CN101025422A - Method for forming film, base plate with film, sensor, and liquid composition - Google Patents
Method for forming film, base plate with film, sensor, and liquid composition Download PDFInfo
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- CN101025422A CN101025422A CN 200710005186 CN200710005186A CN101025422A CN 101025422 A CN101025422 A CN 101025422A CN 200710005186 CN200710005186 CN 200710005186 CN 200710005186 A CN200710005186 A CN 200710005186A CN 101025422 A CN101025422 A CN 101025422A
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Abstract
The film forming method of the invention is a method for forming a detection film including the detection substance for detecting an object on one side of a base plate. The method is characterized in that it includes: a first step for preparing a liquid composition containing a substance for detecting an object and a polyether derivative having a weight-average molecular weight of 2000 or less; a second step for supplying the liquid composition to the said side by using a droplet discharge method to form a liquid film; and a third step for drying the liquid film to form the detection film. Therefore, the invention provides a film forming method which can form a film including protein with excellent film forming accuracy, and can restrain or prevent the reduction of the physiological active of the protein in the film, a base plate with the film formed by using the film forming method and a sensor with high reliability. The invention also provides a liquid composition which can prevent or restrain the reduction of the physiological active of the protein in the film and can form a film including protein with excellent film forming accuracy.
Description
Technical field
The present invention relates to substrate, sensor and the fluid composition etc. of film formation method, band film.
Background technology
In recent years, for example can make protein immobilization on substrate of physiologically actives such as having enzyme or antibody, in the external biosome reaction of carrying out these protein participations of Real Time Observation (detection).
In order to observe this biosome reaction more accurately, must on substrate, support protein with outstanding film-forming accuracy, but as on substrate, fixing this method of protein, widely used is to be provided to ink-jet method on the substrate (for example, with reference to patent documentation 1 with proteinaceous liquid as printing ink and with this printing ink.)。
With regard to ink-jet method, the oscillating plate by piezoelectric element is vibrated, make the volume change of ink chamber (space), the printing ink that provides by ink chamber is provided, from being located at the aperture ejection of above-mentioned oscillating plate opposition side, be provided on the substrate.
In this ink-jet method, requirement is stable from the spray volume of the liquid of aperture ejection, and precision is higher, from the bullet of the drop of the aperture ejection positional precision height that falls, be difficult to produce the obstruction of aperture etc.,, need to adjust the viscosity and the surface tension of printing ink (liquid that contains protein) in order to satisfy this requirement.
At this,, for example use the method for in printing ink, adding the polyethers based compound as viscosity and the capillary method of adjusting printing ink.
Yet if use such printing ink fixing protein on substrate, the polyethers based compound that contains in the printing ink contacts with protein, and the physiologically active that can produce protein thus reduces the problem of (inactivation).
Problem like this except protein, is using ink-jet method can produce too under the situation that forms the film that contains nucleic acid and fluorchrome etc. on the substrate.
Patent documentation 1: the spy opens clear 61-245051 communique
Summary of the invention
One of purpose of the present invention is, provide to form with outstanding film-forming accuracy and contain the detection that detects with material and can suppress simultaneously with film or prevent to detect with detection in the film with the film formation method of the reduction of the activity that material was had, possesses the detection that utilizes this film formation method the to form substrate of the band film of film, the sensor that reliability is high.And then, the present invention also provide can prevent or suppress formed detection with the detection that contains in the film with the reduction of the physiologically active of material and can form the fluid composition that detects with film with outstanding film-forming accuracy.
Such purpose can utilize following the present invention to realize.
Film formation method of the present invention is the film formation method that forms the detection usefulness film that contains the detection usefulness material that is useful on detection detected object thing in the one side side of substrate, it is characterized in that, comprising:
It is the 1st operation of the fluid composition of the polyethers based compound below 2000 with material and weight-average molecular weight that preparation contains described detection;
Use the described one side side of drop ejection normal direction to supply with above-mentioned fluid composition, form the 2nd operation of liquid tunicle; With
Dry described liquid tunicle forms 3rd operation of described detection with film.
Like this, this film formation method contain the detection that detects with material with in the film forming with outstanding film-forming accuracy, can suppress or prevents to detect with the reduction of the detection in the film with the physiologically active that material had.
Promptly, except above-mentioned detection with the material, by adding weight-average molecular weight to the above-mentioned detection that forms in film is the formation of the polyethers based compound 2000 below, can form above-mentioned detection film under the state of above-mentioned detection with the Stability of Substance raising in the above-mentioned detection usefulness film.
In addition, weight-average molecular weight is the polyethers based compound below 2000, has high-affinity owing to protein, nucleic acid etc. is had the material of polar group, so particularly at material that use has a polar group as above-mentioned detection with under the situation of material, as film formation method of the present invention, use that to contain above-mentioned detection be that the above-mentioned fluid composition formation detection of the polyethers based compound 2000 below is effective with the structure of film with material and weight-average molecular weight.
In film formation method of the present invention, be made as A[wt% at content with the above-mentioned polyethers based compound in the above-mentioned fluid composition], above-mentioned detection is made as B[wt% with the content of material] time, A/B preferably satisfies 9～10
5Relation.
By satisfying such relation, the polyethers based compound is set in the preferable range with respect to the adhesive rate that detects with material, can suppress better or prevent to detect the physiologically active of using material to reduce.
In film formation method of the present invention, above-mentioned detection is preferably below the 10wt% with the content of material in the above-mentioned fluid composition.
Be set in this scope by the content that will detect, with in the film, can fully detect reaction with material in the detection that forms with material.
In film formation method of the present invention, the content of polyethers based compound is preferably more than the 90wt% described in the described fluid composition.
If the content of polyethers based compound is too much, according to the polyethers based compound and class, the viscosity of fluid composition can increase, and utilizes drop ejection method to make above-mentioned fluid composition become the drop ejection and can become impossible.In addition, if the content of polyethers based compound is very few, above-mentioned fluid composition may become dry easily.And then, be set in this scope by content the polyethers based compound, the polyethers based compound is set in the preferred scope with respect to the adhesive rate that detects with material, can suppress or prevent to detect the reduction of the physiologically active of using material better.
In film formation method of the present invention, the viscosity under the preferred normal temperature of described fluid composition is below the 20cP.
The ejection stably if viscosity of fluid composition in such scope, then can utilize drop ejection method to make fluid composition become drop.
In film formation method of the present invention, the preferred surface tension at normal temperatures of above-mentioned fluid composition is 20～70mN/m.
Be set in such scope by surface tension, can utilize the liquid tunicle that forms the homogeneous thickness by the drop of drop ejection method ejection fluid composition.
In film formation method of the present invention, when the weight-average molecular weight with the above-mentioned polyethers based compound in the above-mentioned fluid composition was made as C, the deviation of above-mentioned polyethers based compound was preferably in the scope of ± 0.2C.
Thus, with the size of the molecular weight that detects the polyethers based compound that contacts with material by homogenization, so can reduce the probability that contacts with the big polyethers based compound of molecular weight.Its result can prevent better or suppress owing to contact the detection that the causes reduction with the physiologically active of material with the big polyethers based compound of molecular weight.
In film formation method of the present invention, above-mentioned polyethers based compound preferably has the main chain that is made of polyethers and has at least one end of this main chain can be basic with the combination that material combines with described detection.
Like this, in fluid composition, when detection contacted with the polyethers based compound with material, this combined with material with detection in conjunction with base, and the polyethers based compound links with material with detection.Like this, detect connecting airtight property raising with material and polyethers based compound.Its result detects and further improves with Stability of Substance, can suppress or prevent the reduction of its physiologically active better.
In film formation method of the present invention, preferably have described in conjunction with base in two ends of described main chain.
Like this, detect and further improve, can further suppress or prevent the reduction of its physiologically active better with the connecting airtight property between material and the polyethers based compound.
In film formation method of the present invention, in above-mentioned the 1st operation, above-mentioned polyethers based compound preferably combines basic combination with material with above-mentioned by above-mentioned detection, links with material with described detection.
Like this, detect with the connecting airtight property raising between material and the polyethers based compound.Its result detects and further improves with Stability of Substance, can further suppress or prevent the reduction of its physiologically active better.
In film formation method of the present invention, above-mentioned polyethers based compound is a principal ingredient with the polyglycol based compound preferably.
Like this, can be more easily the viscosity and the surface tension of fluid composition be set in the suitable scope.
In film formation method of the present invention, above-mentioned detection is preferably protein with material.
At above-mentioned detection material is under the situation of protein, by using film formation method of the present invention, can suppress or prevent to detect the reduction of the physiologically active of the detection usefulness material in the film more significantly.
In film formation method of the present invention, above-mentioned protein is preferably enzyme.
Like this, can be more stable and repeatability carry out reaction between enzyme and the matrix well.
The substrate of band film of the present invention is characterized in that, possesses the detection film that utilizes film formation method of the present invention to form in the one side side of described substrate.
Like this, can become the substrate that possesses the band film that detects the usefulness film, described detection can suppress with film or prevent to detect with the reduction of the detection in the film with the physiologically active that material had, and can form with outstanding film-forming accuracy.
The substrate of band film of the present invention is to comprise containing being useful on the detection that detects the detected object thing substrate of the detection of material with the band film of film, it is characterized in that,
It is polyethers based compound below 2000 with material and weight-average molecular weight that described detection contains described detection with film.
Like this, can become the substrate that possesses the band film that detects the usefulness film, described detection can suppress with film or prevent to detect with the reduction of the detection in the film with the physiologically active that material had, and can form with outstanding film-forming accuracy.
Sensor of the present invention is characterized in that, possesses the substrate of band film of the present invention.
Like this, can obtain the high sensor of reliability.
In addition, as the present invention, by the weight-average molecular weight that makes the polyethers based compound is below 2000, be used for sensor as described below, the detecting electrode that is used to detect that described sensor utilization is provided with on aforesaid substrate, by in this detecting electrode and above-mentioned detection with giving and accepting of the electronics between material or the detected object thing that detects with material by above-mentioned detection etc., obtain electric signal, at this moment, this detecting electrode and above-mentioned detection can be become be suitable for to obtain the distance of electric signal with the distance setting between material or the above-mentioned detected object thing.
Fluid composition of the present invention is characterised in that, containing detection with physiologically active is polyethers based compound below 2000 with material and weight-average molecular weight.
Like this, can obtain preventing or to suppress formed detection with the detection that contains in the film with the reduction of the physiologically active of material and can form the fluid composition that detects with film with outstanding film-forming accuracy.
Description of drawings
Fig. 1 is the longitudinal diagram of the structure of pattern ground expression sensor of the present invention.
Fig. 2 is the longitudinal diagram that expression is used to illustrate the manufacture method of sensor shown in Figure 1.
Among the figure: 1-sensor, 2-sample receiving space, 20-fluid sample, 3-active electrode, 4-be to electrode, 5-reference electrode, 6-basalis, 7-enzyme electrode layer, 8-substrate, 10-sealing, 101-inlet.
Embodiment
Below, based on preferred implementation shown in the drawings, to film formation method of the present invention, the band film substrate, sensor and fluid composition be elaborated.
＜sensor 〉
At first, the preferred implementation to sensor of the present invention describes.
In addition, in the present embodiment, as containing the detection film that detects with material, giving one example describes the sensor that possesses the enzyme electrode layer that contains the protein with physiologically active.
Fig. 1 is the longitudinal diagram of the structure of pattern ground expression sensor of the present invention.In addition, in the following description, the upside among Fig. 1 be called " on ", downside is called D score.
Sensor 1 shown in Figure 1 has the sample receiving space 2 of accommodating fluid sample 20, active electrode 3, to electrode 4, reference electrode 5, basalis 6 and enzyme electrode layer 7, these each several parts are laminated on the substrate 8 and constitute.This sensor 1 is will be to the amount of the target (detected object thing) 21 in the fluid sample 20 that sample receiving space 2 is supplied with, and as at the sensor that detects to 3,4 current values that produce when applying voltage of pair of electrodes, substrate 8 is provided with a plurality of.
In addition, in the present embodiment, by substrate 8 with constitute the substrate of band film of the present invention at the enzyme electrode layer 7 that the upside of substrate 8 forms.In other words, in the present embodiment, described in detail in the manufacture method of enzyme electrode layer 7 as the sensor that illustrates later, formed by film formation method of the present invention.
At this, as fluid sample 20, for example can enumerate body fluid such as blood, urine, sweat, lymph liquid, spinal fluid, bile, saliva, or these body fluid be implemented liquid etc. after the processing of various processing.
In addition, target is by react ejected electron (e with protein described later
-) material.
As the object lesson of target, for example can enumerate the carbohydrate of glucose and so on, simple protein, glycoprotein and so on protein-based, alcohols, the steroid of cholesterol, steroid hormone, bile acid, bilichol and so on, vitamins, steroids, lactic acid, cholerythrin, uric acid, kreatinin etc.
Constituent material as substrate 8, for example can enumerate the various resin materials of tygon, polypropylene, polystyrene, polyethylene terephthalate (PET), polyacrylic acid, epoxy resin and so on, various glass materials, various stupaliths etc. can be used in combination a kind of in these or more than 2 kinds.In addition, as the constituent material of substrate 8, using under the situation of compound substance, for example the anti-flammability printed circuit board that the compound substance by glass fibre and epoxy resin can be constituted etc. uses as substrate 8.
On substrate 8, be respectively arranged with a plurality of active electrodes 3.
As this active electrode 3, the constituent material to electrode 4 and reference electrode 5 described later, can enumerate for example gold, silver, copper, platinum, platinum respectively or contain the metal material of their alloy and so on, the metal oxide based material of ITO and so on, the carbon-based material of carbon and so on etc.
On active electrode 3, be provided with enzyme electrode layer 7 by basalis (middle layer) 6.
As shown in Figure 1, enzyme electrode layer 7 is configured to its at least a portion (in the present embodiment for top) and exposes to sample receiving space 2, comprise have along with and target between ejected electron (e
-) and the protein of the physiologically active that reacts (is designated hereinafter simply as " protein ".)。
Be not particularly limited as this protein, for example can enumerate the polypeptide of enzyme, antibody and so on or oligopeptides etc., wherein, preferred enzyme.Like this, in the reaction between protein (enzyme) and target (matrix), can be more stable and repeatability ejected electron (e well
-).That is, can be more stable and repeatability carry out reaction between enzyme and the matrix well.
As such enzyme, suitably select according to the kind of the target that becomes determination object, for example can use various oxidase or various dehydrogenasas etc.
As oxidase, for example can use glucose oxidase, galactose oxidase, pyruvate oxidase, D-or L-amino acid oxidase, amine oxidase, cholesterol oxidase, choline oxidase etc.
In addition, as dehydrogenasa, for example can use alcohol dehydrogenase, glutamte dehydrogenase, cholesterin dehydrogenase, aldehyde dehydrogenase, glucose dehydrogenase, Fructose dehydrogenase, SODH, glycerol dehydrogenase etc.
In addition, in sensor of the present invention, it is polyethers based compound below 2000 that enzyme electrode layer (film that forms by film formation method of the present invention) 7 contains weight-average molecular weight.For being the effect that the polyethers based compound below 2000 obtains, describe in detail in the manufacture method of Shuo Ming sensor 1 in the back by in enzyme electrode layer 7, containing weight-average molecular weight.
In addition, preferably in enzyme electrode layer 7, contain the amboceptor (media material) that basad layer 6 side are propagated the electronics of emitting.Like this, amboceptor becomes the media that electronics moves, and electronics is moved to active electrode 3 by basalis 6 from enzyme electrode layer 7.
As amboceptor, for example can enumerate nicotinamide derivates, the phenazine methosulfate, 2 of the flavin derivatives, nicotinamide adenine dinucleotide (NAD), nicotinamide-adenine dinucleotide phosphate (NADP) and so on of ferrocene or ferrocene derivatives, the potassium ferricyanide, dicyclopentadienyl nickel or dicyclopentadienyl nickel derivant, quinone or quinone derivative (for example 1,4-benzoquinone, pyrro-quinoline quinone (PQQ) etc.), flavin adenine dinucleotide (FAD) (FAD) and so on, 6-dichloroindophenol, six cyanogen close iron (III) hydrochlorate, eight cyanogen close tungsten ion etc., can use wherein a kind of or make up more than 2 kinds and use.
Constituent material as basalis 6, be not particularly limited, for example can enumerate natural polymer, synthetic high polymer (synthetic resin) or their modifier etc. of the relevant macromolecule (macromolecule of animal origin) of biosome or plant origin macromolecule and so on, can use wherein a kind or make up use more than 2 kinds.Wherein, be principal ingredient preferably with biosome source macromolecule.By constituting basalis 6 by this material, can transport electronics from enzyme electrode layer 7 to active electrode 3 by basalis 6 reliably, can prevent the physiologically active inactivation of protein simultaneously better.
As the relevant macromolecule of such biosome, for example can enumerate albumin (bovine serum albumin(BSA) for example: BSA), globulin, myoglobin etc., as basalis 6, for example as being the material that principal ingredient contains, preferably use the blended polymer of blended polymer, polyvinyl pyrrolidone and BSA of carboxymethyl cellulose and BSA and the blended polymer of polyglycol and BSA etc. with the relevant macromolecule of such biosome.
In addition, this basalis 6 is by the constituent material of combined protein matter and active electrode 3, even their contacts also can suppress or prevent the reduction of the physiologically active of protein, so also can omit its formation.
Be provided with electrode 4, opposed and at least a portion of itself and active electrode 3 (in the present embodiment for following) is exposed to sample receiving space 2.
This to electrode 4 be and active electrode 3 between apply the electrode of voltage.Under the state of sample receiving space 2 feed fluid samples 20, according to making active electrode 3 just be, electrode 4 is negative mode, to between these electrodes, apply voltage, at this moment, the electronics of emitting (generation) by the reaction between target and the protein is switched at active electrode 3 and between to electrode 4 to active electrode 3 side shiftings.
To active electrode 3 and the voltage that applies between to electrode 4 be preferably below the 1.0V, more preferably about 0.1～0.5V.By making the value that applies voltage in above-mentioned scope, active electrode 3 can be carried out and especially rapidly and reliably to the energising between the electrode 4.In addition, can also prevent the generation of the electronics that (prevention) caused by electrolysis of water etc. reliably.
Reference electrode 5 is fixed in sealing (wall part) 10, and makes its part be positioned at sample receiving space 2.This reference electrode 5 is the electrodes that are used for the normal potential of regulation (setting) relativity electrode 3.By this ginseng reference electrode 5, required standard current potential are set, can improve the mensuration precision or the repeatability (when using different sensors 1 or fluid sample 20, lower measured value and produce deviation) of the amount of target.
As mentioned above, such sensor 1 is used to the amount that tracer liquid sample 20 hits, and for example is used to the monitoring etc. of diabetic's blood glucose value.
In aforesaid sensor 1, if in sample receiving space 2 feed fluid sample 20, diffusion in the targeting enzymes electrode layer that then contains in the fluid sample 20 7.To the target of enzyme electrode layer 7 diffusion by reacting and ejected electron with protein.
For example, be glucose at target, protein is under the situation of glucose oxidase, shown in following formula 1, glucose is oxidized under the catalytic action of glucose oxidase, is broken down into gluconic acid, generation this moment, ejected electron.
C
6H
10O
6+2H
2O+2O
2→C
6H
8O
6+3H
2O
2
→ C
6H
8O
6+ 3O
2+ 6H
++ 6e
-Formula 1
Then, be under the situation of the potassium ferricyanide at amboceptor, by the electronics that produces in the above-mentioned formula 1, shown in following formula 2, fewrricyanic acid ion is reduced to the ferrocyanic acid ion.
[Fe (III) (CN)
6]
3-+ e
-→ [Fe (II) is (CN)
6]
4- Formula 2
At this moment, for make active electrode 3 for just, to electrode 4 for negative, when applying voltage between these electrodes 3,4, then near active electrode 3, the ferrocyanic acid ion is oxidized to fewrricyanic acid ion, it is electric current that the result produces electronics.
Thereby, when applying certain voltage to active electrode 3 with between to electrode 4, by detecting value of current flowing between these electrodes, the target amount that contains in can indirect determination fluid sample 20.
Particularly, prepare the known a plurality of samples of target amount, use each sample to be determined at active electrode 3 and, try to achieve the relation between the current value of target amount and mensuration value of current flowing between the electrode 4.That is, make inspection amount line or form.Then, use, be determined at active electrode 3 and,, be converted into the amount of target from the value of measuring based on inspection amount line or the form made in advance to value of current flowing between the electrode 4 in the fluid sample 20 of practical measurement.Like this, can be in the hope of the amount of the target that contains in the fluid sample 20.
In addition, the program of carrying out these operations is built in reading device (determinator) usually, mensuration person only need be in reading device 1 sensor installation 1, feed fluid sample 20 in the sample receiving space 2 of sensor 1 just can be known the amount of target.
As described above, this sensor 1 can be with the electronics of being emitted by the reaction between target and the protein, as value of current flowing at active electrode 3 and between to electrode 4 and detect (mensuration) and come out, can measure the amount of the target that contains in the fluid sample 20.
In addition, in the present embodiment, to the reaction between sensor 1 target and the protein as current value detect, the mode that goes out the amount of target according to the size detection of this current value is illustrated, but in sensor of the present invention, it also can be sensor as described below, promptly use to show the reagent of color reaction, for example variation indirect determination target of the absorbance by observing this reagent and the reaction between the protein that is to say the sensor of mode of the amount that can be the optical detection target.
The manufacture method of＜sensor 〉
Such sensor 1 for example can as described belowly be made.
Fig. 2 is the longitudinal diagram that is used for illustrating respectively the manufacture method of sensor shown in Figure 1.In addition, in the following description, the upside among Fig. 2 be " on ", downside is a D score.
[1] at first, shown in Fig. 2 (a), prepared substrate 8.
Then, shown in Fig. 2 (b), on this substrate 8, form active electrode 3.This active electrode 3 for example can form by mode as follows.
At first, form top (electrode forming surface) of metal film (metal level) with covered substrate 8.This for example can pass through the chemical vapor deposition method (CVD) of plasma CVD, hot CVD, laser CVD and so on, vacuum evaporation, dry type plating methods such as gunite, ion plating method, wet type plating methods such as plating, dipping plating, electroless plating apply, flame spraying, MOD method, formation such as the joint of metal forming.
Then, on this metal film, after coating (supply) anticorrosive additive material it is solidified, form the shape resist layer corresponding with the shape of active electrode 3.
Then, this resist layer as mask, is removed the part of no use of metal film.Remove this metal film and for example can use that plasma etching, active-ion-etch, light beam etching, light help physical etch methods such as etching, in the chemical method for etching such as wet etching etc. a kind or make up more than 2 kinds.
Then, by removing resist layer, can obtain active electrode 3.
In addition, active electrode 3 for example also can contain after the conductive material of electroconductive particle by coating (supplys) on substrate 8, as required, this enforcement aftertreatment of filming (for example heating, ultrared irradiation, hyperacoustic give etc.) is formed.
At this, in rubbing method, for example can enumerate that spin-coating method, casting method, little gravure coating method, gravure coating method, rod are coated with method, rolling method, wire bar coating process, dip coating, spraying process, silk screen print method, aniline printing method, hectographic printing method, ink-jet method, microcontact print process etc., can use wherein a kind or make up more than 2 kinds.As rubbing method, wherein, especially preferably use ink-jet method.According to ink-jet method, can supply with conductive material to the zone of the needs of substrate 8, form the active electrode 3 that needs shape so can not form aforesaid resist layer.
[2] then, shown in Fig. 2 (c), on active electrode 3, form basalis 6.
State biosome source macromolecule in the use and constitute under the situation of basalis 6, this basalis 6 can as described belowly form.
At first, biosome is originated macromolecule dissolution in solvent, obtain liquid material.
As this solvent, for example can enumerate inorganic solvents such as phosphoric acid, nitric acid, sulfuric acid, ammonia, hydrogen peroxide, water, or N, dinethylformamide (DMF), N, N-dimethyl acetamide (DMA), N-N-methyl-2-2-pyrrolidone N-acid amides series solvents such as (NMP), sulphur compound series solvent such as dimethyl sulfoxide (DMSO), sulfolane or contain these mixed solvent etc.
Biosome in the above-mentioned liquid material high molecular concentration (content) of originating is preferably below the 20wt%, more preferably about 1～10wt%.
Then, can on active electrode 3, supply with the liquid material of preparation, form by dry afterwards.As the method for on active electrode 3, supplying with liquid material, for example in aforesaid rubbing method, especially preferably use ink-jet method.
[3] then, shown in Fig. 2 (d), on basalis 6, form enzyme electrode layer 7.
At this, when the regulation regioselectivity on the relative basalis 6 and so on forms enzyme electrode layer 7 film such as grade, the preferred method from ink-jet method to the zone of regulation that supply with the fluid composition that contains protein by of using.
But, as illustrated in the above-mentioned background technology, if form film by ink-jet method, then the polyethers based compound that contains for the viscosity of adjusting printing ink (fluid composition) or surface tension contacts with protein, produces the physiologically active reduction (inactivation) of protein therefrom.
The inventor etc. concentrate on studies to this problem, and it is closely related that the result is judged as the weight-average molecular weight of polyethers based compound of protein inactivation and contacted protein.
Then, the inventor etc. have further carried out inquiring into repeatedly, found that, be set at below 2000 by weight-average molecular weight polyether compound, the stability of protein can improve, and can prevent or suppress its physiologically active well and reduce, so that finish the present invention.
Below, the method for using film formation method of the present invention to form enzyme electrode layer 7 is described in detail.
[3-1] at first, prepare (preparation) contain protein, and weight-average molecular weight be the fluid composition (fluid composition of the present invention) of the polyethers based compound below 2000.(the 1st operation).
The solvent that uses during as the preparation fluid composition, for example can enumerate inorganic solvents such as phosphoric acid, nitric acid, sulfuric acid, ammonia, hydrogen peroxide, water, or N, dinethylformamide (DMF), N, N-dimethyl acetamide (DMA), N-N-methyl-2-2-pyrrolidone N-acid amides series solvents such as (NMP), sulphur compound series solvents such as dimethyl sulfoxide (DMSO), sulfolane, or contain their mixed solvent etc.
At this, in order to form (film forming) enzyme electrode layer 7 on basalis 6 with outstanding film-forming accuracy, viscosity and surface tension in order to adjust fluid composition contain the polyethers based compound in fluid composition.
It is below 2000, to be preferably about 300～1000 that such polyethers based compound is configured to its weight-average molecular weight.By above-mentioned weight-average molecular weight is set in this scope, the viscosity and the surface tension of fluid composition can be set in the preferred range.And then when polyethers based compound contacted protein, the polyethers based compound is difficult to steric restriction is caused at the activity of proteins position.So, can improve the stability of protein, keep its physiologically active well.
And then when the weight-average molecular weight with this polyethers based compound was made as C, the deviation of above-mentioned polyethers based compound was preferably in the scope of ± 0.2C, more preferably in the scope of ± 0.1C.Like this, the size of the molecular weight of the polyethers based compound that contacts with protein is by homogenization, so can reduce the probability that contacts with the big polyethers based compound of molecular weight.Its result can prevent better or material that inhibition and molecular weight are big contacts the reduction of the physiologically active of the protein that causes.
In addition, the polyethers based compound preferably have the main chain that constitutes by polyethers and have at least one end of this main chain can with the combining base and (be designated hereinafter simply as " in conjunction with base " of protein bound.) compound.Like this, in fluid composition, when protein contacted with the polyethers based compound, this was in conjunction with base and protein bound, and polyethers based compound and protein link.Like this, the connecting airtight property raising between protein and the polyethers based compound.Its result, the stability of protein further improves, and can suppress or prevent the reduction of its physiologically active better.
And then the polyethers based compound more preferably has in above-mentioned two ends in conjunction with base.Like this, the connecting airtight property between protein and the polyethers based compound further improves, and can bring into play aforesaid effect more significantly.
Particularly, the combination base is not particularly limited, for example can enumerates at its end the combination base that contains amino, carboxyl, N-hydroxy-succinamide ester (NHS) base, mercapto, dimaleoyl imino, contains the disulfide bond of followingization 1 and so in conjunction with base and halogen etc.
[changing 1]
For example form amido link, protein is combined with the polyethers based compound by the amino carboxyl that has with protein.The amino that has by carboxyl and protein forms amido link, and protein is combined with the polyethers based compound.The amino that has by N-hydroxy-succinamide ester group and protein replaces, and protein is combined with the polyethers based compound.And then, by replacing of above-mentionedization 1 expression, protein is combined with the polyethers based compound in conjunction with the basic mercapto that has with protein.
In addition, as mentioned above, the polyethers based compound except with can with combine its at least one end of base replacement of protein bound, for example can also be with not replacing its at least one end with the substituting group of protein bound or not having replacement.
Substituting group as such is not particularly limited, and for example can enumerate hydrocarbon cyclic base, hydroxyl of alkyl, phenyl, xenyl, cyclohexyl and so on etc.
As the main chain that constitutes with polyethers, for example can enumerate polyglycol, polypropylene glycol and polymethylene glycol etc., can use wherein a kind or make up use more than 2 kinds, wherein, preferably mainly constitute by polyglycol.In other words, the polyethers based compound is that principal ingredient constitutes with the polyglycol based compound preferably.Like this, can be more easily the viscosity and the surface tension of fluid composition be set in the preferred range.
As the object lesson of aforesaid polyethers based compound, for example can enumerate polyglycol based compound as described below.
[changing 2]
Wherein, above-mentioned various in, n represents the integer more than 1, for example under the weight-average molecular weight with the polyglycol based compound was made as situation about 300～1000, n was set to about 5～20.
In addition, be made as A[wt% at content with the polyethers based compound in the fluid composition], Protein content is made as B[wt%] time, A/B preferably satisfies 9～10
5Relation, more preferably satisfy 20～10
4Relation.Satisfy above-mentioned relation by content and the Protein content that makes the polyethers based compound, the polyethers based compound is set in the preferred range with respect to the adhesive rate of protein, can suppress or prevent the reduction of the physiologically active of protein better.
In addition, the Protein content in the fluid composition is preferably below the 10wt%, more preferably about 0.01～5wt%.Like this, in the enzyme electrode layer 7 that forms, fully reaction takes place between the target that can and contain in fluid sample 20.Its result, more effectively ejected electron.
The content of the polyethers based compound in the fluid composition is preferably more than the 90wt%, more preferably about 95～99.9wt%.If the content of polyethers based compound is too much, according to the kind of polyethers based compound, the viscosity of fluid composition can increase, and makes fluid composition can become impossible as the drop ejection by ink-jet method sometimes.In addition, if the content of polyethers based compound is very few, fluid composition can become dry easily.And then, be set in this scope by content the polyethers based compound, the adhesive rate of polyethers based compound with respect to protein can be set in the preferred scope, the reduction that suppresses or prevent goodly the physiologically active of protein is more arranged.
In addition, also can add amboceptor etc. as required in the fluid composition.
When containing amboceptor in fluid composition, its content is preferably about 0.1～1.0wt%, more preferably about 0.1wt%.Content by making amboceptor can in the formed enzyme electrode layer 7, make the electronics of emitting from target reliably and promptly move to active electrode 3 in back operation [3-3] by basalis 6 in above-mentioned scope.
Viscosity to aforesaid fluid composition (printing ink) is not particularly limited, and viscosity at normal temperatures is preferably below the 20cP usually, more preferably about 4～10cP.If viscosity in such scope, can make fluid composition become drop by ink-jet method and stably spray.
In addition, the surface tension of solvent is not particularly limited, is preferably usually about 20～70mN/m, more preferably about 30～50mN/m.By surface tension being set in such scope, can utilize drop by the ink-jet method ejection, on basalis 6, form the liquid tunicle of homogeneous thickness.
[3-2] then uses (one side side) supply fluid composition on the basad layer 6 of ink-jet method, forms liquid tunicle (the 2nd operation).
At this, the viscosity of fluid composition and surface tension are set in the aforesaid scope, so can form the liquid tunicle of homogeneous thickness simultaneously stably as drop ejection (supply) fluid composition on basalis 6.
[3-3] then by the liquid tunicle that drying forms, forms enzyme electrode layer 7 (the 3rd operation) on basalis 6.
At this, weight-average molecular weight by making the polyethers based compound that contains in the enzyme electrode layer 7 as of the present invention be below 2000, shown in present embodiment, be used to utilize active electrode 3, obtaining by giving and accepting of the electronics between active electrode 3 and protein or the target etc. under the situation of sensor 1 of electric signal, the distance between active electrode 3 and protein or the target is set to the distance that is fit to obtain electric signal.
The temperature of the atmosphere during dry liquid tunicle is preferably about 20～80 ℃, more preferably about 20～45 ℃.
Be preferably drying time about 10～150 minutes, more preferably about 30～100 minutes.
Condition enactment by with the liquid tunicle of drying the time can prevent the reduction of the physiologically active of the protein that contains in the liquid tunicle reliably in this scope, liquid tunicle is formed the enzyme electrode layer 7 of homogeneous thickness by rapidly and dry reliably simultaneously.
In addition, under the end of polyethers based compound has in conjunction with basic situation, condition enactment by with the liquid tunicle of drying the time is in aforesaid scope, can make the protein that contains in the liquid tunicle more reliably and combine basic combination, improve the polyethers based compound more reliably with respect to combination of proteins (binding) rate.
By through aforesaid operation, can suppress or prevent on basalis 6, to form the outstanding enzyme electrode layer 7 of film-forming accuracy under the state that physiologically active that protein has reduces well.
In addition, weight-average molecular weight is the polyethers based compound below 2000, it has high-affinity with respect to the material that the protein with physiologically active of enzyme and so on or nucleic acid etc. have polar group like that, so as present embodiment, using enzyme as detecting with under the situation of material, it is effective especially with film to use film formation method of the present invention to form detection.
In addition, in the present embodiment, to supplying with the method for liquid material, promptly spray method with the drop form as drop as zone to regulation, use the situation of ink-jet method to be illustrated, but be not limited to such situation, as drop ejection method, for example also can use bubble proof (" frothing " is registered trademark) etc.
[4] then, shown in 2 (e), configuration is to electrode 4 and to make it opposed with active electrode 4, at the position configuration reference electrode 5 of regulation, under this state, for example after peripheral part supply bonding agent, makes its curing simultaneously, forms sealing 10 thus.Like this, with enzyme electrode layer 7, electrode 4 and sealing 10 are formed sample receiving space 2.
As bonding agent, for example can enumerate epoxy and be bonding agent, acrylic acid series bonding agent, rubber is bonding agent etc.
In addition, at this moment, be formed for the inlet (supply port) 101 of feed fluid sample 20 in sample receiving space 2 at sealing 10.
Utilize above operation, can obtain sensor shown in Figure 11.
More than based on illustrated embodiment, substrate, sensor and the fluid composition of film formation method of the present invention, band film is illustrated, but the present invention is not limited by these.
For example, film formation method of the present invention also can be appended operation 1 road of any purpose or more than 2 roads.
And then the substrate of band film of the present invention is except can being used for aforesaid sensor, and the protein that for example can also be used for having supported on the substrate protein with physiologically active is with microarray, gene analysis chip etc.
In addition, constitute the each several part of sensor of the present invention, can replace with the part of the arbitrary structures that can bring into play said function.
For example, at each interlayer, in not causing the scope that sensor characteristic reduces, the layer that any purpose (raising of connecting airtight property) also can be set is more than 1 layer.
In addition, in the present embodiment,, describe, but do not limited by such situation so that be representative as the sensor that detects with material, detects the electronics that utilizes the reaction generation between itself and the target with protein with physiologically active.
For example, use material, also can use the material except protein and not have the protein material in addition of physiologically active with physiologically active as detecting.
In addition, as the material beyond the protein with physiologically active, for example can enumerate the nucleic acid of DNA or RNA and so on and steroid hormone etc.
As the material beyond the protein that does not have physiologically active, for example can enumerate fluorchrome etc.
Even under the situation that these materials are used with material as detection, also can obtain the effect identical with the situation of using protein with physiologically active.But, if under detecting, use, can especially significantly see aforesaid effect, so preferred with the situation of material formation detection with film at the protein that uses enzyme to have physiologically active like that.
[embodiment]
Then specific embodiments of the invention are described.
1. the preparation of fluid composition
(sample No.1)
With glucose oxidase (300units/mg; Protein) and with polyglycol based compound (polyethers based compound), ferrocene (amboceptor) that followingization 3 represented be dissolved in phosphate buffer (10mM; PH7.8), preparation fluid composition.
[changing 3]
In addition, the protein in the mixes liquid composition, polyglycol based compound and amboceptor make its concentration in fluid composition be respectively 100units/mL, 1.0mmol/mL and 0.01mmol/mL.
(sample No.2)
As the polyethers based compound, use the polyglycol based compound of followingization 4 expressions, replace the polyglycol based compound of above-mentionedization 3 expressions, in addition, similarly make fluid composition with above-mentioned sample No.1.
[changing 4]
(sample No.3)
As the polyethers based compound, use the polyglycol based compound of followingization 5 expressions, replace the polyglycol based compound of above-mentionedization 3 expressions, in addition, similarly make fluid composition with above-mentioned sample No.1.
[changing 5]
(sample No.4)
As the polyethers based compound, use the polyglycol based compound of followingization 6 expressions, replace the polyglycol based compound of above-mentionedization 3 expressions, in addition, similarly make fluid composition with above-mentioned sample No.1-.
[changing 6]
(sample No.5)
As the polyethers based compound, use the polyglycol based compound of followingization 7 expressions, replace the polyglycol based compound of above-mentionedization 3 expressions, in addition, similarly make fluid composition with above-mentioned sample No.1.
[changing 7]
(sample No.6)
Except the interpolation of omitting polyethers based compound and amboceptor, similarly make fluid composition with above-mentioned sample No.1.
2: the making of sensor
In following each embodiment and comparative example, make 5 sensors respectively.
(embodiment 1)
＜1〉at first, prepares glass substrate, utilize vacuum evaporation, form the Ag electrode (active electrode) of average thickness 200nm.
＜2〉then, will be dissolved in the pure water as the relevant high molecular albumin of biosome, preparation contains albuminous solution.
Then, use ink-jet method on active electrode, to supply with this and contain albuminous solution, form liquid tunicle, under atmospheric pressure atmosphere, carry out drying afterwards, obtain basalis with 60 ℃ * 30 minutes condition.
＜3〉then, to ink-jet printer (Seiko Epson corporate system, " PM700 ") supply with the fluid composition of sample No.1 in the ink chamber that possessed, then by making the piezoelectric element action that is arranged at this ink chamber, to provide on the basad layer of the form of drop.Like this, on basalis, form liquid tunicle.In addition, the viscosity of fluid composition and surface tension are respectively 3.6cP and 32mN/m, and the vibration of piezoelectric element is carried out under the condition of driving voltage 28V, frequency 10KHz.
Then, should the liquid state tunicle in drying under the atmospheric pressure atmosphere, under 50 ℃ * 40 minutes conditions, forming average thickness on basalis is the enzyme electrode layer of 50nm.
＜4〉then, configuration platinum system to electrode and to make it opposed with active electrode, simultaneously the reference electrode of carbon system is disposed at the position of regulation, under this state, for example after peripheral part supply epoxy is bonding agent, make its curing, form sealing thus.Like this, with responding layer, electrode and sealing are formed the sample receiving space.
In addition, at this moment, be formed for the inlet of feed fluid sample in the sample receiving space at sealing.
Utilize above operation, obtain sensor.
(embodiment 2)
In above-mentioned operation＜3〉in, as fluid composition, use sample No.2 to replace sample No.1, in addition, make sensor in the same manner with embodiment 1.
(embodiment 3)
In above-mentioned operation＜3〉in, as fluid composition, use sample No.3 to replace sample No.1, in addition, make sensor in the same manner with embodiment 1.
(embodiment 4)
In above-mentioned operation＜3〉in, as fluid composition, use sample No.4 to replace sample No.1, in addition, make sensor in the same manner with embodiment 1.
(comparative example)
In above-mentioned operation＜3〉in, as fluid composition, use sample No.5 to replace sample No.1, in addition, make sensor in the same manner with embodiment 1.
3. estimate
3-1. the evaluation of fluid composition
The fluid composition 1mL of each sample No. adds the evaluation reagent 1mL that contains HRP (horseradish peroxidase (Horse radish peroxidase)), 4-amino-antipyrine (4-AA) and phenol relatively.
In addition, mix HRP, 4-AA and the phenol estimated with in the reagent, and make it be respectively 100units/mL, 1.0mmol/mL and 1.0mmol/mL with the concentration in the reagent in evaluation.
In having added the fluid composition of such evaluation, add 0.001mmol/mL D/W 1mL with each sample No. of reagent.
Like this, utilize the hydrogen peroxide that generates in the 1st stage of above-mentioned formula 1, the reaction shown in following formula 3 is carried out, producing red quinone is pigment.
[changing 8]
Quinone is a pigment molecular
Then, by measuring the absorbance under the wavelength 505nm, determine the growing amount that this redness quinone is a pigment, thus the activity degree of the glucose oxidase (protein) that contains in the fluid composition of each sample No. of indirect determination.
In addition, to the fluid composition of each sample No., carry out the mensuration of 5 absorbances (activity degree) respectively.
Then, be reference value with the activity degree of the glucose oxidase in sample No.6, measured, the activity degree of in each embodiment, measuring according to the benchmark evaluation in following 4 stages respectively.
◎: with respect to the activity degree of sample No.6 is more than 0.80 times below 1.00 times
Zero: the activity degree with respect to sample No.6 is more than 0.60 times and less than 0.80 times
△: the activity degree with respect to sample No.6 is less than 0.60 times more than 0.40 times
*: the activity degree with respect to sample No.6 is less than 0.4 times.
Respectively these evaluation results are shown in table 1.
[table 1]
| The polyglycol based compound | Current value | |
| Sample No.1 (the present invention) | |
○ |
| Sample No.2 (the present invention) | |
○～◎ |
| Sample No.3 (the present invention) | Change 5 | ○～◎ |
| Sample No.4 (the present invention) | |
◎ |
| Sample No.5 (comparative example) | |
× |
As shown in table 1, the fluid composition of sample No.1～4 (fluid composition of the present invention) all can obtain comparing with the fluid composition (comparative example) of sample No.5, and the activity degree of the glucose oxidase that contains in the fluid composition (protein) becomes big result.
Thus can be clear and definite, in fluid composition of the present invention, the reduction (inactivation) of the physiologically active of protein (enzyme) is suppressed well or is prevented.
In addition, the fluid composition that contains sample No.2～4 of the glucose oxidase (protein) that links with the polyglycol based compound, compare with the fluid composition of the sample No.1 that contains not the protein that links with the polyglycol based compound, the activity degree of glucose oxidase (protein) has the trend that becomes bigger.
Further can be clear and definite, the material that the weight-average molecular weight of polyglycol based compound is remained on more appropriate size is the fluid composition of sample No.4, can obtain the activity degree result about equally of the fluid composition of its activity degree and sample No.6, so the glucose oxidase in the fluid composition (protein) inactivation hardly.
3-2. the evaluation of sensor
For the sensor of each embodiment and comparative example,, apply voltage to active electrode and between to electrode in the sample receiving space, supplying with 0.001mmol/mL D/W (fluid sample) afterwards.Then, the maximal value that is determined between these electrodes the electric current that flows (is designated hereinafter simply as " current value ".)(μA)。
In addition, current value is measured 5 sensors respectively in each embodiment and comparative example.
In addition, the mensuration of current value by to active electrode and the voltage that applies 0.5V between to electrode carry out.
Its result, the current value of measuring in each embodiment is all big than the current value of measuring in comparative example, and its size is a backward with respect to the order of embodiment 1～4.
This result has reflected activity degree poor of the glucose oxidase that contains in the fluid composition of each the sample No. that measures in the evaluation of fluid composition.
Claims (17)
1. a film formation method is the film formation method that forms the detection usefulness film that contains the detection usefulness material that is useful on detection detected object thing in the one side side of substrate, it is characterized in that, comprising:
It is the 1st operation of the fluid composition of the polyethers based compound below 2000 with material and weight-average molecular weight that preparation contains described detection;
Use the described one side side of drop ejection normal direction to supply with described fluid composition and form the 2nd operation of liquid tunicle; With
Dry described liquid tunicle and form 3rd operation of described detection with film.
2. film formation method according to claim 1, wherein,
The content of the described polyethers based compound in establishing described fluid composition is A[wt%], to establish described detection be B[wt% with the content of material] time, A/B satisfies 9～10
5Relation.
3. film formation method according to claim 1 and 2, wherein,
Shown in described detection in the fluid composition be below the 10wt% with the content of material.
4. according to any described film formation method in the claim 1～3, wherein,
The content of the described polyethers based compound in the described fluid composition is more than the 90wt%.
5. according to any described film formation method in the claim 1～4, wherein,
Described fluid composition viscosity at normal temperatures is below the 20cP.
6. according to any described film formation method in the claim 1～5, wherein,
Described fluid composition surface tension at normal temperatures is 20～70mN/m.
7. according to any described film formation method in the claim 1～6, wherein,
When the weight-average molecular weight with the described polyethers based compound in the described fluid composition was made as C, the deviation of described polyethers based compound was in the scope of ± 0.2C.
8. according to any described film formation method in the claim 1～6, wherein,
Described polyethers based compound has the main chain that constitutes with polyethers and has at least one end of this main chain can be basic with the combination that material combines with described detection.
9. film formation method according to claim 8, wherein,
Have described in conjunction with base in two ends of described main chain.
10. according to Claim 8 or 9 described film formation methods, wherein,
In described the 1st operation, described polyethers based compound, links with material with described detection with material and the described combination that combines base by described detection.
11. according to any described film formation method in the claim 1～10, wherein,
Described polyethers based compound is a principal ingredient with the polyglycol based compound.
12. according to any described film formation method in the claim 1～11, wherein,
Described detection material is a protein.
13. film formation method according to claim 12, wherein,
Described protein is enzyme.
14. the substrate with film is characterized in that,
Its one side side at described substrate possesses by any detection film that described film formation method forms in the claim 1～13.
15. the substrate with film is to comprise to contain to be useful on the detection that detects the detected object thing substrate of the detection of material with the band film of film, it is characterized in that,
It is polyethers based compound below 2000 with material and weight-average molecular weight that described detection contains described detection with film.
16. a sensor is characterized in that,
The substrate that possesses claim 14 or 15 described band films.
17. a fluid composition is characterized in that,
Containing detection with physiologically active is polyethers based compound below 2000 with material and weight-average molecular weight.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2006045937 | 2006-02-22 | ||
| JP2006045937 | 2006-02-22 | ||
| JP2006335122 | 2006-12-12 |
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| Publication Number | Publication Date |
|---|---|
| CN101025422A true CN101025422A (en) | 2007-08-29 |
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|---|---|---|---|
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