Summary of the invention
The present invention is directed to the deficiency that glucose sensor bio-compatibility in the prior art is poor, its blood glucose value at that time can not be provided to user in real time, good needle amperometric determination type glucose sensor for subcutaneous tissue real-time monitoring of a kind of bio-compatibility and preparation method thereof is provided.In order to solve the problems of the technologies described above, the present invention is able to solve through following technical proposals:
Needle amperometric determination type glucose sensor for subcutaneous tissue real-time monitoring; Comprise a needle-like formula reference electrode and at least one needle-like formula working electrode; Working electrode and reference electrode can be fixed through plastic feet, and plastic feet designs for circular arc, the vertical base circular arc of electrode; Wireless launcher embeds in the base and with electrode and links to each other; Working electrode comprises conductive layer and enzyme membrane layer, and said working electrode also comprises macromolecular material theca interna and macromolecular material control diffusion layer, and conductive layer, macromolecular material theca interna, enzyme membrane layer and macromolecular material control diffusion layer cover successively from the inside to the outside and form.
The enzyme membrane layer contains glucoseoxidase and catalase, is one deck enzyme glue-line.The enzyme membrane layer can oxidizing glucose and hydrogen peroxide.
When needle amperometric determination type glucose sensor for subcutaneous tissue real-time monitoring of the present invention adopts a plurality of working electrode, a plurality of working electrodes parallel connections.One of them is as main working electrode, and main working electrode adopts optimal way to choose, and all the other working electrodes are the back work electrode.Working electrode and reference electrode constitute the loop and form electrochemical sensor.
Working electrode and reference electrode can be fixed through plastic feet, and plastic feet designs for circular arc, and the vertical base circular arc of electrode, wireless launcher embed in the base and with electrode and link to each other.The material of plastic feet can be polypropylene, gather any one material in imines, polrvinyl chloride, polyethers, polyester, Merlon, polyethylene, polyamide, polyurethane, polrvinyl chloride, poly terephthalic acid butenylidene ester, PETG, polyoxygenated benzene or the acrylonitrile-butadiene resin, preferably polyethylene.
As preferably, the conductive layer of working electrode is made up of metallic matrix, intermediate metal, layer of precious metal from inside to outside successively.
As preferably, the material of the metallic matrix of the conductive layer of described working electrode can be any one of rustless steel, copper or titanium, preferred rustless steel;
The material of intermediate metal can be in titanium, nickel, gold, platinum, palladium, rhodium, iridium or the chromium any one, preferred palladium;
The material of layer of precious metal can be any one in palladium, rhodium, iridium, chromium or the platinum, preferred palladium and platinum.
Conductive layer need have enough rigidity, can thrust skin at an easy rate.
As preferably; The material of macromolecular material theca interna can be any one in poly terephthalic acid butenylidene ester, PETG, polyoxygenated benzene, acrylonitrile-butadiene resin, silane resin, epoxy resin or the acetate fiber; According to the film forming characteristics of material on conductive layer; Preferred epoxy and acetate fiber consider that glucoseoxidase adopts cross-linking method to be linked on the macromolecular material theca interna, preferred especially acetate fiber;
The material of macromolecular material control diffusion layer can be any one in cellulose acetate, Merlon, polyvinyl alcohol, polyurethane, polyurethane, politef or the polrvinyl chloride; The polyurethane good biocompatibility; Good filming performance is arranged, so optimization polyurethane;
The hydrophilic hydration layer of the many space lattices of macromolecular material control diffusion layer surface parcel one deck; Material can be the chitosan of porous polyvinyl alcohol, Polyethylene Glycol, carboxylation; In carboxymethyl cellulose, hydroxyethyl-cellulose, low oligosaccharide or the dioxygen starch any one; Preferred material is a porous polyethylene alcohol, the porous polyethylene alcohol of elite 60 μ m.
The effect of macromolecular material theca interna is to insulate and exclude the micromolecule chaff interference to comprise ascorbic acid and a spot of amino uric acid, bilirubin, catecholamine, medicine and other various metabolite; Only allow hydrogen peroxide to get into aciculiform metal microelectrode surface, can reduce the electrochemistry of working electrode and disturb.
Glucoseoxidase is learned the response power of glucose, and only in lower concentration of glucose scope, glucoseoxidase is only linear response to the response of concentration of glucose.Thereby the present invention covers one deck macromolecular material control diffusion barrier at the enzyme membrane laminar surface.The macromolecular material control diffusion layer of working electrode has the selection permeability; Make the glucose molecule diffusion in the blood see through this floor height molecular film arrival glucose oxidase film surface; Reduce the diffusion velocity of glucose molecule; Make the control procedure of the diffusion process of glucose molecule as entire reaction, the concentration of glucose in the enzyme membrane remains in the lower concentration range, reaches the purpose that enlarges the range of linearity.
Macromolecular material control diffusion layer can stop the loss of enzyme; Select impregnability can slow down diffusion than macromole such as albumen etc.; Electrode interference can be reduced, the detection range of linearity of pick off can also be increased through the amount that the restriction glucose is diffused into working electrode.If the impregnability of this tunic oxygen greater than the impregnability of glucose, can also be improved the dependence of electrode pair oxygen.Macromolecular material control diffusion layer can filter macromole and cell, and entire reaction is played the DIFFUSION CONTROLLED effect.
If have the fibrous tissue of enriching vascularity on the glucose sensor surface, blood just can be easier to be diffused into the surface of glucose sensor, and then glucose sensor just can be measured the variation of blood-glucose preferably.This has just shortened the response time of glucose sensor, thereby reaches real-time monitoring.And if had abundant vascular tissue on glucose sensor surface, could make glucose sensor work can be under not gauged situation steady in a long-term.The fixing hydrophilic high molecular material layer of the many space lattices of one deck on the surface of glucose sensor; Can promote the formation of neovascularity around pick off; Thereby shortened the response time of glucose sensor, can also effectively increase the glucose and the oxygen concentration of glucose sensor surrounding tissue simultaneously.
As preferably, reference electrode is silver/silver chloride electrode, comprises three layers of conductive layer, silver/silver chloride layer and macromolecular material theca externas from inside to outside successively.
As preferably, the conductive layer of described reference electrode is made up of metallic matrix and silver layer from inside to outside successively, and the material of metallic matrix can be any one in rustless steel, copper or the titanium, preferred rustless steel; The material of the macromolecular material theca externa of reference electrode can be any one in cellulose acetate, Merlon, polyvinyl alcohol, polyurethane, polyurethane, politef or the polrvinyl chloride, preferably polyethylene alcohol.
As preferably, the metallic matrix diameter R7 of the conductive layer of described working electrode is 0.15~0.25mm, preferred 0.2mm, and the metal base surface granular size is 30nm;
Metal transfer layer thickness R6 is 0.05 μ m~1 μ m, and granular size is 100nm;
Layer of precious metal thickness R5 is 1 μ m~3 μ m, and granular size is 30nm;
Enzyme membrane layer thickness R3 is 0.05 μ m~1 μ m;
Macromolecular material control thickness of diffusion layer R2 is 5 μ m~10 μ m.
The hydrophilic hydration layer of one deck space lattice of macromolecular material control diffusion layer surface parcel, its thickness R1 is 3 μ m~6 μ m.
The silver thickness R10 of reference electrode is 0.05 μ m~1 μ m, and silver-colored granular size is about 100nm.
The invention also discloses a kind of manufacture method of needle amperometric determination type glucose sensor for subcutaneous tissue real-time monitoring:
The macromolecular material theca interna is combined in the surface of conductive layer through a kind of mode of coating or dipping, preferred coating process;
The enzyme membrane layer is with glutaraldehyde as cross linker, is carrier with bovine serum albumin or cyclodextrin or gelatin, through cross-linking method with covalent bond is legal is fixed on the macromolecular material theca interna surface;
Macromolecular material control diffusion layer adopts the mode of coating or dipping to be fixed on the enzyme membrane laminar surface, preferred coating process;
The conductive layer surface of working electrode carries out the surface active pretreatment with the oxonium ion etching method.Metal base surface at conductive layer deposits one deck gold layer as intermediate metal; The platinum black granule of the loose shape of gold electrode surfaces electro-deposition one deck; Amboceptor and enzyme directly are adsorbed on the platinum black electrode surface, to satisfy the electrode requirement of electrochemical oxidation hydrogen peroxide under the physiological environment of pH=7.4.Platinum black porous microparticle has that specific surface area is big, electro-catalysis ability, good characteristic such as strong; The gold electrode of modifying through platinum black is in the enzyme immobilization process; Utilize the particulate porosity characteristic of platinum black; The enzyme molecular energy is adsorbed on the platinum black electrode surface securely, through changing electro-deposition voltage, can control the size and the sedimentation of platinum black electrode specific surface area.Thereby platinum black provides enough big area to make electrode process be the control of glucose diffusion process, and the glucose sensor for preparing like this has the characteristics of little, the highly sensitive and high conformity of volume.The platinum black electrode surface energy of high loose shape effectively increases the signal to noise ratio of sensor current signal output, improves the hydrophilic of electrode surface, thereby reduces the enzyme loss.
Before the deposition platinum black, carry out pretreatment, can effectively improve the resistance to corrosion of electrode, reduce electrode impedance, strengthen the degree of cross linking of platinum black granule and gold electrode with the method counter electrode surface of physics or chemistry.The present invention adopts the method for oxygen rie that gold electrode surfaces is carried out pretreatment, can remove the pollutant of electrode surface, can also play the effect on activated electrode surface simultaneously.The condition of etching can be done suitable adjustment according to the situation of electrode surface, and electroplating voltage, sedimentation time and different electrolytes proportioning all will influence the thickness and the density of film.
The advantage of enzyme membrane layer combined techniques of the present invention is the bonded fastness of enzyme, good stability, and sensitivity, selectivity and repeatability are high.
The hydrophilic hydration layer of the many space lattices of macromolecular material control diffusion layer surface parcel one deck.
As preferably, the conductive layer of working electrode is made up of metallic matrix, intermediate metal, layer of precious metal from inside to outside successively;
The intermediate metal of conductive layer can pass through any one method in chemical deposition, magnetron sputtering or the electro-plating method attached to metal base surface, preferred magnetically controlled sputter method;
The layer of precious metal of conductive layer can pass through any one method in chemical deposition, magnetron sputtering or the electro-plating method attached to the metal transfer laminar surface.
As preferably, reference electrode is silver/silver chloride electrode, comprises three layers of conductive layer, silver/silver chloride layer and macromolecular material theca externas from inside to outside successively; The conductive layer of reference electrode is made up of metallic matrix and silver layer from inside to outside successively, and the material of metallic matrix can be any one in rustless steel, copper or the titanium;
The metal base surface of reference electrode crosses one deck silver through any one method in electro-plating method, chemical deposition or the magnetron sputtering method, and preferred electro-plating method carries out chlorination and forms silver/silver chloride layer, and granular size is 100nm;
The material of the macromolecular material theca externa of reference electrode can be any one in cellulose acetate, Merlon, polyvinyl alcohol, polyurethane, polyurethane, politef or the polrvinyl chloride;
The macromolecular material theca externa of reference electrode is fixed on silver/silver chloride layer surface through any one method in coating or the dipping.
According to glucoseoxidase the response power of glucose is learned, only in lower concentration of glucose scope, glucoseoxidase is only linear response to the response of concentration of glucose.Thereby the present invention covers one deck macromolecular material control diffusion layer in glucose oxidase layer.Macromolecular material control diffusion layer makes the glucose molecule diffusion in the blood see through the macromolecule membrane arrival glucose oxidase layer surface that this layer has the selection permeability; Thereby reduce the diffusion velocity of glucose molecule; Make the control procedure of the diffusion process of glucose molecule as entire reaction; Concentration of glucose in the glucose oxidase layer remains in the lower concentration range, reaches the purpose that enlarges the range of linearity.
According to clinical requirement, the glucose sensor range of linearity upper limit must reach 30mmol.Under this upper limit, the analytical characteristics of pick off such as sensitivity, linear zone etc., stability is by the thickness and the preparation method decision of macromolecular material control diffusion barrier.The glucose sensor of the present invention range of linearity upper limit reaches more than the 30mmol.
The present invention can be used for the detection of human body or animal skins undertissue liquid glucose, and implant site is that subcutaneous tissue can be arm or abdominal part position, and preferred implant site is a subcutaneous tissue of abdomen.Glucose sensor continuous operation of the present invention is no less than 72 hours.
It is subcutaneous that detecting head of the present invention is implanted the tester; Glucose in the subcutaneous intercellular fluid of tester carries out dialysis the working sensor electrode surface through semipermeable membrane; Carry out dialysis working sensor electrode surface glucose and enzyme and react, the signal of telecommunication reflection tissue liquid glucose level of generation.
The decay of implanting the back transducer sensitivity is relevant with the structure of pick off, and subject matter is that the catalytic reaction of enzyme when hypoxgia is suppressed the electrochemistry interference with some little endogenous molecules.The problem of hypoxgia can reduce the area of enzyme reaction through the infiltration area that relatively increases oxygen, uses the film of high-solubility oxygen or use the hydrophobicity outer membrane optionally to reduce glucose to be diffused in the enzyme membrane layer and to solve.Electrochemistry is disturbed can have optionally that film and non-conductive polymeric membrane solve through use.
According to manufacture method of the present invention; But the range of linearity of glucose sensor good stability, susceptiveness height, output current and concentration of glucose is extensive, the short continuous real-time monitoring of response time; The bonded fastness of enzyme, good stability, sensitivity, selectivity and repeatability are high.
The specific embodiment
Below in conjunction with the accompanying drawing and the specific embodiment the present invention is described in further detail:
Embodiment 1; Needle amperometric determination type glucose sensor for subcutaneous tissue real-time monitoring; Like Fig. 1,2, shown in 3; Comprise a needle-like formula reference electrode 2 and at least one needle-like formula working electrode 1; Working electrode 1 comprises conductive layer 8 and enzyme membrane layer 6, it is characterized in that: said working electrode 1 also comprises macromolecular material theca interna 7 and macromolecular material control diffusion layer 5, and conductive layer 8, macromolecular material theca interna 7, enzyme membrane layer 6 and macromolecular material control diffusion layer 5 cover successively from the inside to the outside and form.
Working electrode 1 can pass through plastic feet 3 with reference electrode 2, and the fixed wireless discharger embeds in the plastic feet 3 and with working electrode 1 and links to each other with reference electrode 2.
In the present embodiment:
The metallic matrix 83 material selection rustless steels of conductive layer 8;
The intermediate metal 82 material selection palladiums of conductive layer 8;
The layer of precious metal 81 material selection palladiums of conductive layer 8;
The material selection acetate fiber of macromolecular material theca interna 7;
Enzyme membrane layer 6 contains glucoseoxidase and catalase;
The material selection polyurethane of macromolecular material control diffusion layer 5;
The hydrophilic hydration layer 4 of one deck space lattice of macromolecular material control diffusion layer 5 surface parcels, the porous polyethylene alcohol of hydrophilic hydration layer 4 material selection 60 μ m;
The material of plastic feet 3 is a polyethylene;
Wherein 9 is the centrage of pick off.
According to 8 identical glucose sensors of following thickness R parameter preparation.
Show 1:8 glucose sensor dimensional parameters table
These 8 glucose sensors are in the glucose solution test of external phosphate-buffered reagent, and test voltage both positive and negative polarity voltage difference is 0.55V, and test result is following:
Show the glucose solution table with test results (unit: nA) of 2:8 pick off at external phosphate-buffered reagent
Guaranteeing under the clinical service condition that the linear test of glucose sensor glucose of the present invention is greater than 30mmol/L, linearity reaches 99.9%, and sensitivity is greater than 25nA/ (mmol/L), and sensitivity is better; The performance of each pick off is consistent, stable.
Embodiment 2, needle amperometric determination type glucose sensor for subcutaneous tissue real-time monitoring, structure roughly is identical with experiment condition, different places be merely as follows.
The layer of precious metal 81 material selection platinum of conductive layer 8;
The material selection epoxy resin of macromolecular material theca interna 7;
Also prepare 8 identical glucose sensors (being pick off 9 to pick off 16) according to following thickness R.
Table 3: the dimensional parameters table of pick off 9 to pick off 16
These 7 glucose sensors are in the glucose solution test of external phosphate-buffered reagent, and test voltage both positive and negative polarity voltage difference is 0.55V, and test result is following:
Show the glucose solution table with test results (unit: nA) of 4:8 pick off at external phosphate-buffered reagent
Guaranteeing under the clinical service condition that the linear test of glucose sensor glucose of the present invention is greater than 27mmol/L, the linearity test height of reaching the standard grade, linearity reaches 99.9%; Sensitivity is 28nA/ (mmol/L), and sensitivity is better; The performance of each pick off is consistent, stable.
Embodiment 3; Make the method for the needle amperometric determination type glucose sensor for subcutaneous tissue real-time monitoring of embodiment 1: glucose sensor has two working electrodes 1 and a reference electrode 2; Working electrode 1 can be fixed on the plastic feet 3 with reference electrode 2, and working electrode 1 constitutes the loop with reference electrode 2 through lead.
The conductive layer 8 of working electrode 1 is made up of metallic matrix 83, intermediate metal 82, layer of precious metal 81 from inside to outside successively.
Intermediate metal 82 passes through magnetically controlled sputter method attached to metallic matrix 83 surfaces;
Layer of precious metal 81 passes through electro-plating method attached to intermediate metal 82 surfaces;
Conductive layer 8 surfaces adopt the oxygen rie method to make surface active.
Macromolecular material theca interna 7 is combined in the surface of conductive layer 8 through the mode of coating;
Enzyme membrane layer 6 is carrier with glutaraldehyde as cross linker with the bovine serum albumin, through cross-linking method with covalent bond is legal is fixed on macromolecular material theca interna 7 surfaces;
Macromolecular material control diffusion layer 5 adopts the mode of coating to be fixed on enzyme membrane layer 6 surface;
One deck silver is crossed through the electro-plating method method in metallic matrix 122 surfaces of reference electrode 2, carries out chlorination and forms silver/silver chloride layer 11, and granular size is 100nm;
Macromolecular material theca externa 10 is fixed on silver/silver chloride layer 11 surfaces through coating process.
Pick off with this method preparation can reach embodiment 1 described effect.
Embodiment 4; Make the method for the needle amperometric determination type glucose sensor for subcutaneous tissue real-time monitoring of embodiment 2: different with embodiment 3 is: enzyme membrane layer 6 is with glutaraldehyde as cross linker; With the gelatin is carrier, through cross-linking method with covalent bond is legal is fixed on macromolecular material theca interna 7 surfaces;
Macromolecular material control diffusion layer 5 adopts the mode of dipping to be fixed on enzyme membrane layer 6 surface;
Other step is identical with embodiment 3, and the pick off for preparing with this method can reach embodiment 2 described effects.
Embodiment 5; The needle amperometric determination type glucose sensor for subcutaneous tissue real-time monitoring 1 of embodiment 2 is implanted arm position continuous operation be no less than 72 hours, the current value that Fig. 6 measures for the needle amperometric determination type glucose sensor for subcutaneous tissue real-time monitoring continuous real-time of using present embodiment 2.
In a word, the above is merely preferred embodiment of the present invention, and all equalizations of doing according to claim of the present invention change and modify, and all should belong to the covering scope of patent of the present invention.