is uncoated (tester= Untreated cell) sensor be incubated after expression of the THP-1 cells to surface protein CD54.

Figure 12 a and 12b are shown with being coated or uncoated (right with barrier film (Adapt and Lipidure CM5206) According to thing=untreated cell) sensor be incubated after the secretion of THP-1 cells respectively to cell factor IL-8 and MCP-1.

Figure 13 show with barrier film (Adapt, Lipidure CM5206 and Eudragit E100) coating or Uncoated (Polyst.) and with extra fibrinogen layer tissue culturing plate be incubated after the THP-1 cell by cell factors IL-8 secretion.

Figure 14 shown compared with the isolation polymer Adapt without sensor, with barrier film (Adapt and Lipidure CM5206) coating or uncoated sensor be incubated after haemolysis (negative control thing=be only incubated medium；It is positive The osmotic lysis of tester=100%).

Show to be described in detail

Fig. 1 shows an exemplary of electrode system, and it is used to insert in the body tissue of human or animal, for example, insert Enter in corium or subcutaneus adipose tissue.The partial enlarged drawing that B is shown in A partial enlarged drawing, Fig. 3 is shown in Fig. 2.Fig. 4 Show the corresponding sectional view along the section line CC in Fig. 2.

Shown electrode system has working electrode 1, counterelectrode 2 and reference electrode 3.Electric conductor 1a, 2a, 3a of electrode are with gold Belong to conductor path, preferably by palladium or the path that is made of gold, form be arranged in substrate 4.In shown exemplary In, substrate 4 is flexible plastic sheet, for example, be made up of polyester.The thickness of substrate 4 be less than 0.5mm, for example, 100-300 microns, and because This is readily bent so that its movement for being adaptable to surrounding body tissue after insertion thereof.Substrate 4, which has, to be used to insert patient's body group Narrow bar (narrow shaft) in knitting and the wide head for being connected in the electronic system of setting in vitro.The bar of substrate 4 is excellent Elect at least 1cm length, particularly 2cm-5cm as.

In the illustrated exemplary embodiment, a part for facility is measured, i.e. the head of substrate, in use Stretched from patient's body.Or, it is also possible that whole test facilities are implanted into and wirelessly measurement data are passed The defeated receiver to setting in vitro.

Working electrode 1 carries enzyme layer 5, and it includes the enzyme molecule of the fixation for catalyzed conversion analyte.Enzyme layer 5 can example Such as applied in the form of carbon particle, polymer adhesive, redox mediator or elctro-catalyst and the solidification pastel of enzyme molecule Plus.The details for producing such enzyme layer 5 is disclosed in such as WO 2007/147475, and it is in this context as ginseng Examine.

In the illustrated exemplary embodiment, enzyme layer 5 is continuously applied on the conductor 1a of working electrode 1, and It is to be applied in the form of single piece be intervally installed.Single piece of enzyme layer 5 in the illustrated exemplary embodiment It is in a series of arrangements to be.

The conductor 1a of working electrode 1 has narrow positions between enzyme layer block, and it is especially clear visible in fig. 2.Anti- electricity The conductor 2a of pole 2 has the profile of the route (course) for the conductor 1a for following working electrode 1.This means generate The insertion or arranged in a crossed manner of the working electrode 1 and counterelectrode 2 of short current path and low current density sharply.

In order to increase its active surface, counterelectrode 2 can have porous conductive layer 6, and it is located at anti-in the form of single piece On the conductor 2a of electrode 2.Similar to the enzyme layer 5 of working electrode 1, this layer 6 can be with the solidification of carbon particle and polymer adhesive The form of pastel applies.The block of layer 6 preferably has the block identical size with enzyme layer 5, although this is not essential.But That the measure on increase counterelectrode surface may also be precognition, and counterelectrode 2 be also designed to without any types coating, Or the linear conductor path with the coating that is made up of the block copolymer and optional spacer.

Reference electrode 3 is arranged between the conductor 1a of working electrode 1 and the conductor 2a of counterelectrode 2.Reference shown in Fig. 3 Electrode is made up of conductor 3a, and the block 3b of silver/silver chlorate pastel of conduction is provided with conductor 3a.

Fig. 4 shows the schematic sectional view along the section line CC in Fig. 2.Section line CC passes through the enzyme layer of working electrode 1 Between the block of the conductive layer 6 of one of block 5 and counterelectrode 2.Between the block of enzyme layer 5, the conductor 1a of working electrode 1 can be covered There is electric insulation layer 7, as the conductor 2a of the counterelectrode 2 between the block of conductive layer 6, so that prevent otherwise may be by conductor road The disturbing reaction of footpath 1a, 2a metal catalytic.The block of enzyme layer 5 is therefore in the opening of insulating barrier 7.Equally, counterelectrode 2 is led The block of electric layer 6 can also be placed on the opening of insulating barrier 7.

Enzyme layer 5 is coated to cap rock 8 and covered, and it provides diffusional resistance for analyte to be measured, therefore act as diffuser screen Barrier.The diffusion barrier 8 is made up of the above-mentioned single copolymer with alternate hydrophily and hydrophobic block.

The useful thickness of coating 8 is, for example, 3-30 μm, particularly from about 5-10 μm or about 10-15 μm.Because its diffusion Resistance, coating 8 causes analyte molecule less in each unit interval to reach enzyme layer 5.Therefore coating 8 reduces analysis The conversion rate of thing molecule, and thus counteract the exhaustion of the analyte concentration around working electrode.

Coating 8 continuously substantially extends on the conductor 1a of working electrode 1 whole area.It is biological on coating 8 Compatibility film can be set as spacer 9, and it establishes minimum range between enzyme layer 5 and the cell of surrounding body tissue.This Mode advantageously generates the memory for analyte molecule, the fluid communication (fluid around enzyme layer block 5 Exchange in the case of) occurring of short duration disturbance, analyte molecule can reach corresponding enzyme layer block 5 from the memory.If Body fluid around electrode system, which is exchanged, temporarily to be limited or is even prevented from, and the analyte molecule being stored in spacer 9 continues The enzyme layer 5 of working electrode 1 is diffused into, they are converted herein.Spacer 9 is hence in so that only during the significantly longer time The obvious exhaustion of analyte concentration and corresponding measurement result distortion just occur afterwards.In example shown embodiment, shape Film into spacer 9 also cover counterelectrode 2 and reference electrode 3.

Barrier film 9 may, for example, be dialysis membrane.In this context, dialysis membrane is understood to be greater than maximum sized The impervious film of molecule.The dialysis membrane can be prefabricated in single manufacture method, then can be made in the electrode system During apply.Select the full-size for the molecule that the dialysis membrane can pass through so that analyte molecule can pass through, and more Big molecule is trapped.

Or, instead of dialysis membrane, by the coating that there is the polymer of high osmosis to be made to analyte and water, for example based on The coating of polyurethane or acrylate, can be applied on the electrode system as barrier film 9.

It is preferred that the spacer is made up of the copolymer of (methyl) acrylate.It is preferred that the barrier film is at least 2 or 3 kind The copolymer of (methyl) acrylate.More preferably the barrier film, which is included, is more than 50mol%, at least 60mol% or at least 70mol%'s Hydrophilic monomer unit, such as HEMA and/or 2-HPMA, and at most 40mol% or most 30mol% hydrophobic unit, for example BUMA and/or MMA.The spacer can be random or block copolymer.Particularly preferred barrier film includes MMA or BUMA conducts Hydrophobic parts and 2-HEMA and/or 2-HPMA are used as hydrophilic parts.Preferably, hydrophilic monomer HEMA and/or HPMA Measure as 80 mol-% to 85 mol-%, and hydrophobic components MMA and/or BUMA amount are 15 mol-% to 20 mol-%.

The present invention highly preferred barrier film by copolymer A dapt (BioInteractions Ltd, Reading, England) it is made.Adapt includes the BUMA as the hydrophobic parts and 2-HEMA as hydrophilic parts And 2-HPMA, wherein the amount of the 2-HEMA hydrophilic monomers is about 80 mol-%.

The barrier film is high osmosis to analyte, i.e. it significantly reduces the spirit of each area working electrode really Sensitivity, such as 20% or lower, or 5% or lower, and thickness degree is less than about 20 μm, preferably less than about 5 μm.It is particularly preferred every It is about 3 μm of about 1- from film thickness.

The enzyme layer 5 of the electrode system can include metal oxide particle, and preferred manganese dioxide particle is used as catalysis oxidation Reducing medium.Manganese dioxide catalyzed conversion hydrogen peroxide, it is for example by glucose and the oxydasis of other biological analytes Formed.During hydrogen peroxide degradation, manganese dioxide particle transfers an electron to the conductive component such as enzyme of working electrode 1 Graphite particle in layer 5.The catalytic degradation of hydrogen peroxide counteracts any reduction of oxygen concentration in enzyme layer 5.Advantageously, this causes The conversion of analyte to be measured can not be limited by local oxygen concentration in enzyme layer 5.The use of catalytic oxidation-reduction medium because This counteract due to oxygen concentration it is low caused by measurement result distortion.Another advantage of catalytic oxidation-reduction medium is that it is prevented Produce the hydrogen peroxide of cytoclasis concentration.

Preferred isolation membrane polymer described herein may be used as the external coating of the diffusion barrier of the present invention, but can also External coating as general electrode system, particularly for measure in vivo under the conditions of analyte concentration electrode system outer painting Layer, the system includes the electrode with fixed enzyme molecule and diffusion barrier, and the barrier controls analyte from the electrode system The outside diffusion to the enzyme molecule of system.Therefore, barrier film can be arranged in diffusion barrier, still, and barrier film can be with It is set directly in enzyme layer.In the case where this is last, barrier film can also serve as diffusion barrier in itself and slow down analyte Diffusion from molecule to enzyme layer.

When the present invention electrode system be inserted into or implant when, barrier film be implantation sensor and ambient body fluid or Interface between tissue.Therefore, when in body fluid or tissue, barrier film of the invention must be it is mechanically firm, So that it is both indeformable or without departing from sensor.Therefore, barrier film copolymer water absorption rate and the adjoint of the copolymer are swelled It must limit, although the copolymer has intrinsic hydrophily.

Total speed of copolymer is preferably based on, the relative water-intake rate of barrier film copolymer should be no more than 50 weight %, It is preferred that 40 weight %, more preferably 30 weight %.In the present case, the measurement of relative water-intake rate is by that will dry copolymer Excessive phosphate buffer (pH 7.4) is subjected at a temperature of 37 DEG C to continue 48 h and carry out.Relative water-intake rate (WU%) It is preferred that being determined according to below equation：

WU% = (m₂-m₁)/m₁ x 100,

Wherein m₁And m₂The quality of the copolymer after the drying copolymer and aquation according to above-mentioned measuring condition is represented respectively.

Present inventor determined that, absorbed by the copolymer A dapt preferred barrier films being made at 37 DEG C through 48 h Relative to 33 ± 1.8 weight % of its own weight phosphate buffer (pH 7.4).Under the same conditions, polymer Lipidure CM5206 (NOF Corporation, Japan) film is absorbed relative to the 157 ± 9.7 of its own weight Weight % phosphate buffer.The relatively low water absorption of polymer advantageously increases the mechanical stability of the barrier film of the present invention. By contrast, Lipidure CM5206 are shown compared with high water absorbing capacity, and are swelled as water that is more fragile, easily deformable or leaving Gel, particularly when being applied on the electrode system of enzyme vivo sensing device.

In addition, during inserting and implementing electrode system, sept and tissue and/or body fluid, such as interstitial fluid or Blood etc. (containing biomolecule such as albumen) and cell are directly contacted.Preferably, the barrier film must protective tissue and/or body The sensor for inserting and being implanted into pendular ring border, and therefore minimize the tissue reaction of body and implant.In fact, body pin The reaction for being implanted into material is referred to as " external precursor reactant " (FBR).By FBR, body attempts to destroy implant, or, if It is impossible, then generates capsule to separate it with surrounding tissue (allochthon granuloma).The first step of FBR reactions It is the table that albumen (for example, fibrinogen, albumin, immunoglobulin, complement) is bound to the former material (i.e. implant) On face.Binding site is in the acceptor being handed on immunocyte by albumen coating.For example, fibrinogen, which contains, is bound to monokaryon Cell receptor MAC-1 structural motif.When fibrinogen is bound to the surface of implant, it changes its conformation and exposure pair In MAC-1 binding site.Therefore, immunocyte, such as monocyte, are recruited to implant, and are activated, secretase and from By base to attack implant.In addition, immunocyte secretes soluble factor, i.e. cell factor, it is other immune to recruit and activate Cell, and thus expand immune response.If implant can not be removed, fibrous capsule is formed by phoirocyte and albumen. But, the capsule is the diffusion barrier that analyte reaches sensor.In a word, the event of external precursor reactant as described above may interfere with Internal electrode system function and its life-span.

Therefore, the improved barrier film on the electrode system of enzyme vivo sensing device further provides tissue to implant The reduction of reaction, and suppress the formation of capsule for separating sensor with surrounding tissue and body fluid.

Therefore, the electrode system of the analyte concentration under the conditions of another object of the present invention is to provide for measuring in vivo, its Including with fixed enzyme molecule and the preferably electrode of diffusion barrier, the diffusion barrier controls analyte from the electrode system The outside diffusion to the enzyme molecule, is characterised by that barrier film forms at least a portion of the outer layer of the electrode system, wherein should Hydrophilic copolymers of the barrier film comprising acrylic acid and/or methacrylic acid monomer, wherein the polymer, which is included, is more than 50mol% Hydrophilic monomer.

As described above, the barrier film of the present invention has limited protein binding capacity really, to protect the electrode of sensor System may then trigger the response of immunocyte and may limit or disturb it to show in vivo from protein adsorption.The He of embodiment 5 The preferred barrier film of the 6 display present invention provides the weak binding with fibrinogen, and prevents that the conformation of fibrinogen from becoming Change, the conformation change will cause exposure of the MAC-1 binding motifs to monocyte.Advantageously, the barrier film copolymer material Material will not activating immune cell in itself.In embodiment 6, may indicate that the barrier film copolymer of the present invention can weaken implantation Activation of the sensor to immunocyte.Advantageously, moreover, barrier film is the material of bio-compatible, especially and body fluid, such as blood Liquid, it is compatible.Embodiment 7 shows that barrier film copolymer of the invention is prevented from the haemolysis and complement activation of the sensor of implantation. Therefore, barrier film of the invention advantageously not only shows high mechanical stability, and with optimal biocompatibility, this is It is surprising, because there is low water absorption when moistened.

The feature of this embodiment, particularly in terms of the structure, analyte and enzyme molecule of electrode system, such as this paper institutes State.The diffusion barrier is preferably as described herein, but it can also have different composition or can be not present.It is excellent according to one The embodiment of choosing, diffusion barrier preferably includes have at least one hydrophilic block and at least one hydrophobicity embedding as described above The block copolymer of section.

According to a further preferred embodiment, diffusion barrier includes hydrophilic polyurethane.It is used as diffusion barrier Hydrophilic polyurethane can by (poly-) diisocyanate, preferably 4-4- methylene-bis- (cyclohexyl isocyanates) and polyalcohol, It is prepared by the addition polymerization (polyaddition) of preferred diol mixture.

The component of diol mixture is preferably PAG, such as polyethylene glycol (PEG) and polypropylene glycol (PPG) and Aliphatic diol, such as ethylene glycol.Preferably, the hydrophilic polyurethane includes 45-55 mol-%, preferably 50 mol-% isocyanides Acid esters and 25-35 mol-%, preferably 30 mol-% ethylene glycol.Then the degree of hydrophiling is adjusted by PEG and PPG ratio It is whole.Preferably, the polyurethane includes 2-3 mol-%, more preferably 2.5 mol-% PEG and 17-18 mol-%, preferably 17.5 mol-% PPG.In order to increase the hydrophily of polyurethane, PEG ratio can be increased, for example, to 4.5-5.5 mol-%, It is preferred that 5 mol-% PEG, to obtain extremely hydrophilic polyurethane.The different hydrophily variants of polyurethane can also be mixed, To optimize the characteristic of diffusion barrier.

The preferred acrylic acid and methacrylic acid monomer of the barrier film copolymer are as described herein.

The hydrophilic monomer unit is preferably selected from hydrophily (methyl) acrylate, i.e. have pole in the alcohol part of ester Property group (that is, OH, OCH₃Or OC₂H₅) ester, with acid amides (NH₂) or N- alkyl-or N, N- dialkylamide groups is hydrophilic Property (methyl) acrylamide, wherein the alkyl include 1-3 C atom and optional hydrophilic radical, such as OH, OCH₃Or OC₂H₅, and suitably have charged group, such as anion or cation group, (methyl) acrylic acid units, such as propylene Sour (acrylate) or methacrylic acid (methacrylate).In addition it is possible to use the combination of monomeric unit.

Acrylic acid 2- hydroxyl ethyl esters,

HEMA (HEMA),

Acrylic acid 2- methoxy acrylates,

Methacrylic acid 2- methoxy acrylates,

Acrylic acid 2- ethoxy ethyl esters,

Methacrylic acid 2- ethoxy ethyl esters,

Acrylic acid 2- or 3- hydroxypropyl acrylate,

Methacrylic acid 2- or 3- hydroxypropyl acrylate (2- or 3-HPMA),

Acrylic acid 2- or 3- methoxyl group propyl ester,

Methacrylic acid 2- or 3- methoxyl group propyl ester,

Acrylic acid 2- or 3- ethyoxyl propyl ester,

Methacrylic acid 2- or 3- ethyoxyl propyl ester,

Acrylic acid 1- or 2- glyceride,

Methacrylic acid 1- or 2- glyceride,

Acrylamide,

Methacrylamide,

N- alkyl-or N, N- dialkylacrylamides, and

Acrylic acid (acrylate),

Methacrylic acid (methacrylate) and combinations thereof.

It is preferred that hydrophilic monomer be HEMA (HEMA) and/or methacrylic acid 2- or 3- hydroxypropyl acrylate (2- or 3-HPMA).

The hydrophobic monomer units are preferably selected from hydrophobic acrylic acid and/or methacrylic acid unit or its combination.It is preferred that The hydrophobic monomer units are selected from hydrophobicity (methyl) acrylate, such as with containing 1-3 C atom but without hydrophilic radical Alcohol part ester.Instantiation for the monomeric unit of the hydrophobic block is selected from：

Methyl acrylate,

Methyl methacrylate (MMA),

Ethyl acrylate,

EMA (EMA),

Acrylic acid just or isopropyl ester,

Methacrylic acid just or isopropyl ester,

N-butyl acrylate,

N-BMA (BUMA),

Acrylic acid peopentyl ester,

Methacrylic acid peopentyl ester and combinations thereof.

In a preferred embodiment, the hydrophobic block includes methyl methacrylate (MMA) and metering system Sour N-butyl (BUMA).

The outer barrier film preferably covers at least part comprising enzyme molecule of the working electrode and optionally also covers it Its part, such as counterelectrode.If it does, barrier film also covers reference electrode.Barrier film preferably covers the whole of electrode system Implant surface.Barrier film preferably covers working electrode, optional counterelectrode, and reference electrode (if deposited in the form of pantostrat ).

The electrode system of improved barrier film comprising the present invention can be a part for sensor, such as by being connected to Voltage-stablizer and for the amplifier for the measurement signal for amplifying the electrode system.The sensor is preferably enzyme nonfluid (ENF) sensing Device, more preferably electrochemistry ENF sensors.The electrode of the electrode system can be arranged in the substrate for carrying voltage-stablizer or be attached to On the circuit board for carrying voltage-stablizer.Preferably, sensor is used to measure glucose.

The further theme of the present invention is related to the hydrophilic copolymers of acrylic acid and/or methacrylic acid monomer as enzyme electricity The purposes of the barrier film of pole, wherein hydrophilic copolymers, which are included, is more than 50 mol-% hydrophilic monomers.Hydrophilic copolymers are preferred As described above.Preferably, barrier film is used to make the external precursor reactant when it is inserted into or is implanted to internal for enzyme electrode (FRB) minimize.

Embodiment

Embodiment 1Enzyme nonfluid (ENF) glucose sensor with the distributed electrode for being percutaneously implantable oozes Permeability, the electrode has the diffusion layer being made up of a kind of single block copolymer.

The sensor is building up on the prefabricated palladium bar conductor structure in the polyester base of 250 μ m-thicks.By working electrode (WE) set (as shown in Figure 1-2) with counterelectrode (CE) distribution ground.

With carbon pastel double exposure CE block, the remainder of bar conductor is insulated.With the glucose oxidase (enzyme) of crosslinking, The mixture double exposure WE of conducting polymer pastel and elctro-catalyst (being manganese oxide (IV) (Technipur) herein) block. To remaining path of bar conductor minor insulation again.Reference electrode (RE) is made up of Ag/AgCl pastel.These electrodes are covered about 1cm rodmeter.

The WE blocks are coated with the block copolymer diffusion layer being made up of HEMA blocks and BUMA blocks.The thickness of this layer is 7 μ m。

Produce four sensor batches, each with specific block copolymer as diffusion layer (referring to following List).All block copolymers are all obtained from Polymer Source, Montreal, and are listed in the table below in 1.

Title	Molecular proportion/%	Monomeric unit	Molecular weight
				Copolymer	BUMA/HEMA	HEMA	Copolymer [kD]
C	73/27	92	47
				F	60/40	108	37
D	48/52	162	44
				B	62/38	169	61

By the dissolving of respective block copolymer in organic solvent (25% concentration), and with it sensor is coated.By In after band drier drying (2min, 30-50 DEG C), the sensor of the coating is carried out in the glucose solution of various concentrations Testing in vitro.In each sensor batch, 10 sensors are measured as chance sample.It is used as the amount for external sensitivity Degree, by the mathematic interpolation signal of the measurement electric current in 10mM and 0mM concentration of glucose, itself and then divided by 10mM (ginsengs See embodiment 4).

All the sensors are all worked under 350mV polarizing voltage (relative to Ag/AgCl), and the temperature of measurement is kept into permanent It is scheduled on 37 DEG C.Sensor for measurement series does not include spacer described in WO2010/028708, it is contemplated that test Signal level, it does not have any difference.Fig. 5 shows the sensor spirit under the standard deviation for four different diffusion layers Sensitivity.

On block copolymer C, D and F, in vitro between the mol ratio of sensitivity and hydrophobic block/hydrophilic block It there is clear relation.In about identical copolymer overall chain length, the sensitivity is with the increasing of the amount of hydrophilic block (HEMA) Plus and increase.

The sensor of diffusion layer with block copolymer B is an exception.Even if polymer B, which has, is similar to polymer F hydrophobicity and the relative ratios of hydrophily amount, sensitivity and thus the permeability for glucose is also reduced.Empirically may be used To say in the case of polymer B, overall chain length (molecular weight (total molecular weight) for corresponding to copolymer molecule) is so large that, with As for the permeability reduction of the layer.This can also with remaining polymer phase ratio, the weight analysis determining of block copolymer B Water absorption rate in see.Polymer B has the water absorption rate of 10.6% ± 1.5% (percentage by weight refers to polymer dry weight).It is poly- Compound C is 15.6% ± 0.0%, and polymer F is 16.5 ± 3.1%, and polymer D is 27% ± 1.7%.

Embodiment 2The machinery of the diffusion layer of ENF glucose sensorsIt is softToughness.

Sensor is manufactured as described in WO 2010/028708, but with the diffusion layer of the present invention.It is assumed that gamma transition temperature Degree (Tg) is the alternate parameter of mechanical flexibility.Furthermore, it is assumed that the glass transition temperature, it can distribute to hydrophobic block, Determine the mechanical flexibility in applying in vivo.It should be noted that a kind of block copolymer may identify several Tg, its Corresponding to the number of block.

The sensor is coated with electrode paste same as Example 1.Then, some sensors are used and is selected from MMA- HEMA (being produced by the Polymer Source in Montreal) copolymer is coated.The total molecular weight of the polymer (being referred to as E) is 41kD, MMA (hydrophobicity amount) and HEMA mol ratio are 60%：40%.The glass transition temperature of hydrophobic block is 111 DEG C, its It is to be determined by the DSC and 10 DEG C/min rate of heat addition.

In addition, other sensors are provided with block copolymer (being referred to as A) diffusion layer of the present invention.The copolymer A dredge Aqueous block includes the MMA and BUMA of the equimolar amounts in random order.Equally, hydrophobic parts and hydrophilic parts are rubbed Your ratio is 60%：40%.Molecular weight is 36 kD.The Tg of hydrophobic block is reduced to 73 DEG C, and this is attributed to MMA and BUMA, and (Tg is about 45 DEG C) random order.

Two kinds of diffusion layers are produced by solution (25%) of the respective copolymer in ether, and such as embodiment 1 Dry like that.The thickness of diffusion layer is 7 μm.Then apply separation layer via dip-coating, and in drying at room temperature 24h.The separation layer It is that the Lipidure CM5206 produced by Japanese NOF are made.

After being removed from tissue, the sensor with copolymer E diffusion layers shows sporadicly splitting in the diffusion layer Line.This is considered as the effect of mechanical load.In contrast, the sensor with copolymer A diffusion layer is under identical load Any crackle is not shown.This is substantially attributed to Tg reduction, and which raises the mechanical stability of copolymer.No longer need as The physical mixture of two kinds of copolymers disclosed in WO 2010/028708.

Embodiment 3According to the optimization of the ENF glucose sensors with distributed electrode and diffusion layer of the present invention Permeability behavior.

Sensor is manufactured as described in Example 1, but there is other separation layer on whole rodmeter.For implementing The copolymer A of example 1 and 2, C, D and F, are prepared for the sensor with respective diffusion layer.For this purpose, generating the copolymerization 24% ethereal solution of thing.Each solution is applied on one group of sensor (N=10), then dried in band drier.Thus Obtain the diffusion layer that thickness is 7 μm.

Thereafter, the separation layer described in embodiment 2 is provided to these sensors.

Sensor is connected with the measuring system on sensor head, measurement data is delivered to data storage by it.As implemented In-vitro measurements are carried out like that in example 1, but are carried out in the measurement period of 7 days.From the measurement data, in respective measurement The sensitivity drift of each sensor is calculated on cycle.Fig. 6 is shown for each sensor variant, i.e. with diffusion layer The sensor of variant, the average value of described group of external drift value.It is described to calculate the starting stage (first for eliminating measurement 6h, so-called startup stage).

For all copolymer C, D and F with hydrophobic block BUMA, there is positive drift, i.e. sensitivity according to when Between and increase.In contrast, the copolymer A of the hydrophobic block of the random copolymer with MMA and BUMA result in very low Slightly negative drift.

These differences can be responded to explain by the prolonged permeation of respective diffusion layer, and it is surveyed in other experiment .With polymer solution coating palladium sensor above (no WE pastel, but with the active surface limited, i.e. also do not have Enzyme layer, eliminates its influence of the swelling behavior for result), and the thickness of this layer is measured after the drying.Then, containing sodium Electrical conductivity is measured with the cushioning liquid of chloride.

Fig. 7 shown after short startup stage, and the electrical conductivity of copolymer A remains close to constant.

As can be seen from Fig. 8, it is really not so for copolymer F, or even under the same measuring conditions.In this feelings Under condition, it was observed that the long-term and strong permeability response of copolymer F diffusion layer, it is actually unrelated with thickness degree.For For copolymer F and copolymer C and D (not shown) with BUMA hydrophobic blocks, or even cause permeability in long-term Increase.When measuring, if diffusion layer is applied on the sensor for carrying distributed enzyme layer, which results in continuing for sensitivity Increase.This explains observed positive sensor drift.

Vice versa, and the sensor sheet with block copolymer A reveals negligible drift, and this is attributed in electrical conductivity Low-down permeability changes in measurement.But it is an immediately proceeding at and starts after measurement (about 1h after continueing to), to see in copolymer A Observe the fierce increase of electrical conductivity.Here, it was observed that very fast startup, it is terminated after about 1 hour.Now diffusion layer is complete Wetting, and terminate due to water suction its structural rearrangement.The degree of structure change probably depends on Tg.Appear to tool The copolymer for the Tg being improved has passed through restructuring, and compared with the copolymer with the Tg in ambient temperature range, it is in the time With it is limited in amplitude.

Further it is necessary to recognize that the sensor with copolymer A is shown with having copolymer F diffusion layers when measuring and starting Sensor compare suitable high sensitivity.Due to identical relative ratios between hydrophobicity and hydrophilic block, this is can be with It is expected.The range of sensitivity 1-1.5nA/mM (referring to embodiment 1) reached is considered as preferable.With by copolymer A The sensor of the diffusion layer of composition equally obtains the sensitivity.

The summation responded on three kinds of physicochemical property-permeability, mechanical stability and permeability, preferable sensor can be with Preferably obtained with the diffusion layer of block copolymer, the block copolymer, which has, carries at least two different random settings The hydrophobic block of hydrophobic monomer units, such as block copolymer A.(their hydrophobic block is only for other block copolymers Only be made up of single monomeric unit) be all not reaching to can be compared with copolymer A in all three parameter quality.

Embodiment 4The sign of block copolymer.

Many-block sensor (working electrode and counterelectrode distinguish 10 blocks) for continuously measuring glucose is produced, and And characterized in vitro.

The sensor has the diffusion layer being made up of block copolymer, and the block copolymer includes the methyl-prop of random copolymerization The hydrophobic block and HEMA (HEMA) of e pioic acid methyl ester (MMA) and n-BMA (BUMA) Hydrophilic block.These polymer (being appointed as G and H) are produced via Polymer Source, Montreal, and than real Applying the polymer A of a 1-3 has bigger permeability, and it is incorporated herein by reference.

In table 2 below, these copolymers are described：

Polymer	G	H	A
				Molecular weight Mn [kD]	23.5-b-29	21-b-20.5	21-b-15
Weight %HEMA	55.2	49.4	41.6
				Mole %HEMA (stoichiometry)	53.5	47.4	40
Mole %HEMA (passes through¹H,¹³C NMR are measured)	51	46	32.6
				Tg [DEG C] hydrophobic block	65	68	86
HEMA monomeric units	223	157	115
				MMA monomeric units	194	174	174

The molecular weight Mn of each block is illustrated respectively in upper table 2, and represents average value, because it is known that be polymerization Thing has the molecular chain length distribution near specific intermediate value.This is also applied for the derived quantity in table 2.

The glass transition temperature of the hydrophobic block of display is in desired scope, to ensure mechanical flexibility.

Diffusion barrier is that every square measure working electrode area is (i.e. several for the infiltrative conclusive parameter of analyte What area) sensitivity.For the sensor of each analysis, sensitivity S E be by phosphate buffer solution (pH7.4) 10mM and electric current (I) measured value under 0mM concentration of glucose are come what is calculated, and unit is nA/mM：

SE = [I(10 mM) - I(0 mM)]/10

From each measured value (N=8), it is determined that average sensitivity SE_m.By the Sensitirity va1ue obtained divided by many-block sensing The geometry gross area F being measured microscopically of all working electrode points on device.It thereby is achieved sensitivity density SE_m/F。

The linearity Y of external function curve is the finger of the diffusion control function of the polymer covering on working electrode Show.For the sensor of each analysis, it is calculated by the current measurement value in 20mM, 10mM and 0mM concentration of glucose, Unit is %：

Y^20mM = 50·[I(20mM) - I(0mM)]/[I(10mM) - I(0mM)]

Average linear angle value and its standard deviation is determined from these each measured values (referring to table 3).

Finally, for each polymer, the thickness L of the diffusion barrier of sensor is determined by optical measurement.Calculate With identical polymer >=the corresponding average value of the samples of 23 sensors.It is possible thereby to calculate effective diffusion of coating Coefficient D_eff, unit cm²/s：

D_eff=SE_m/F·L_m·5.182∙10^-8

Wherein SE_mAnd L_mIt is the respective average value of sensitivity and thickness, F is the gross area of all working electrode points.

During sensor drift is the in-vitro measurements by 7 days, what the concentration of glucose stage repeated calculated.Show basic The polymer H of constant electrical conductivity result is described in fig .9.

Table 3 below shows the result that function is characterized：

Polymer	G	H
			SE_m/F [nA/mM*mm²)]	1.85	1.25
Drift about [%d]	-1.5±0.2	0.3±0.1
			Y^20mM[%]	88.2±0.7	88.6±0.3
Thickness L_m [µm]	11.61	12.69
			D_eff [cm²/s]	1.11305*10^-9	8.22019*10^-10

For more hydrophilic polymer G (it is bigger for the permeability of glucose), the expansion is also determined with alternative Coefficient is dissipated, such as glucose is from the room with glucose solution by polymer film to the room with the buffer solution without glucose Infiltration.According to this method, similar diffusion coefficient value (1.1710 is obtained^-9 cm²/s)。

Embodiment 5The combination of albumen and insolated layer materials.

In order to assess the combination of albumen and insolated layer materials, by AdaptTM (Biointeractions Ltd, Reading, England) or Eudragit E100 (Evonik Industries) ethanol solution insert incubation plate (FluoroNunc Maxisorp, Thermo Scientific).Eudragit E100 are to be based on dimethylaminoethyl acrylate methyl base The cation copolymer of amino ethyl ester, butyl methacrylate and methyl methacrylate.Polymer is dried overnight at 40 DEG C. Hereafter, isolated material is covered with fibrinogen solution.The solution contains (to be purchased from fluorescent dye Alexa488 Invitrogen) the conjugated fibrinogen from human plasma.It is incubated after 4 h, vacuum fibers albumen original solution, and uses boron Phthalate buffer washing separation layer eight times.Using fluorescence reader (Synergy4, BioTek Instruments) with 485 nm Excitation wavelength and 528 nm launch wavelength measurement be incubated plate in fluorescence intensity and analyze spacer combination albumen amount. Calibration curve is prepared using the labelled protein (6.25-500 ng) of concentration known, so as to which fluorescence reading is converted into albumen Amount.

As expected, fibrinogen is bound to uncoated incubation plate (blank), the albumen (figure for causing 390 ng to combine 10).The protein binding of 60 ng reduction is shown with the Eudragit E100 plates coated.In the plate that AdaptTM is coated almost Any protein binding is not detected.Caused by reading before incubation is due to background fluorescence.These results are clearly illustrated, are used The surface of spacer material coating, the surface particularly coated with AdaptTM has been obtained well for fibrinogen adhesive Protection.

Embodiment 6The cytokine release of cell after being contacted with separation layer.

Sensor is manufactured as described in example 2 above.Afterwards, the sensor with separation layer is provided as described in example 2 above. Separation layer is made up or by AdaptTM of Lipidure CM 5206 (NOF Corporation, Japan) (Biointeractions Ltd, Reading, England) is made.

By the sensor without separation layer, with the sensor by the separation layers being made of Lipidure CM 5206 and tool There are the sensor and monokaryon THP-1 cell incubations by the AdaptTM separation layers being made, and analyze the induction of Inflammation Marker.

THP-1 cells are cultivated into 24 h at 37 DEG C in the presence of sensor.Then by the way that cell is collected by centrifugation.On Clear liquid is used for the release for determining cell factor, and cell precipitation is resuspended in into the PBS containing 1 % bovine serum albumin(BSA)s (BSA) In, and for analyzing cell surface protein CD54 (also referred to as ICAM-1, inflammatory biomarker) expression.By THP-1 cells With the anti-CD54 antibody incubation conjugated with fluorescent dye phycoerythrin (BD Bioscience).After 4 DEG C of 45 min of incubation, Cell is washed in the % BSA of PBS/ 1, and uses flow cytometer (nm of excitation wavelength 532, the nm of launch wavelength 585) (BD FACSArray, BD Bioscience) determine 10000 cells average fluorescent strength (MFI).It is thin with untreated THP-1 Born of the same parents compare, as shown in high MFI readings, cause increased relative CD54 expression (6 times of inductions) with not having cated sensor to be incubated (Figure 11).Cell is incubated with the sensor of the separation layer covering with CM 5206 or AdaptTM causes CD54 expression decay respectively 45% or 41%.

Supernatant is used for using the specification (Flex sets, BD Bioscience) according to manufacturer based on bead Immunoassay and subsequent flow cytometry (BD FACSArray, BD Bioscience) to determine cell factor white The amount of interleukin -8 (IL-8) and " monocyte chemoattractant protein-1 " (MCP-1).Use FCAP array software v1.0.1 (Soft Flow Hungary Ltd.) carry out data analysis.

Compared with untreated THP-1 cells, uncoated sensor is induction of IL-8 (49 vs.197 pg/ml) (figures 12a) with MCP-1 (6 vs.48 pg/ml) (Figure 12 b) strong release.Covered when with CM 5206 or AdaptTM separation layer During sensor, IL-8 and MCP-1 release are reduced.The sensor covered with AdaptTM induction of 100 pg/ml IL-8 and 25 pg/ml MCP-1 release.125 pg/ml IL-8 and 18 pg/ml MCP- are result in the sensors covered of CM 5206 1 secretion.

In a word, these as shown by data, the separation layer has been decayed three kinds of well-known inflammation biomarkers, i.e. CD54, IL-8 and MCP-1 induction.

In order to which analyzing proteins adsorb the effect for activating THP-1 cells, with CM 5206, AdaptTM or Eudragit E100 coats tissue culturing plate.Then spacer and human fibrinogen (Sigma-Aldrich) are incubated.By THP-1 cells It is incubated from different spacers+fibrinogen layer.After 37 DEG C of 48 h of incubation, by centrifugal sedimentation cell, and analyze The IL-8 releases of clear liquid.As control, by cell in no spacer but with fibrinogen (Polyst.=cultivates plate material) Grown in the culture plate of coating.As shown in Figure 13, these cells discharge 89 pg/ml IL-8.In CM 5206+fiber eggs The cell cultivated on white former upper or AdaptTM+fibrinogen discharges 68 or 49 pg/ml respectively.By contrast, it is grown in Cell on Eudragit E100+fibrinogen discharges 206 pg/ml IL-8.It is worth noting that, without fibrin The cell grown on the Eudragit E100 of former coating only secretes 59 pg/ml IL-8.Fibrinogen is on polymer surfaces Absorption and albumen in conformation change may expose MAC-1 binding sites.Via and its MAC-1 acceptor combination activation THP-1 cell cytokine releases such as IL-8, and thus trigger inflammatory response.Therefore, it is made up of AdaptTM or CM 5206 Separation layer avoid on surface (such as sensor) proteinosis and structural motif exposure, and thus make for implant Inflammatory response is minimized.

Embodiment 7The limited haemolysis of the sensor coated with separation layer.

Analyze the sensor without separation layer, with the sensor by the separation layers being made of Lipidure CM 5206 or Hemolytic potential with the sensor by the AdaptTM separation layers being made.Therefore, by total surface area for 6 cm2 sensor with Red blood cell is incubated, and is then cracked by measuring hemoglobin to the release measure of supernatant.By centrifuging from people's new blood Separating red corpuscle (using citrate to avoid blood coagulation).Then them are washed with phosphate buffered saline (PBS) (PBS), and then existed 1 in PBS:40 dilutions.Red blood cell suspension and sensor are incubated 24 in the dark on rotation platform (350 rpm) at 37 DEG C h.Afterwards, by centrifugal sedimentation cell, and the spectrographic determination supernatant by the absorption with 575 nm wavelength measurement supernatant The content of hemoglobin of liquid.As a result the cracking index in terms of % is expressed as, this is the release of hemoglobin in sample divided by positive right According to the hemoglobin release in thing (the complete osmotic lysis of red blood cell in=distilled water).As a result it is shown in Figure 14.

Sensor without separation layer significantly causes haemolysis, as shown in 47.4% high haemolytic index.Use Lipidure CM 5206 separation layer coating sensor reduces the haemolysis potential of sensor, as indicated in 14.7 % cracking index.With AdaptTM separation layer coating sensor somewhat cause haemolysis, cause 7.5% cracking index, this negative control thing (= There is no the red blood cell in the PBS that is incubated in the case of any test material) or individually in the range of AdaptTM.These result tables Bright separation layer reduces the defencive function of haemolysis.

Claims

1. the electrode system of the analyte concentration under the conditions of for measuring in vivo, it includes the electrode with fixed enzyme molecule, The diffusion barrier for optionally controlling the analyte to be spread outside the electrode system to enzyme molecule,

It is characterized in that barrier film forms at least a portion of the outer layer of the electrode system, wherein the barrier film includes acrylic acid And/or the hydrophilic copolymers of methacrylic acid monomer, wherein the hydrophilic copolymers include more than 50mol% hydrophily list Body.

2. the electrode system of claim 1, wherein the barrier film is to come from least two kinds of or 3 kinds of acrylic acid and/or metering system The hydrophilic copolymers of acid monomers.

3. the electrode system described in claim 1 or 2, wherein the hydrophilic monomer, which is selected from, has polar group such as OH, OCH₃ Or OC₂H₅Hydrophily (methyl) acrylate, hydrophily (methyl) acrylamide, (methyl) acrylic acid or its combination.

4. the electrode system of claim 3, wherein the hydrophilic monomer is selected from：

Acrylic acid 2- hydroxyl ethyl esters,

HEMA (HEMA),

Acrylic acid 2- methoxy acrylates,

Methacrylic acid 2- methoxy acrylates,

Acrylic acid 2- ethoxy ethyl esters,

Methacrylic acid 2- ethoxy ethyl esters,

Acrylic acid 2- or 3- hydroxypropyl acrylate,

Methacrylic acid 2- or 3- hydroxypropyl acrylate (2- or 3-HPMA),

Acrylic acid 2- or 3- methoxyl group propyl ester,

Methacrylic acid 2- or 3- methoxyl group propyl ester,

Acrylic acid 2- or 3- ethyoxyl propyl ester,

Methacrylic acid 2- or 3- ethyoxyl propyl ester,

Acrylic acid 1- or 2- glyceride,

Methacrylic acid 1- or 2- glyceride,

Acrylamide,

Methacrylamide,

N- alkyl-or N, N- dialkylacrylamides, and

N- alkyl-or N, N- dialkyl methyl acid amides,

Wherein alkyl includes 1-3 C atom,

Acrylic acid,

Methacrylic acid and combinations thereof,

It is preferably selected from HEMA (HEMA), methacrylic acid 2- hydroxypropyl acrylates (2-HPMA) and combinations thereof.

5. any one of claim 1-4 electrode system, wherein the hydrophilic copolymers of the barrier film include at least 60 Mol-% or at least 70 mol-% hydrophilic monomer.

6. any one of claim 1-5 electrode system, wherein the copolymer of the barrier film is further contained up to up to 40 Mol-% or up to 30 mol-% hydrophobic monomer.

7. the electrode system of claim 6, wherein the hydrophobic monomer is selected from：

Methyl acrylate,

Methyl methacrylate (MMA),

Ethyl acrylate,

EMA (EMA),

Acrylic acid just or isopropyl ester,

Methacrylic acid just or isopropyl ester,

N-butyl acrylate,

N-BMA (BUMA),

Acrylic acid peopentyl ester,

Methacrylic acid peopentyl ester and combinations thereof,

It is preferably selected from methyl methacrylate (MMA), n-BMA (BUMA) and combinations thereof.

8. the electrode system of claim 6 or 7, wherein the hydrophobic monomer is methyl methacrylate (MMA) or methyl-prop Olefin(e) acid N-butyl (BUMA), and the hydrophilic monomer is HEMA (HEMA) and/or methacrylic acid 2- Hydroxypropyl acrylate (2-HPMA).

9. any one of claim 6-8 electrode system, wherein the barrier film, which is included, comes from n-BMA (BUMA), the copolymer of HEMA (2-HEMA) and methacrylic acid 2- hydroxypropyl acrylates (2-HPMA), wherein institute State the 2-HEMA monomers that copolymer includes 80 mol-%.

10. any one of claim 1-9 electrode system, wherein the hydrophilic copolymers are that random copolymer or block are common Polymers.

11. any one of claim 1-10 electrode system, wherein the thickness of the barrier film is less than about 20 μm, it is preferably small In 5 μm, more preferably from about 1 to about 3 μm.

12. any one of claim 1-11 electrode system, wherein, based on the gross weight of the copolymer, the hydrophily The relative water-intake rate of copolymer is no more than 50 weight %, more preferably preferably 40 weight %, 30 weight %.

13. any one of claim 1-12 electrode system, wherein the diffusion barrier is comprising hydrophilic polyurethane or has The block copolymer of at least one hydrophilic block and at least one hydrophobic block.

14. any one of claim 1-13 electrode system, it includes the counterelectrode (2) with electric conductor (2a), with electricity The working electrode (1) of conductor (1a), and barrier film (9), set fixed enzyme molecule (5) and optionally on the electric conductor (1a) Diffusion barrier (8), the barrier film (9) covering at least working electrode (1) and optionally also counterelectrode (2).

15. the sensor in pluggable or implantable, it includes any one of claim 1-15 electrode system.

16. the sensor of claim 15, it is used to measure glucose.

17. the hydrophilic copolymers of acrylic acid and/or methacrylic acid monomer are as the purposes of the barrier film of enzyme electrode system, its Described in hydrophilic copolymers include be more than 50 mol-% hydrophilic monomers.

18. the purposes of claim 17, it is used to minimize the external precursor reactant (FRB) for enzyme electrode system.