CA2412302A1 - Chemical sensor device - Google Patents
Chemical sensor device Download PDFInfo
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- CA2412302A1 CA2412302A1 CA002412302A CA2412302A CA2412302A1 CA 2412302 A1 CA2412302 A1 CA 2412302A1 CA 002412302 A CA002412302 A CA 002412302A CA 2412302 A CA2412302 A CA 2412302A CA 2412302 A1 CA2412302 A1 CA 2412302A1
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- Prior art keywords
- sensor device
- region
- detected
- subject
- reaction
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
- G01N33/5438—Electrodes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
Abstract
A sensor device is provided for performing rapid and high-sensitivity detection. The sensor device comprising at least one support for immobilizing a subject to be detected and a cell for containing a solution in which a reaction product generated from the subject to be detected is diffused.
At least one reaction region having a constant concentration of the reaction product is formed by diffusion of the reaction product into the solution, and the reaction region is formed in such a manner that the reaction product is specifically detected in a predetermined measurement time.
At least one reaction region having a constant concentration of the reaction product is formed by diffusion of the reaction product into the solution, and the reaction region is formed in such a manner that the reaction product is specifically detected in a predetermined measurement time.
Description
.- CA 02412302 2002-10-30 . P25162 DBBCRIPTION
CHEMICAL SENSOR DBYICE
S ' TECHNICAL. FIELD
The present invention relates to a sensor devise for dateating rapidly end spec~.fically.a very small~amount of .
an analyte; such as,a chemical subetance,'a protein, a microorganiem,~ a virus, or the likd, and a detection method wing the lama. ~ ' ~ ' BACKGROUND ART ' ' ~ As a convent3~onal sensor devise for detesting a very small amount of ,an nnalyte, an envyme electrode immunosensor is known. ~ Ths sensor uses. an immobilized antibody, a labeled antibody to which an enzyme is linked, and an sleatrode,' to detest an analyte,~whiah is linked to the immobilized antibody, where, the labeled antibody linked to the analyte . ' is a sub~eat to be detected. The amount of tha analyte is deteoted as a change in the current produced by oxidation and reduction of a product by an enzyme xeaotion using the . elect=ode.. Figurs Z shows a conventional enayme ele~s~trode immunosanevr. As shown in Figure ~, in the oonventional enzyme electrode, immunosensar, antibodies ioa are immobilized an the entirety of a bottom surface of a detection ~asll~l0l. Analyte molecules (antigens) i03 in a specimen . are diffused in a solution within the cell, and~reaoh and speaifi.cally bind ~ta ~ the immobilized antibodies 10~. 14 labeled antibody 105 linked to an enzyme 104 is allowed to bind additionally to the analyte 103, resulting information of n sandwich complex. Excess antigens and labeled antibodies are washed out before adding n reaction 'solution 106 containing a ~subatra~e 107 for at1 enzyme reaction. ~Th~ substrate 107 is converted to an enzym~
reaction product x08 by, aot~.on of the eazymt. ~ The .enzyme ' reaction product 108 i~ further sub jested to oxidation and ' reduction by an electrode 3, thereby deteat~.ng the analyta.~
The potential of the. electrode needs to be held at a predetermined potential for the purpose of eleetroehemiaal oxidation and reduction of the enzyme reaction product 10a (not ahovvn in Figure Z). Thexofors. the potential of the electrode for oxidation and reduction is sot to be a potential '.
based on the potential of ~a .reference electrode placed is the.reaction solution se a.referonae potential.
'~ . . . ' , ' ,Although ~uah a conventional. enaym~ electrode inununosensor device advt~ntagsously hasp ~x relatively simple structure, detiation rate and detection sensitivity may not ~ .
be obtained to sufficient extent in a practical sense for .
Z0 some s~bjeats to be detected. Soma sensor devices employ, a labeled antibody labeled arith a fluorescent dye instead . of an enzyme. ~Onfortunately, detection of ~fluoresaence eompliaates the procedure. The present invention is v ~ provided to solve the problems of thd above-described conventional seneo~ dev3.ess . . Tk~e object of the present invention isv to eliminate the above-desaxibed problems.
,~ , the potential of the electrode for oxidation and reduction '~ is set to ba the potential of a reference electro$e placed in~the reaction solution as a reference potential.
,..... . _ .._. . . ..t: ~zz=::,-r,...... ~ . . .. . . . .. .... . . .. .. . .
. ~. . .. ,.,"_ .. .. ..
DIBCLOBURE OF THE INVENTION
Although such ei conventional enzyme electrode immunosensor advantageously has a relatively simple devise structure, a detection rate end a doteetion sensitivity cannot be obtained to sufficient extent in a practical sense, depending on the subject to be detected. Some censor device 'employs a. labeled antibody labeled with a fluorescent dye ir~staad of an enzyme.' Unfortunately, detection of 10~ fluorescence causes the proaedura to be complicated. The present invention is provided to solve the problems of the above-described tsonveritional sensor devices. , The~objeCt of the present invention is to eliminate the nbov~-described~~ ' problems.
The preseht invention relates to a sensor devise, comprising at, least one support, for fixing $ subject to be detected, and a sell for~aontaining a solution in which a reaction product, generated from the sub jest to bs detested '. 'ie diffused. At least one reaotlon rogion having a eon~stant ooneentxation of the reaction product is formed by diffusion of the reaction product into the solution, and the reaat~.on region is formed in such ~a manner that the reaction product . 'isspecifiaeliydetootedinapredetexminet~measuremdnttime.
2S . . . . . , Preferably, the sensor device comprises a p~.ux~ality of reaction regions. The plurality of reaction regions may be sepdrately farmed by different reaction products, . . Preferably, the sensor device may further comprise detetstion means .
The aubjeat to be detected may be immobilized by - . pasisa fixing mean.
The detection roeax~s mayy have a detection legion, aed the detection region may include the xeaction region.
Alternatively, the dete.otion meaae May have a detection region, az~d the d4tectiori region may overlap the reaction region. . .
10Alternatively, the detection, meanw may have 'a.
' detection region; and the detection region mey~ be included in the .reaction region. ' Preferably, the support may be a ~partieular region on a. bare plate. ' ' Alternatively, the support may ba a particulate o.r a rod-shaped member. .~
a0 ~ Preferably, the measurement time may be 30 minutes;
more preferably.l0 minutes, oven more~pre~erab,ly 5 minutes,.
still more. preferably 3 minutes, and most preferably 1 minute. . ~ ~ .
a5 . . The detecstion means may maasurs light or heat.
Preferably, the detection means may comprise at least one el~otxo$e. ~ .
3 Q Preferably, the e7.e~strode max sot on . tho reaction product and generate an eleatrioal eigrtal corresponding to the amount of the reaction praduCt. ~ ' ,... . . . . .... ,.~r., .~......, . . .... . .. . . . , . , .._.... . . .. ~
.,.. . . ..
- . P2~162 The sub~eot to be detected may be an.enzyme, and the reaction produat~may be an en$yme.reaatioW product.
. . The. subject to be detected may be an antibody or an 5 enzyme linked to a peptide.
.f w ~ The . f~.xinQ meetnl~ may , , be ~ an antiQea dr an antigen-antibody complex.
In one embodiment, the subject to be detected may bs an enzyme linked to a first antibody li.naced to an antigen, and the fixing means may be.a second antibo~.y, ~ .
y ' . Preferably, the subjeat~to bs detected may b~
1S . immobilized in.a region having a diameter of i~everal tens to several hundreds of.Erm, and the d~tsatiow means may be an ele~strode having a diameter of l~mm or less.
In one embodiment, when the subject to be deteoted-is measured, the electrode may approach the region in which the subject to be detected is immobilised. _ . Prafsrably, the electrode may be provided on the support, and tha subject to be detected may be immobilized 28 in a region having a diameter of several tens to several hundreds of dun. , ". Preferably, tile eub~oct to ba detected may be immobilized ~.n a region having a diameter ,of several tens to several hundreds of fun, and.the e~.ec~trode'may surround the region.
Preferably, the support may be a material selected ..
8 ' . ' PZS162 from the group consisting of glass, aeramica, noble metals;
and r~sins: .
. In one embodiment, the deviao of the present invention , may further campriee means for yromoting the fixing of the ' sub~eat to be detected.
In one embodiment,. the promoting means may be used tn stir the solution in the 0~11.~ .
In one embodiment, the promoting means may be used to. exchange the solution in the.Qell.
. In ono embodiment, the promoting means malr be used 1S to supply the solution in the sell.
In one embodim~nt, the. promoting means may be used to allow the aolution~to flow through th~~cell.
~ BRIEF DESCRIPTION OF TTiB DRA1~INCYB
F~.gura 1 schematically shows asensor device according to the pr~ssnt invention.
2S . Figure 2 schematically shows a conventional i~cQnunosansor device.
" Figure 3 schematically shotws the prinaipl~, of the present invention. .
Figure, X11 schematically shows the principle of the .
present invention. ..
. ~ ' ' ~ P2S16Z
' Figure ~H ~.s, e~ graph showing the ~ principle of the pre~ent,invention. ~ w Figure 4C is ,~ gxaph showing the prs.nciple of the ' . present invention. ' Figure g sahematioally shows ari embodimsnt of the praoant invention. ~ ~ .
' Figure b schematically shows an amboditnant of the present invention.
Figure '7 sahamatiaally shoora art embodiment of the ~ ' praerent invention. . . , Figure a show: an exomplary support used in the .
present invention.
Figure 9 shows'an exemplary support used in the ' present invention. , ' ~ . ~ ' Figurs .1o schwmat~.cnll.y ahoaPS an embodiment of the present invention. ..
FlQure ii schematically shows an embodiment of the present invention. ' w ~ . .
Figure 1Z schematically e~ows an embodiment of the pre~ant~invention. . ' ~ .
, Figure 13 sohematioally,showe an.embodiment of the present invention.
.. . .. . . ...... ",: da:...... ~ ... . t, .... ......._. .......
pasisa F~.gure 14. s~chematicaliy shows an embodiment of the present invention. ' ' . ' .
Figure 15 s~chaa~atiaally shove an embodiment of the present ~~.nvention.
Figure 16 schematically shove an embodiment of the present invention. ' ~ .
' ~ Figure 17 sohematica.~.ly shows the principle of measurement performed by the' sensor device of the present invention. . ' Figure .1e shows a graph indicating the relationship betwaea the width of an electrode arid the time requ~rad to stabilise the electrode.
.Figure i~ shows a. graph indicating the relationship between the diameter of an electrode and the sensitivity of the electrode.
Figure 20 ~showe a graph indicating the relationship between the time: requ~.xeQ for measurement and.the output currents of electrodes having different widths, Figure 2111 is a ~ plan vises, showing a sensor devitse according to the present invention.
Y
Figure a1H 1e an enlargdd v3,av~ showing an sleatrode portion of the sensor devise of thi present inventioa~
Figure Z~ is a. plan view showing .a sensor devie7e aaoording to the present invention. .
9 ~ . P2516Z
Figure ~3 ie a diagram showing a resist pattern used 3x1 preparation of the aeneor device of the present invention.
S , Figure.Z~ 1e n cross-:eotional view showing the . eens~or.deviae of the presa»t invention. ' Figure 2s ~.~. a plan view Showing a sensor deQiae accorb.ing to the pr~eent invention.
The present invention relates to~ ti sensor device comprising at least one support .for immobilizing a sub jsat to be 8etaoted: arid a~ cell for containing a aolutiori i.n rvhiah a xeaction produot generated from the subjeot to be detected, is diffused, wherein at,~least one reaction region having a constant aoT~oentration of the reaction produot ins farmed by diffusion of the raaatian product into the solution, and the xeACtion region is formed 1n such a mannor that the reaotion product is specifically det~eted in a predotermined measurement tuns. .. ' Figure 1 shows an example. according to Embadimont 1 of the present invention. Nots.that~in explaining Figure 1 below, for the sake of simpiiaity, xeference numerals 17a ,. .
the figure; which correspond to the same portions as tho8e in a oohventional example ( Figure 2 ) , axe indicated by the same numerals . . ' As shown.in,Figure 1, antibodies. , ~,O.Z era immobilized 'on a portion of a bottom.surface of~a dateation cell 101, In this embodiment, the size of the region in which the . . . . . . . .. . .......___...._ ..... . ' . . . . . ... . . ._ . . . ... ..
.. . . . .. . . _. .... . _. _ . _:._ .. . _ . . '. ' . ~ 10 - ~ 128162 antibodies are immobilized is limited to a'diameter of s~veral tcns of.~,utt to several, hun0,reds of Eun.~ For this reason, an electrode can be use$ to detaat an enzymes reaction product rapidly and apeci,fically.
3.
In gener$1. a key favtor for achieving rapid and .apeoifio detection is to efficiently conduct a xeaction of a labeled enzyme, which is immobilized by a complex formation reaot~.bt~ among an aaalyte, an immobilized antibody and an , eazyme.labeled antibody, with an en~tyma substrtite contained in a solution. ' ' In this embodiment, the size .of ~th~ region in which ' the .antibodies .are immob~.ltzed is limited to a small range 33 . having ~ .diameter of several tens of ~m to several huadreda of Vim. Thereby, reaction of the labeled enzyme with the substrate.. and oxidation ,and reduction reactions of the ent~yme reaotiori product by tho electrode, do not depend on the diffusion rats of each substanes in the .solution, tend reactions.. ~arhich sequentially cyels .from the reaction of the enzyme with the substrate to the oxidation and reduction reactions of 'the eM$yme. reaction. product, arc aonductad within the . very smaxx ragivn. the a result, the onzyme ~reaation product iii dstaatad rdpidly and specifically.
Moreover, the region, in which detention is performed ,-by the electrode, has.substantially the same sine as that of the ~ region in , which, the . antibodies . are immobilized.
Thereby,. a noise component generated, outside the antibody-immobilized region is not aubste~ntially detec~t~rd..
As~ a result, only enzyme reactions occurring in the vicinity .
of the antibody-immobilized region arc sffiCiently deteotecl, thereby t't~ajcing it posaible~to perform measurement at a high - 11 - , paslsa sjN ('signal. to noise) ratio.
In practical. situations, is or8ex_to prepare the antibody-iirimobilizad region with precieios~ and to pos3.tion S the electrode with respect to the region with preaisioa~, ~' an appropxi~.te size of the antibody-immobilized region is a diameter of, several tens o~ ~tm to several hundreds of Vim: .
. . y~lhen the antibody-imatiobilizad region is smaller than that , size, it is difficult to accurately popition the electrode . 10 with respect to the region. when the erntibody-ft~mobilised region is larger than that siz~,. it tal~ea f~rvm seviral tens of m~.nutes to. ~saveral~ hours for a' re~acstion region of. the enzyme reaat~.on~ produot to 'be formed, as desrar~.be~cl below ' with' reference to F~.gu~e~ 3.' Therefore, as shown int this 1S embodiment., in order to achieve rapid and specific.
measurement while keeping the advantnges of the .enmyme .
eleetrode.immunosensor device, i.e., "simple structure and rapid detetstivn" , the size of the antibody-immobilized region is limited to a diameter of severed tens of E,vm to sevexal .
20 ~ hundreds of ~." ' ~n the above-des~oribed example, the antibodies are immobilized on the support. Either antigens or antibodies are irrunobilizsd err ~.tha support, depending on the purpose.
2Z A subject ~o be detested is a labeled antibody bound to an .
analyte, or an enzyme wh3ch~ is linked ~v,i~i~ an erntibody to as , analyte .
. Figure 3 i~s a schematic diagram showing the principle 30 of the present. invention. The present invantio~l is.~ not vong.trained'by~any principle. Far the sake o~ simplicity of understanding of the present invention, the present invention will b~ described with reference to figure 3. The , CA 02412302 2002-10-30 .12 - pz5lsx column to the left of .Figure 3 .shows a time course of nom . ' a ree~ation product ( e. g. , an ensyme Faction prvdubt~.j , ~tvhich is generated in a region a on a base plate 1 in whi.ch.a sub jest tv be detected i~ inunobilized. is diffused in a solution v to form' a reaction region S ( after 1 second, ' 100 seconds, wnd 1,Q00 seconds of reaation)~. .
The reaction region s beoomQe~ a layer having a thickness of about 10 Wra after 1 second o~ the reaction, a laxsr having:a thickness of about 100 dun after 10 seaor~de of the ~reaatlon, 'and a layer having a thiokne~x~ of . abaut 1000 ~uri after 1, 000 seconds of the reaction, ,for. example.
The reaction within the reaction region is~ aorltinued as long . ' as a.~reaation substrate is supplied from the avlutian. The production of the reaction product within the reaction reg3.on anti the diffusion of the reaction product from the reaativn region ' tv , the ~ solution ~ reach equilibrium after a predetermined times (typiealXy palled 8 measurement time).
As a result, a region 5' having a sub~tantie~lly constant . :ize and a aon~tant concentration of the .reaativn product is formed. As used herein, .tha.term "reaction. rs;~ion"
representatively refers to a region 5' having a,generally constant concentration of a rsact~.on prvduot at a predetermined .time after the start of a reaction beginning with the addition of the reaction substrate (i.e., the start of the diffusion of the reaction product).
," Figure ~C is a diagram representing the si:e o! the . reaction region theor~tiaally as a distance from the.reaation site which is a furivtioW of time. ' Figure ~C shows the size o~ the reaction, region when hydrogen ion is dif fused semi-infinitely in the solution,.for exampl~. . The reaction region, can be cvnsidared to~be a region enclosed with a surface . . . ... . . . . .. . .. . . . . . ...,..... ......._..~. . .., . . . .. ..
... . . . . ..: ..._ . . ... .. .. . ..:.:' . . : . . . .., ,~ . . .... _. .
~
- 1~ - . pasisz having a ocnstant oonoentration of hydrogen ion at a predetermined t~.me after the start of the. diffusion.
In general, hydrogen inn aanos#~tration at distance X
~ from the reaotion site (the start3.rig point of the ~iiffus~:on) Wfter aims ~ T ~ie xepresented by the ~squationi C
Caxerf (X/2xDTQ'r) : where Co repxesents an ~.nitial concentration of hydrogen ion at the diffusion starting ~ ' point; C represents a hydrogen ion concentration (:urfacia.~
~ concentration) at distance X and time Ts D represents the . diffusion coefficient of a hydrogen inn ( 9. 31x10' mm~/seo) f and erf represents ari error function. Tha~plat of the equation substantially forms ~Figuxe Wli1'. ~ .
Assuming that the surface concentration of hydrvgon inn is 10 ( the hydrogen~ion aonoentratian within the reaction region is considered to be oonstant and in an equilibrium .
state ) , changes. Aver time of the hydrogen ion coaoentration at predetermined ~distanaes (1 ~, 0.5 mm, 0.1 mm, and 0.05 mm) are .plotted in . Figure 4a aocvrdiiig to tha "
above-described ec~ustivn. In Figure 4H, a diamond represents a plot of the hydrog$n inn concentration at a distance of 1. mm, a square xeprssent~e a plat at a distance of 0.5 mm, a triangle represents a plot at a distance of 0.1 mm, and x represents a plot eat a distance of O.OS mm.~
Next, the time at which the hydrogen ibn concentration xe4ahes ~ (1.a.., the.hydrogen ion aoncentrativn is substantially in an equilibrium state ) is plotted with respect to a' distanoe in the graph of Figure ~C. As can be understood from this graph, when the measurement time is 30 minutes, thi reaction xegiori is 0.6. to 0. 7 mm think with reference to the reaatiort s~.te, i.e., the diffusion starting point:. when tha measurement time is l0 minutes, the reaction region is 0.3 . . . .. . . .. . ....,_...",r__m .., ...~. . . . . _ .....,.. . , . .
- 24 - P2816~
. to 0.4 mm thick; wham the measurement time is !3 minutes, the reaction ra9lon is 0.2 to . 0. 3 mm ~thio~c; when the mea~aur~ment time is 3 minutes, the reaction region ie 0.1 to 0.2 mm thickl and when the measurement time is 1 ritinute, 5~ . the reaction region is about 0.1 mm thick.
Referring again to Figure 3, the column to the right ' of Figure ~3 shows. a t3.ms course of hoot/ a detection meane9 3 (e. g., an electrode) reaches a stable operational state, corresponding to the column to the loft of Figure 3: Wh~n the detection means 8 is eatuated, the thickness of a deteoti;on region ?, iu which a eub~eet to be dsteated is ~~
detected, becomes larger over time . It takes a predetermined time'~uhtil the~deteotion means 3 operates atably. In the ' . example shosat7, ~.n .~ the column to the right of Figure 8, the thickness of the reaction region ? reaexhes a constant state about 100 seconds after the stert of the aatue~tiart ( indicated by ?' in the column to the right of Figure 3).
As used herein, the term 'detection region" of'a.
~deteotion means refers to tho detection rigion~? 'having a generally constant'sise.
. In the present invention, . th~ slew of the region 2 Z5 in which the antibodie~3 are immobiliz~d is s~t to a diameter of sevgral tens of dun to several hundreds of dun, so that the detaetion reg3.on ?' and the ret~otion region 5' are arrangid in.n rsXati,va poe~.tiorit~l relationship such that a reaction product can be detected rapidly and specifically.
Therefore, the dataation .region ?' may be disposed .in such a manner as to include or ovsxl~tp the reaction region Alternatively, the detection rsg~.oli ~?' i13 di~9pdsed in such a manner as to be inaludad in the reaction region 5'.' ' - 1' - P2S16~
Antibodies used in the present invention may b~
immobilized do the support by a method.oPel1 known to~ those.
skilled in the art. The,antibodies may bg immobilized in 3 ~a predetermine'd region, in high density pithaut impairing their functions . The density of immobilized antibodies may be increased by roughening the smoothness o~ the s~urfaoe .
of .the support by physical or chemical means. . Hy treating , the support with an appropriate surface treatment reagent, the density of immobilized antibodies may bd increased.
In' the example of .Embodiment 1 shaven in Figure 1, the reaction region faces trie electrode. The, reaction region ' ~ and the electrode may be prov~.ded an the same support, for example. ~ ~ . ~ ..
zn the example of ,Embodiment 1 shown in Figure l, an immunoeansor is provided, in wh3.ah nn antigen-antibody roaotion is detected by an en2yme reaction. The present ZO invention is not. eo limited. Far example, the sensor devise of the, present invention may be a,sensor device for detecting . the activity of microorganisms, in which the cells of the miarooxganit~ms are immobilized on a support and their activities are detested based an changes in oxygen .25 concentration around the microorganism. Further, the sensor device of the present invention may be et protein sensor device in which . an analyte is speaifieally captured or detected.with a reaction other than~an antigen-antibody reaatian.~ Moreover, the sensor devise cf the present 30 invention may be a toxin sensor for detesting a toxin or the like in a specimen, in which a support is made of a synthetic ligid membrane or the like.
~
' 1b ' P25162 Figure d schematically shows, embodim~nts of the present invention. Acs shown in Figurse 8(a), (b) and (a), . ,in the present invention, as long as the reaatfon~region 5~' and the detection reQion~ 7' are disposed ire such a manner 3 that a reaction, product can bb detested rapidly and ixpecif~aally, the detection means 3 may be diepooed in such a manner as to face the immobilized region Z on the support t Figure S ( o ) : Embodiment 1 ) , the detection means ' 9 msy be disposed on or within the ~suppvrt t (Figure 6(b)i 10, Embodimel~t. 2 j , or the dstsation moans 3 may bb dispo~ed on .the . . support and ad~eaent to . ~ths reaction ~ region 3 (Figure 5(n); 8mbodiment 3j.
. Figure b is a schematic diagram showing 8mbodiment Z
1S of the present invention (the lower device.in Figure. 6) in comparison with Embodiment 1 of the present invention ( the upper devise in. Figure b). Tn embodiment 2, a detection means 113 is disposed with in a base plate, and a subject to be detected i13 ~.s immobilized, on the detention means 113 20 via ~t fixing means 119. Concentric ellipses shoarxi. in Figures 8 represent detention ,regions of the detention means 113.
Figure .7 is a schematic diagxam showing Embodiment 4 25 of the present invention ( a sensor devise employing e~ support.
,other than abase platet a device at a lower~portion~ of . Figure .7 ) in aompaxison with Example 1 of the preseut~
invention. In Embodiment 9, the'sub3eat to be detected ilS
is immobilized via a .fixing means 119 an a support 120 in 30 the shape ~of, for example, a particulate other .thah .et~ base plate. Ellipses shooon in the figure represent detection regions of the detection means .113. In the figuxe, the particulate support 120 is shown as a single partioulatG
- 1~ - ~ PZ516.2 .disposed an a bottom surface iZi of a cell, or alternatively, there.may be a plurality of particulate supports . For example ,.
Embodiment 4 of. the present invention may oompxise a tubular cull containing a plurality of particulate euppox~ta~ therein.
Alternatively, ae. shown in Figure 8, a clustex of particulate supports may be disposed on the cell ( to the left of Figure .a ) , or a means 116 for ooilecting.particulate supports iritv a speoific region in the device may be provided ( to, the right of Figure b).
The support used in Embodiment 4 . of, the . present invention may be optionally in any shape: Figure 9 . s~ahematioally.'shows a particulate support i20 and a ~ ' xod~shaped support lid with a subj~ot to be detected 115 affixed via a fixing means ii7. .
The device of the present invention may optionally comprise any detectian means knv~tn to thosd skilled in the art. . ' ZO .' ' . Figure 10 sahamatieally shows Embodiment 5 of the present invention employing et CCD as a detection means . In this embodiment, a reaction product in a.reaotion region ' is detected by .irradiating the reaction pxodutxt with 2S ultraviolet light from a light source X41, oolleating light emitted ~rom the reaction product using a glass fiber sod, and detesting the light using e~ CCp 204.
Figure 11. aahe~riatice~Xly shows Embodiment 6 ~ of the 30 present.invention. In this embodiment, a reaction product in, a reaction region is dsteoted by oollectirig light emitt~d by the reaction produot itself using a glass fiber 20~ and a lane 203, and detesting the light using a CCD ~0~.
..... . ...._... .. '. .: . .., ~, ~_.::.__.' . . . . . . .. .... . . .r,,, ".
-.18 - ' ' p~5162 Figure.l2 achematioally shows Embodiment 7 of the present iri~rention. . In this embodiment, a renetion product in a relation region is detected by oolleeting heat ~infrar~d llg~ht ) emitted by the reaction product itself , and deteetine the 'heat using a pyroeleatric ~3o~rsor 20!i .
Figure 13 shows a vax'iant of Embodiment 2~ot~the present invention. Figure 13(a) shoyPS, a plan v~,ew and a ~aross-sectional view~of a devise in which a~region Z, in which a aubjeet to be, detecsted~ is immobilised, is provided .
on a portion of an electrode 3 disposed on a base plate 1.
Figure 13(b) is a plan view and n cross-eectional view of a d~ir~.oe in which a region 3, in which a subject to be detected i~a immobilized, is.providad at the middle of a doughnut-shaped electrode 3 disposed on a base plate 1, and on a support structure 209 of the electrode 3. figutre 13(0) is.a plan view and a arose - sectional view of a devise in which a ~egioa 3, .
in which a subject to be detected is immobilized, is provided ~
at the middle of a doughnut-shop~d electrode 3 disposed ca a bass plats'i, aal~ o» the baso plats 1.
Note that materials fox the members of the devises according to the above-described embodiments of the present' ~ invention,may be any material used in conventional sensor devises, which are known to those skilled in the art', unless , otherwise specified. Members known in the art can be used ae the ~ devices and members used. in the above-de~c~ibed embodiments of the present invention, such as a CCD, a glal~s fiber, an. ultraviolet light source, and a pyroeleatxic sensor. . ~ . .
Figure 14 is- a conceptual diagram showing .. . . .. .~ .. .,. ., ..t . ,..~~~~-.:.: ., ~... . . . ... ...~~ . . ' ~.e.~
:{::~::.':°.. . . . . :ww,n :....... d. .....~~.:.,~.: ~. ~ . ~.tr,:
- 19., PZb162 Embodiment s of the present invention. Ia this embodiment;
a detect~,on mean8 209 is allowed to approach a support 1.
in a direction ir~$icated by an arro'v when detecting a re4t~tion product _ Any arrangement knocai~ in the. art may be -uaad so as to eilciwwthe detection means 209 to approach the bd,se ' plate ~. ~ .
Figure 15 is '.a conceptual ' diagram shoorinQ
Embodiment 9 of ~tha present invent~,on. In this embodiment, a means 215 (not shown) is provided for promoting the fixing' of a subjeat.~to be detgated 115 ~Co a support 1. As 'the means 215, a means for stixring or vibratting.'a solution.
'such as a stirring rod ,(Figures 15(a) yr (b)), or a means ' for .Vibrating a support ( Figure 15 ( a ) ) may b~ used. Arty' ~ arrangement known in the art may be used Qie the ~ means ~ill5 .
for promoting the fixing of the sub jest to bs dsteatad 11S , to the support 1. , Figure . i6 is a conceptual $iagram shov~rinQ a variant of Embodiment 9.. of the present invention. In this variant, as the; means 215 ~ ~. ( not shown ) fQr promoting the f axing of the sub~eat to be detected 115 to the support ~., a means fox loading and unloading a solution in a call, a meaner for exaha~nging a solution~in a cell, a means for keeping on supply~.ng .a solution to a cell, end a means for flowing a solution through s sell, may be used. These means are knoovn in the ' .art .
H ~
Examples The ~preerent in~srentioa pill be described by way of examples . The ~xamples below are o~lly illustrations of the present invention. The pz'esent invention is not so limited.
: , .~:;. w~::: w;; ~~~:..;; ' w ~ ~~ w::. ~ v ~ ~:: :~.: . ..r....... . . .
.: ,:
'. ~- 20 ~ ~ P2516Z
(Example 1: Optimization of Detection means) Firstly, as the above-described mlectrodes 9, a sat 'of elebtrodes having a shape shown in Figure 21H(a) were prepared, where they are different, from each other only in the electrode width (W) (by depo~iting gold on~a base plate)'.
Each o~ the prepared electrodes , was set in a sensor device having 'a structure as schematically shown in Figure 1? (note that secondary antibodies for ELISA were immobilized) . The aurrer~t responss~ time of each eleotro$o was mensursd under the following conditions. ~ ' ' . ' (Menaurement Conditions) , hrunobilized antibodya seacndary nr~tibady for ELrSA (labeled with horseradish peroxidase.(HRP)).
Immobilized region a diameter 100 stn,' 0 . 5 mg/ml of~a solution tsontaining s~condary antibodies for ELISl~r was dropped and immobili$ad.
'Composition of reaction solutions ferrooenemsthanol.(FMA).
0 . 5 mMr HzOz, 5 mM; lcCl, 0 .1 M: disodium hydrogen phosphate, 0.f M. ~ .
As shown in Fieure~l?, 1n this evaluation system, a current generated by an oxidat~.on-reduction reaction of FMA coupled with an enzyme reaction wee detected to evaluates Z5, the performance of each~elactrode. Tha test results nxe shown ' in Figure 1a. In Figure 10. the horizontal~axi: represents electrode width ( Vim) ; which is the width W of the doughnut of the doughnut-shaped electrode shoran in ~'igura 21H(a).
. In Figure la , the vertical axis represents the stabilizhtion . ~ time of the electrode. Notes that the stabilization time of the electrode is defined as a time in which 90~r of the maximum current ~talue is obtained. As indicated in Figure 1~, when the elaatrode hgd d width of 100 Win, ~ths stabilization .time - zi - ~ Pz5lsx was about 300 second9~. The greater the electrode width, the greater the time reduired for stabilization. The eleotrode having a radius of 1,000 N,m (1 mm) required. about 10,000~seconds (about Z.8 hours)..
. . . .
Practically speaking, it is ooasidar~d that an appropriate~measurement~time~is ty~iaallp~ within about 1, 000 . seconds. Therefore, the electrode width needs to be less than about 500 Wn.
. Next, the same set of electrodes as above were used to aoadur~t measurement under the same conditions a,s abo~re, except that the measurement time oval fixed to about 100 .seconds, thereby determining the ae~naitivity of the aloctrodee. Tha~resulte are shown in Figuri i9. In . Figure 19,, the horizontal axis indicates' the electrode diameter (~tm) and the vertical axis indicates the electrode , sensitivity. The~eleatroda.sensitivity is represented by the difference between an output aurrentwvalue when the ZO secondary antibody for ELISA was 3.m~mobilized 8nd 'an output ' cux~x~ent value before the. immobilization of the antibody.
As shown. in Figure ~.9, whets, th~a diameter of the electrode is smaller than or equal to about 500 Er,m, the electrode.sensitivity increases with an inereasa is the electrode diameter. Nhen thd eleatrod~e diameter is about 500 Eun, the eleotrode sensitivity reaches a maximum value.
Aver about 500 ~cn,, the eleotrode sensitivity deoreases.
Further, under the same conditions. the measurement time was elongated. The measurement results are summnrized in Figure a0.
.:.. ~... . , . .... .,.,.~~,;;~T",.",:: ' ... ......::.:. .,. ~
..,.;,__,:.;.:" ,:-: :~~ . :".:~~... . ...:..,~y.....,....;.:., In Figure 20, the horizontal axis indicates the ' measurement time, and the .vertical axis indicates an output current of the electrode. Aoavrding to~the results shown in Figure Z0. the greater thw electrode width, the greater the output current . Tn this case, however, thb responwe tithe is increneod. The smaller the wloatrode width, the smaller the obtained output currant . It is. .understood that if the . measurement time af~d the electrode width are specified, am eleotrode width is uniquely determined.
~ ' ' According to the test xe~aults using the censer devi~ae shown in'this example, it is found that when the device had a raaotion region having a diameter of about 100 Vim, the optimum measurement aenaitivity was obtained in the i5 measurement taking about 1,000 seoonde or less if an' electrode having a w~.dth of about . 500 y~s~ was ussd~.
(Example Zs Preparation of Sensor Chip) Figure ,21A shows an exemplary sensor ship prepared according to the pregnant invention. ' Dashed lines Hl and H2 surrounding eubstantially oiraulax regions at the middle of Figure 2i11~ indicate regions in whioh the antibodies ørs immobil.izsd. In the figures, 'two thick .lines 30Z'indicate electrode lines, whose tips form a measuring electrode and a blank measuring alsotrods, re~speofively. Typically, these electrodes are formed by depositing gold or the like , on the base plate 1: Note that numsrioal values in the figure represent dimensions of parts in units-of ate.
. FiQure'21H is a diagram :hewing detailed structures of the measuring electrode and the blank mwasurit~g el,a~trode, which. can be used in the deviad shown in F~.gurw 2171.
Figure a1H(a) shows a structure of a sensor chip in~whiah .. .... . - ..._ "-.. ... . . .._~ , ~...~,r.~,. m.» :.. __..._. . . .. _ ......... . .. , ......... . --. , . ..,_. _ .._.....
- a~ ~ ~asisa BhawinQ a resist pattern used in production of the sensor ohip. Figure-,~4 is a cross-sectional view showin0 a situntion in which the resist .pattern was provided on the sensor chip. indicating a arose section fl~ thv el~otrods 3.
Figure $S is a plan view showing a situation in which the resist:pattern was provided on the sensor ship. .
INDUSTRIAL APPLICABILITY
A rwgion in which a subject to be' dsteatsd~ la imrnobilizsd is l.imitid to a v4ry smnll prize; and a d~tection region of a detection means and a reaction region in which a reaction product is formed ere disposed in a relative posi~'tional rela~tionah~.p such that the reaction product can is . . be detected rapidly and speoifioally,' thereby providing a sensor devise which has a relativellr ~simplo~ struaturel Bind rapidly detests avert' small amount of an analyte in a epsoimen, such as an imtnunogenie substanow (e.g., a protein, a microorganism, and a virus) or a ahemioai substance.
CHEMICAL SENSOR DBYICE
S ' TECHNICAL. FIELD
The present invention relates to a sensor devise for dateating rapidly end spec~.fically.a very small~amount of .
an analyte; such as,a chemical subetance,'a protein, a microorganiem,~ a virus, or the likd, and a detection method wing the lama. ~ ' ~ ' BACKGROUND ART ' ' ~ As a convent3~onal sensor devise for detesting a very small amount of ,an nnalyte, an envyme electrode immunosensor is known. ~ Ths sensor uses. an immobilized antibody, a labeled antibody to which an enzyme is linked, and an sleatrode,' to detest an analyte,~whiah is linked to the immobilized antibody, where, the labeled antibody linked to the analyte . ' is a sub~eat to be detected. The amount of tha analyte is deteoted as a change in the current produced by oxidation and reduction of a product by an enzyme xeaotion using the . elect=ode.. Figurs Z shows a conventional enayme ele~s~trode immunosanevr. As shown in Figure ~, in the oonventional enzyme electrode, immunosensar, antibodies ioa are immobilized an the entirety of a bottom surface of a detection ~asll~l0l. Analyte molecules (antigens) i03 in a specimen . are diffused in a solution within the cell, and~reaoh and speaifi.cally bind ~ta ~ the immobilized antibodies 10~. 14 labeled antibody 105 linked to an enzyme 104 is allowed to bind additionally to the analyte 103, resulting information of n sandwich complex. Excess antigens and labeled antibodies are washed out before adding n reaction 'solution 106 containing a ~subatra~e 107 for at1 enzyme reaction. ~Th~ substrate 107 is converted to an enzym~
reaction product x08 by, aot~.on of the eazymt. ~ The .enzyme ' reaction product 108 i~ further sub jested to oxidation and ' reduction by an electrode 3, thereby deteat~.ng the analyta.~
The potential of the. electrode needs to be held at a predetermined potential for the purpose of eleetroehemiaal oxidation and reduction of the enzyme reaction product 10a (not ahovvn in Figure Z). Thexofors. the potential of the electrode for oxidation and reduction is sot to be a potential '.
based on the potential of ~a .reference electrode placed is the.reaction solution se a.referonae potential.
'~ . . . ' , ' ,Although ~uah a conventional. enaym~ electrode inununosensor device advt~ntagsously hasp ~x relatively simple structure, detiation rate and detection sensitivity may not ~ .
be obtained to sufficient extent in a practical sense for .
Z0 some s~bjeats to be detected. Soma sensor devices employ, a labeled antibody labeled arith a fluorescent dye instead . of an enzyme. ~Onfortunately, detection of ~fluoresaence eompliaates the procedure. The present invention is v ~ provided to solve the problems of thd above-described conventional seneo~ dev3.ess . . Tk~e object of the present invention isv to eliminate the above-desaxibed problems.
,~ , the potential of the electrode for oxidation and reduction '~ is set to ba the potential of a reference electro$e placed in~the reaction solution as a reference potential.
,..... . _ .._. . . ..t: ~zz=::,-r,...... ~ . . .. . . . .. .... . . .. .. . .
. ~. . .. ,.,"_ .. .. ..
DIBCLOBURE OF THE INVENTION
Although such ei conventional enzyme electrode immunosensor advantageously has a relatively simple devise structure, a detection rate end a doteetion sensitivity cannot be obtained to sufficient extent in a practical sense, depending on the subject to be detected. Some censor device 'employs a. labeled antibody labeled with a fluorescent dye ir~staad of an enzyme.' Unfortunately, detection of 10~ fluorescence causes the proaedura to be complicated. The present invention is provided to solve the problems of the above-described tsonveritional sensor devices. , The~objeCt of the present invention is to eliminate the nbov~-described~~ ' problems.
The preseht invention relates to a sensor devise, comprising at, least one support, for fixing $ subject to be detected, and a sell for~aontaining a solution in which a reaction product, generated from the sub jest to bs detested '. 'ie diffused. At least one reaotlon rogion having a eon~stant ooneentxation of the reaction product is formed by diffusion of the reaction product into the solution, and the reaat~.on region is formed in such ~a manner that the reaction product . 'isspecifiaeliydetootedinapredetexminet~measuremdnttime.
2S . . . . . , Preferably, the sensor device comprises a p~.ux~ality of reaction regions. The plurality of reaction regions may be sepdrately farmed by different reaction products, . . Preferably, the sensor device may further comprise detetstion means .
The aubjeat to be detected may be immobilized by - . pasisa fixing mean.
The detection roeax~s mayy have a detection legion, aed the detection region may include the xeaction region.
Alternatively, the dete.otion meaae May have a detection region, az~d the d4tectiori region may overlap the reaction region. . .
10Alternatively, the detection, meanw may have 'a.
' detection region; and the detection region mey~ be included in the .reaction region. ' Preferably, the support may be a ~partieular region on a. bare plate. ' ' Alternatively, the support may ba a particulate o.r a rod-shaped member. .~
a0 ~ Preferably, the measurement time may be 30 minutes;
more preferably.l0 minutes, oven more~pre~erab,ly 5 minutes,.
still more. preferably 3 minutes, and most preferably 1 minute. . ~ ~ .
a5 . . The detecstion means may maasurs light or heat.
Preferably, the detection means may comprise at least one el~otxo$e. ~ .
3 Q Preferably, the e7.e~strode max sot on . tho reaction product and generate an eleatrioal eigrtal corresponding to the amount of the reaction praduCt. ~ ' ,... . . . . .... ,.~r., .~......, . . .... . .. . . . , . , .._.... . . .. ~
.,.. . . ..
- . P2~162 The sub~eot to be detected may be an.enzyme, and the reaction produat~may be an en$yme.reaatioW product.
. . The. subject to be detected may be an antibody or an 5 enzyme linked to a peptide.
.f w ~ The . f~.xinQ meetnl~ may , , be ~ an antiQea dr an antigen-antibody complex.
In one embodiment, the subject to be detected may bs an enzyme linked to a first antibody li.naced to an antigen, and the fixing means may be.a second antibo~.y, ~ .
y ' . Preferably, the subjeat~to bs detected may b~
1S . immobilized in.a region having a diameter of i~everal tens to several hundreds of.Erm, and the d~tsatiow means may be an ele~strode having a diameter of l~mm or less.
In one embodiment, when the subject to be deteoted-is measured, the electrode may approach the region in which the subject to be detected is immobilised. _ . Prafsrably, the electrode may be provided on the support, and tha subject to be detected may be immobilized 28 in a region having a diameter of several tens to several hundreds of dun. , ". Preferably, tile eub~oct to ba detected may be immobilized ~.n a region having a diameter ,of several tens to several hundreds of fun, and.the e~.ec~trode'may surround the region.
Preferably, the support may be a material selected ..
8 ' . ' PZS162 from the group consisting of glass, aeramica, noble metals;
and r~sins: .
. In one embodiment, the deviao of the present invention , may further campriee means for yromoting the fixing of the ' sub~eat to be detected.
In one embodiment,. the promoting means may be used tn stir the solution in the 0~11.~ .
In one embodiment, the promoting means may be used to. exchange the solution in the.Qell.
. In ono embodiment, the promoting means malr be used 1S to supply the solution in the sell.
In one embodim~nt, the. promoting means may be used to allow the aolution~to flow through th~~cell.
~ BRIEF DESCRIPTION OF TTiB DRA1~INCYB
F~.gura 1 schematically shows asensor device according to the pr~ssnt invention.
2S . Figure 2 schematically shows a conventional i~cQnunosansor device.
" Figure 3 schematically shotws the prinaipl~, of the present invention. .
Figure, X11 schematically shows the principle of the .
present invention. ..
. ~ ' ' ~ P2S16Z
' Figure ~H ~.s, e~ graph showing the ~ principle of the pre~ent,invention. ~ w Figure 4C is ,~ gxaph showing the prs.nciple of the ' . present invention. ' Figure g sahematioally shows ari embodimsnt of the praoant invention. ~ ~ .
' Figure b schematically shows an amboditnant of the present invention.
Figure '7 sahamatiaally shoora art embodiment of the ~ ' praerent invention. . . , Figure a show: an exomplary support used in the .
present invention.
Figure 9 shows'an exemplary support used in the ' present invention. , ' ~ . ~ ' Figurs .1o schwmat~.cnll.y ahoaPS an embodiment of the present invention. ..
FlQure ii schematically shows an embodiment of the present invention. ' w ~ . .
Figure 1Z schematically e~ows an embodiment of the pre~ant~invention. . ' ~ .
, Figure 13 sohematioally,showe an.embodiment of the present invention.
.. . .. . . ...... ",: da:...... ~ ... . t, .... ......._. .......
pasisa F~.gure 14. s~chematicaliy shows an embodiment of the present invention. ' ' . ' .
Figure 15 s~chaa~atiaally shove an embodiment of the present ~~.nvention.
Figure 16 schematically shove an embodiment of the present invention. ' ~ .
' ~ Figure 17 sohematica.~.ly shows the principle of measurement performed by the' sensor device of the present invention. . ' Figure .1e shows a graph indicating the relationship betwaea the width of an electrode arid the time requ~rad to stabilise the electrode.
.Figure i~ shows a. graph indicating the relationship between the diameter of an electrode and the sensitivity of the electrode.
Figure 20 ~showe a graph indicating the relationship between the time: requ~.xeQ for measurement and.the output currents of electrodes having different widths, Figure 2111 is a ~ plan vises, showing a sensor devitse according to the present invention.
Y
Figure a1H 1e an enlargdd v3,av~ showing an sleatrode portion of the sensor devise of thi present inventioa~
Figure Z~ is a. plan view showing .a sensor devie7e aaoording to the present invention. .
9 ~ . P2516Z
Figure ~3 ie a diagram showing a resist pattern used 3x1 preparation of the aeneor device of the present invention.
S , Figure.Z~ 1e n cross-:eotional view showing the . eens~or.deviae of the presa»t invention. ' Figure 2s ~.~. a plan view Showing a sensor deQiae accorb.ing to the pr~eent invention.
The present invention relates to~ ti sensor device comprising at least one support .for immobilizing a sub jsat to be 8etaoted: arid a~ cell for containing a aolutiori i.n rvhiah a xeaction produot generated from the subjeot to be detected, is diffused, wherein at,~least one reaction region having a constant aoT~oentration of the reaction produot ins farmed by diffusion of the raaatian product into the solution, and the xeACtion region is formed 1n such a mannor that the reaotion product is specifically det~eted in a predotermined measurement tuns. .. ' Figure 1 shows an example. according to Embadimont 1 of the present invention. Nots.that~in explaining Figure 1 below, for the sake of simpiiaity, xeference numerals 17a ,. .
the figure; which correspond to the same portions as tho8e in a oohventional example ( Figure 2 ) , axe indicated by the same numerals . . ' As shown.in,Figure 1, antibodies. , ~,O.Z era immobilized 'on a portion of a bottom.surface of~a dateation cell 101, In this embodiment, the size of the region in which the . . . . . . . .. . .......___...._ ..... . ' . . . . . ... . . ._ . . . ... ..
.. . . . .. . . _. .... . _. _ . _:._ .. . _ . . '. ' . ~ 10 - ~ 128162 antibodies are immobilized is limited to a'diameter of s~veral tcns of.~,utt to several, hun0,reds of Eun.~ For this reason, an electrode can be use$ to detaat an enzymes reaction product rapidly and apeci,fically.
3.
In gener$1. a key favtor for achieving rapid and .apeoifio detection is to efficiently conduct a xeaction of a labeled enzyme, which is immobilized by a complex formation reaot~.bt~ among an aaalyte, an immobilized antibody and an , eazyme.labeled antibody, with an en~tyma substrtite contained in a solution. ' ' In this embodiment, the size .of ~th~ region in which ' the .antibodies .are immob~.ltzed is limited to a small range 33 . having ~ .diameter of several tens of ~m to several huadreda of Vim. Thereby, reaction of the labeled enzyme with the substrate.. and oxidation ,and reduction reactions of the ent~yme reaotiori product by tho electrode, do not depend on the diffusion rats of each substanes in the .solution, tend reactions.. ~arhich sequentially cyels .from the reaction of the enzyme with the substrate to the oxidation and reduction reactions of 'the eM$yme. reaction. product, arc aonductad within the . very smaxx ragivn. the a result, the onzyme ~reaation product iii dstaatad rdpidly and specifically.
Moreover, the region, in which detention is performed ,-by the electrode, has.substantially the same sine as that of the ~ region in , which, the . antibodies . are immobilized.
Thereby,. a noise component generated, outside the antibody-immobilized region is not aubste~ntially detec~t~rd..
As~ a result, only enzyme reactions occurring in the vicinity .
of the antibody-immobilized region arc sffiCiently deteotecl, thereby t't~ajcing it posaible~to perform measurement at a high - 11 - , paslsa sjN ('signal. to noise) ratio.
In practical. situations, is or8ex_to prepare the antibody-iirimobilizad region with precieios~ and to pos3.tion S the electrode with respect to the region with preaisioa~, ~' an appropxi~.te size of the antibody-immobilized region is a diameter of, several tens o~ ~tm to several hundreds of Vim: .
. . y~lhen the antibody-imatiobilizad region is smaller than that , size, it is difficult to accurately popition the electrode . 10 with respect to the region. when the erntibody-ft~mobilised region is larger than that siz~,. it tal~ea f~rvm seviral tens of m~.nutes to. ~saveral~ hours for a' re~acstion region of. the enzyme reaat~.on~ produot to 'be formed, as desrar~.be~cl below ' with' reference to F~.gu~e~ 3.' Therefore, as shown int this 1S embodiment., in order to achieve rapid and specific.
measurement while keeping the advantnges of the .enmyme .
eleetrode.immunosensor device, i.e., "simple structure and rapid detetstivn" , the size of the antibody-immobilized region is limited to a diameter of severed tens of E,vm to sevexal .
20 ~ hundreds of ~." ' ~n the above-des~oribed example, the antibodies are immobilized on the support. Either antigens or antibodies are irrunobilizsd err ~.tha support, depending on the purpose.
2Z A subject ~o be detested is a labeled antibody bound to an .
analyte, or an enzyme wh3ch~ is linked ~v,i~i~ an erntibody to as , analyte .
. Figure 3 i~s a schematic diagram showing the principle 30 of the present. invention. The present invantio~l is.~ not vong.trained'by~any principle. Far the sake o~ simplicity of understanding of the present invention, the present invention will b~ described with reference to figure 3. The , CA 02412302 2002-10-30 .12 - pz5lsx column to the left of .Figure 3 .shows a time course of nom . ' a ree~ation product ( e. g. , an ensyme Faction prvdubt~.j , ~tvhich is generated in a region a on a base plate 1 in whi.ch.a sub jest tv be detected i~ inunobilized. is diffused in a solution v to form' a reaction region S ( after 1 second, ' 100 seconds, wnd 1,Q00 seconds of reaation)~. .
The reaction region s beoomQe~ a layer having a thickness of about 10 Wra after 1 second o~ the reaction, a laxsr having:a thickness of about 100 dun after 10 seaor~de of the ~reaatlon, 'and a layer having a thiokne~x~ of . abaut 1000 ~uri after 1, 000 seconds of the reaction, ,for. example.
The reaction within the reaction region is~ aorltinued as long . ' as a.~reaation substrate is supplied from the avlutian. The production of the reaction product within the reaction reg3.on anti the diffusion of the reaction product from the reaativn region ' tv , the ~ solution ~ reach equilibrium after a predetermined times (typiealXy palled 8 measurement time).
As a result, a region 5' having a sub~tantie~lly constant . :ize and a aon~tant concentration of the .reaativn product is formed. As used herein, .tha.term "reaction. rs;~ion"
representatively refers to a region 5' having a,generally constant concentration of a rsact~.on prvduot at a predetermined .time after the start of a reaction beginning with the addition of the reaction substrate (i.e., the start of the diffusion of the reaction product).
," Figure ~C is a diagram representing the si:e o! the . reaction region theor~tiaally as a distance from the.reaation site which is a furivtioW of time. ' Figure ~C shows the size o~ the reaction, region when hydrogen ion is dif fused semi-infinitely in the solution,.for exampl~. . The reaction region, can be cvnsidared to~be a region enclosed with a surface . . . ... . . . . .. . .. . . . . . ...,..... ......._..~. . .., . . . .. ..
... . . . . ..: ..._ . . ... .. .. . ..:.:' . . : . . . .., ,~ . . .... _. .
~
- 1~ - . pasisz having a ocnstant oonoentration of hydrogen ion at a predetermined t~.me after the start of the. diffusion.
In general, hydrogen inn aanos#~tration at distance X
~ from the reaotion site (the start3.rig point of the ~iiffus~:on) Wfter aims ~ T ~ie xepresented by the ~squationi C
Caxerf (X/2xDTQ'r) : where Co repxesents an ~.nitial concentration of hydrogen ion at the diffusion starting ~ ' point; C represents a hydrogen ion concentration (:urfacia.~
~ concentration) at distance X and time Ts D represents the . diffusion coefficient of a hydrogen inn ( 9. 31x10' mm~/seo) f and erf represents ari error function. Tha~plat of the equation substantially forms ~Figuxe Wli1'. ~ .
Assuming that the surface concentration of hydrvgon inn is 10 ( the hydrogen~ion aonoentratian within the reaction region is considered to be oonstant and in an equilibrium .
state ) , changes. Aver time of the hydrogen ion coaoentration at predetermined ~distanaes (1 ~, 0.5 mm, 0.1 mm, and 0.05 mm) are .plotted in . Figure 4a aocvrdiiig to tha "
above-described ec~ustivn. In Figure 4H, a diamond represents a plot of the hydrog$n inn concentration at a distance of 1. mm, a square xeprssent~e a plat at a distance of 0.5 mm, a triangle represents a plot at a distance of 0.1 mm, and x represents a plot eat a distance of O.OS mm.~
Next, the time at which the hydrogen ibn concentration xe4ahes ~ (1.a.., the.hydrogen ion aoncentrativn is substantially in an equilibrium state ) is plotted with respect to a' distanoe in the graph of Figure ~C. As can be understood from this graph, when the measurement time is 30 minutes, thi reaction xegiori is 0.6. to 0. 7 mm think with reference to the reaatiort s~.te, i.e., the diffusion starting point:. when tha measurement time is l0 minutes, the reaction region is 0.3 . . . .. . . .. . ....,_...",r__m .., ...~. . . . . _ .....,.. . , . .
- 24 - P2816~
. to 0.4 mm thick; wham the measurement time is !3 minutes, the reaction ra9lon is 0.2 to . 0. 3 mm ~thio~c; when the mea~aur~ment time is 3 minutes, the reaction region ie 0.1 to 0.2 mm thickl and when the measurement time is 1 ritinute, 5~ . the reaction region is about 0.1 mm thick.
Referring again to Figure 3, the column to the right ' of Figure ~3 shows. a t3.ms course of hoot/ a detection meane9 3 (e. g., an electrode) reaches a stable operational state, corresponding to the column to the loft of Figure 3: Wh~n the detection means 8 is eatuated, the thickness of a deteoti;on region ?, iu which a eub~eet to be dsteated is ~~
detected, becomes larger over time . It takes a predetermined time'~uhtil the~deteotion means 3 operates atably. In the ' . example shosat7, ~.n .~ the column to the right of Figure 8, the thickness of the reaction region ? reaexhes a constant state about 100 seconds after the stert of the aatue~tiart ( indicated by ?' in the column to the right of Figure 3).
As used herein, the term 'detection region" of'a.
~deteotion means refers to tho detection rigion~? 'having a generally constant'sise.
. In the present invention, . th~ slew of the region 2 Z5 in which the antibodie~3 are immobiliz~d is s~t to a diameter of sevgral tens of dun to several hundreds of dun, so that the detaetion reg3.on ?' and the ret~otion region 5' are arrangid in.n rsXati,va poe~.tiorit~l relationship such that a reaction product can be detected rapidly and specifically.
Therefore, the dataation .region ?' may be disposed .in such a manner as to include or ovsxl~tp the reaction region Alternatively, the detection rsg~.oli ~?' i13 di~9pdsed in such a manner as to be inaludad in the reaction region 5'.' ' - 1' - P2S16~
Antibodies used in the present invention may b~
immobilized do the support by a method.oPel1 known to~ those.
skilled in the art. The,antibodies may bg immobilized in 3 ~a predetermine'd region, in high density pithaut impairing their functions . The density of immobilized antibodies may be increased by roughening the smoothness o~ the s~urfaoe .
of .the support by physical or chemical means. . Hy treating , the support with an appropriate surface treatment reagent, the density of immobilized antibodies may bd increased.
In' the example of .Embodiment 1 shaven in Figure 1, the reaction region faces trie electrode. The, reaction region ' ~ and the electrode may be prov~.ded an the same support, for example. ~ ~ . ~ ..
zn the example of ,Embodiment 1 shown in Figure l, an immunoeansor is provided, in wh3.ah nn antigen-antibody roaotion is detected by an en2yme reaction. The present ZO invention is not. eo limited. Far example, the sensor devise of the, present invention may be a,sensor device for detecting . the activity of microorganisms, in which the cells of the miarooxganit~ms are immobilized on a support and their activities are detested based an changes in oxygen .25 concentration around the microorganism. Further, the sensor device of the present invention may be et protein sensor device in which . an analyte is speaifieally captured or detected.with a reaction other than~an antigen-antibody reaatian.~ Moreover, the sensor devise cf the present 30 invention may be a toxin sensor for detesting a toxin or the like in a specimen, in which a support is made of a synthetic ligid membrane or the like.
~
' 1b ' P25162 Figure d schematically shows, embodim~nts of the present invention. Acs shown in Figurse 8(a), (b) and (a), . ,in the present invention, as long as the reaatfon~region 5~' and the detection reQion~ 7' are disposed ire such a manner 3 that a reaction, product can bb detested rapidly and ixpecif~aally, the detection means 3 may be diepooed in such a manner as to face the immobilized region Z on the support t Figure S ( o ) : Embodiment 1 ) , the detection means ' 9 msy be disposed on or within the ~suppvrt t (Figure 6(b)i 10, Embodimel~t. 2 j , or the dstsation moans 3 may bb dispo~ed on .the . . support and ad~eaent to . ~ths reaction ~ region 3 (Figure 5(n); 8mbodiment 3j.
. Figure b is a schematic diagram showing 8mbodiment Z
1S of the present invention (the lower device.in Figure. 6) in comparison with Embodiment 1 of the present invention ( the upper devise in. Figure b). Tn embodiment 2, a detection means 113 is disposed with in a base plate, and a subject to be detected i13 ~.s immobilized, on the detention means 113 20 via ~t fixing means 119. Concentric ellipses shoarxi. in Figures 8 represent detention ,regions of the detention means 113.
Figure .7 is a schematic diagxam showing Embodiment 4 25 of the present invention ( a sensor devise employing e~ support.
,other than abase platet a device at a lower~portion~ of . Figure .7 ) in aompaxison with Example 1 of the preseut~
invention. In Embodiment 9, the'sub3eat to be detected ilS
is immobilized via a .fixing means 119 an a support 120 in 30 the shape ~of, for example, a particulate other .thah .et~ base plate. Ellipses shooon in the figure represent detection regions of the detection means .113. In the figuxe, the particulate support 120 is shown as a single partioulatG
- 1~ - ~ PZ516.2 .disposed an a bottom surface iZi of a cell, or alternatively, there.may be a plurality of particulate supports . For example ,.
Embodiment 4 of. the present invention may oompxise a tubular cull containing a plurality of particulate euppox~ta~ therein.
Alternatively, ae. shown in Figure 8, a clustex of particulate supports may be disposed on the cell ( to the left of Figure .a ) , or a means 116 for ooilecting.particulate supports iritv a speoific region in the device may be provided ( to, the right of Figure b).
The support used in Embodiment 4 . of, the . present invention may be optionally in any shape: Figure 9 . s~ahematioally.'shows a particulate support i20 and a ~ ' xod~shaped support lid with a subj~ot to be detected 115 affixed via a fixing means ii7. .
The device of the present invention may optionally comprise any detectian means knv~tn to thosd skilled in the art. . ' ZO .' ' . Figure 10 sahamatieally shows Embodiment 5 of the present invention employing et CCD as a detection means . In this embodiment, a reaction product in a.reaotion region ' is detected by .irradiating the reaction pxodutxt with 2S ultraviolet light from a light source X41, oolleating light emitted ~rom the reaction product using a glass fiber sod, and detesting the light using e~ CCp 204.
Figure 11. aahe~riatice~Xly shows Embodiment 6 ~ of the 30 present.invention. In this embodiment, a reaction product in, a reaction region is dsteoted by oollectirig light emitt~d by the reaction produot itself using a glass fiber 20~ and a lane 203, and detesting the light using a CCD ~0~.
..... . ...._... .. '. .: . .., ~, ~_.::.__.' . . . . . . .. .... . . .r,,, ".
-.18 - ' ' p~5162 Figure.l2 achematioally shows Embodiment 7 of the present iri~rention. . In this embodiment, a renetion product in a relation region is detected by oolleeting heat ~infrar~d llg~ht ) emitted by the reaction product itself , and deteetine the 'heat using a pyroeleatric ~3o~rsor 20!i .
Figure 13 shows a vax'iant of Embodiment 2~ot~the present invention. Figure 13(a) shoyPS, a plan v~,ew and a ~aross-sectional view~of a devise in which a~region Z, in which a aubjeet to be, detecsted~ is immobilised, is provided .
on a portion of an electrode 3 disposed on a base plate 1.
Figure 13(b) is a plan view and n cross-eectional view of a d~ir~.oe in which a region 3, in which a subject to be detected i~a immobilized, is.providad at the middle of a doughnut-shaped electrode 3 disposed on a base plate 1, and on a support structure 209 of the electrode 3. figutre 13(0) is.a plan view and a arose - sectional view of a devise in which a ~egioa 3, .
in which a subject to be detected is immobilized, is provided ~
at the middle of a doughnut-shop~d electrode 3 disposed ca a bass plats'i, aal~ o» the baso plats 1.
Note that materials fox the members of the devises according to the above-described embodiments of the present' ~ invention,may be any material used in conventional sensor devises, which are known to those skilled in the art', unless , otherwise specified. Members known in the art can be used ae the ~ devices and members used. in the above-de~c~ibed embodiments of the present invention, such as a CCD, a glal~s fiber, an. ultraviolet light source, and a pyroeleatxic sensor. . ~ . .
Figure 14 is- a conceptual diagram showing .. . . .. .~ .. .,. ., ..t . ,..~~~~-.:.: ., ~... . . . ... ...~~ . . ' ~.e.~
:{::~::.':°.. . . . . :ww,n :....... d. .....~~.:.,~.: ~. ~ . ~.tr,:
- 19., PZb162 Embodiment s of the present invention. Ia this embodiment;
a detect~,on mean8 209 is allowed to approach a support 1.
in a direction ir~$icated by an arro'v when detecting a re4t~tion product _ Any arrangement knocai~ in the. art may be -uaad so as to eilciwwthe detection means 209 to approach the bd,se ' plate ~. ~ .
Figure 15 is '.a conceptual ' diagram shoorinQ
Embodiment 9 of ~tha present invent~,on. In this embodiment, a means 215 (not shown) is provided for promoting the fixing' of a subjeat.~to be detgated 115 ~Co a support 1. As 'the means 215, a means for stixring or vibratting.'a solution.
'such as a stirring rod ,(Figures 15(a) yr (b)), or a means ' for .Vibrating a support ( Figure 15 ( a ) ) may b~ used. Arty' ~ arrangement known in the art may be used Qie the ~ means ~ill5 .
for promoting the fixing of the sub jest to bs dsteatad 11S , to the support 1. , Figure . i6 is a conceptual $iagram shov~rinQ a variant of Embodiment 9.. of the present invention. In this variant, as the; means 215 ~ ~. ( not shown ) fQr promoting the f axing of the sub~eat to be detected 115 to the support ~., a means fox loading and unloading a solution in a call, a meaner for exaha~nging a solution~in a cell, a means for keeping on supply~.ng .a solution to a cell, end a means for flowing a solution through s sell, may be used. These means are knoovn in the ' .art .
H ~
Examples The ~preerent in~srentioa pill be described by way of examples . The ~xamples below are o~lly illustrations of the present invention. The pz'esent invention is not so limited.
: , .~:;. w~::: w;; ~~~:..;; ' w ~ ~~ w::. ~ v ~ ~:: :~.: . ..r....... . . .
.: ,:
'. ~- 20 ~ ~ P2516Z
(Example 1: Optimization of Detection means) Firstly, as the above-described mlectrodes 9, a sat 'of elebtrodes having a shape shown in Figure 21H(a) were prepared, where they are different, from each other only in the electrode width (W) (by depo~iting gold on~a base plate)'.
Each o~ the prepared electrodes , was set in a sensor device having 'a structure as schematically shown in Figure 1? (note that secondary antibodies for ELISA were immobilized) . The aurrer~t responss~ time of each eleotro$o was mensursd under the following conditions. ~ ' ' . ' (Menaurement Conditions) , hrunobilized antibodya seacndary nr~tibady for ELrSA (labeled with horseradish peroxidase.(HRP)).
Immobilized region a diameter 100 stn,' 0 . 5 mg/ml of~a solution tsontaining s~condary antibodies for ELISl~r was dropped and immobili$ad.
'Composition of reaction solutions ferrooenemsthanol.(FMA).
0 . 5 mMr HzOz, 5 mM; lcCl, 0 .1 M: disodium hydrogen phosphate, 0.f M. ~ .
As shown in Fieure~l?, 1n this evaluation system, a current generated by an oxidat~.on-reduction reaction of FMA coupled with an enzyme reaction wee detected to evaluates Z5, the performance of each~elactrode. Tha test results nxe shown ' in Figure 1a. In Figure 10. the horizontal~axi: represents electrode width ( Vim) ; which is the width W of the doughnut of the doughnut-shaped electrode shoran in ~'igura 21H(a).
. In Figure la , the vertical axis represents the stabilizhtion . ~ time of the electrode. Notes that the stabilization time of the electrode is defined as a time in which 90~r of the maximum current ~talue is obtained. As indicated in Figure 1~, when the elaatrode hgd d width of 100 Win, ~ths stabilization .time - zi - ~ Pz5lsx was about 300 second9~. The greater the electrode width, the greater the time reduired for stabilization. The eleotrode having a radius of 1,000 N,m (1 mm) required. about 10,000~seconds (about Z.8 hours)..
. . . .
Practically speaking, it is ooasidar~d that an appropriate~measurement~time~is ty~iaallp~ within about 1, 000 . seconds. Therefore, the electrode width needs to be less than about 500 Wn.
. Next, the same set of electrodes as above were used to aoadur~t measurement under the same conditions a,s abo~re, except that the measurement time oval fixed to about 100 .seconds, thereby determining the ae~naitivity of the aloctrodee. Tha~resulte are shown in Figuri i9. In . Figure 19,, the horizontal axis indicates' the electrode diameter (~tm) and the vertical axis indicates the electrode , sensitivity. The~eleatroda.sensitivity is represented by the difference between an output aurrentwvalue when the ZO secondary antibody for ELISA was 3.m~mobilized 8nd 'an output ' cux~x~ent value before the. immobilization of the antibody.
As shown. in Figure ~.9, whets, th~a diameter of the electrode is smaller than or equal to about 500 Er,m, the electrode.sensitivity increases with an inereasa is the electrode diameter. Nhen thd eleatrod~e diameter is about 500 Eun, the eleotrode sensitivity reaches a maximum value.
Aver about 500 ~cn,, the eleotrode sensitivity deoreases.
Further, under the same conditions. the measurement time was elongated. The measurement results are summnrized in Figure a0.
.:.. ~... . , . .... .,.,.~~,;;~T",.",:: ' ... ......::.:. .,. ~
..,.;,__,:.;.:" ,:-: :~~ . :".:~~... . ...:..,~y.....,....;.:., In Figure 20, the horizontal axis indicates the ' measurement time, and the .vertical axis indicates an output current of the electrode. Aoavrding to~the results shown in Figure Z0. the greater thw electrode width, the greater the output current . Tn this case, however, thb responwe tithe is increneod. The smaller the wloatrode width, the smaller the obtained output currant . It is. .understood that if the . measurement time af~d the electrode width are specified, am eleotrode width is uniquely determined.
~ ' ' According to the test xe~aults using the censer devi~ae shown in'this example, it is found that when the device had a raaotion region having a diameter of about 100 Vim, the optimum measurement aenaitivity was obtained in the i5 measurement taking about 1,000 seoonde or less if an' electrode having a w~.dth of about . 500 y~s~ was ussd~.
(Example Zs Preparation of Sensor Chip) Figure ,21A shows an exemplary sensor ship prepared according to the pregnant invention. ' Dashed lines Hl and H2 surrounding eubstantially oiraulax regions at the middle of Figure 2i11~ indicate regions in whioh the antibodies ørs immobil.izsd. In the figures, 'two thick .lines 30Z'indicate electrode lines, whose tips form a measuring electrode and a blank measuring alsotrods, re~speofively. Typically, these electrodes are formed by depositing gold or the like , on the base plate 1: Note that numsrioal values in the figure represent dimensions of parts in units-of ate.
. FiQure'21H is a diagram :hewing detailed structures of the measuring electrode and the blank mwasurit~g el,a~trode, which. can be used in the deviad shown in F~.gurw 2171.
Figure a1H(a) shows a structure of a sensor chip in~whiah .. .... . - ..._ "-.. ... . . .._~ , ~...~,r.~,. m.» :.. __..._. . . .. _ ......... . .. , ......... . --. , . ..,_. _ .._.....
- a~ ~ ~asisa BhawinQ a resist pattern used in production of the sensor ohip. Figure-,~4 is a cross-sectional view showin0 a situntion in which the resist .pattern was provided on the sensor chip. indicating a arose section fl~ thv el~otrods 3.
Figure $S is a plan view showing a situation in which the resist:pattern was provided on the sensor ship. .
INDUSTRIAL APPLICABILITY
A rwgion in which a subject to be' dsteatsd~ la imrnobilizsd is l.imitid to a v4ry smnll prize; and a d~tection region of a detection means and a reaction region in which a reaction product is formed ere disposed in a relative posi~'tional rela~tionah~.p such that the reaction product can is . . be detected rapidly and speoifioally,' thereby providing a sensor devise which has a relativellr ~simplo~ struaturel Bind rapidly detests avert' small amount of an analyte in a epsoimen, such as an imtnunogenie substanow (e.g., a protein, a microorganism, and a virus) or a ahemioai substance.
2 0 ,;
As described above, the present invention is .desaxibed with reference to the axamplvs. The present invention is not. aa. limited. The present invention can bs implemented'in embodiments additionally having variations, 25 modifications, and alterations based on knowledge of those skilled. iiz the art .without departing from the spirit and saopa of the present invention., ~ , y . . . . .. .. _ . . . , . . ~ . . , ..~;w~~... . .. ~ . . .. , .... . .... , , . . , , ... . . . .. , ,., r . ~ . .. ..
As described above, the present invention is .desaxibed with reference to the axamplvs. The present invention is not. aa. limited. The present invention can bs implemented'in embodiments additionally having variations, 25 modifications, and alterations based on knowledge of those skilled. iiz the art .without departing from the spirit and saopa of the present invention., ~ , y . . . . .. .. _ . . . , . . ~ . . , ..~;w~~... . .. ~ . . .. , .... . .... , , . . , , ... . . . .. , ,., r . ~ . .. ..
Claims (30)
1. A sensor device, comprising: at least one support for immobilizing a subject to be detected; and a cell for containing a solution in which a reaction product generated from the subject to be detected is diffused, wherein at least one reaction region having a constant concentration of the reaction product is formed by diffusion of the reaction product into the solution.
2. A sensor device according to claim 1, wherein a plurality of reaction regions are formed.
3. A sensor device according to claim 2, wherein the plurality of reaction regions are separately formed by different reaction products.
4. A sensor device according to claim 1, further comprising detection means.
5. A sensor device according to claim 1, wherein the subject to be detected is immobilized by fixing means.
6. A sensor device according to claim 4, wherein the detection means has a detection region, and the detection region includes the reaction region.
7. A sensor device according to claim 4, wherein the detection means has a detection region, and the detection region overlaps the reaction region.
8. A sensor device according to claim 4, wherein the detection means has a detection region, and the detection region is included in the reaction region.
9. A sensor device according to claim 1, wherein the support is a particular region on a base plate.
10. A sensor device according to claim 1, wherein the support is a particulate.
11. A sensor device according to claim 1, wherein the support is a rod-shaped member.
12. A sensor device according to claim 4, wherein the detection means measures light.
13. A sensor device according to claim 4, wherein the detection means measures heat.
14. A sensor device according to claim 4, wherein the detection means comprises at least one electrode.
15. A sensor device according to claim 14, wherein the electrode acts on the reaction product and generates an electrical signal corresponding to the amount of the reaction product.
16. A sensor device according to claim 1, wherein the subject to be detected is an enzyme, and the reaction product is an enzyme reaction product.
17. A sensor device according to claim 1, wherein the subject to be detected is an antibody or an enzyme linked to a peptide.
18. A sensor device according to claim 17, wherein the subject to be detected is immobilized by an antigen.
19. A sensor device according to claim 17, wherein the subject to be detected is immobilized by an antigen-antibody complex.
20. A sensor device according to claim 1, wherein the subject to be detected is an enzyme linked to a first antibody linked to an antigen, and is immobilized by a second antibody.
21. A sensor device according to claim 4, wherein the subject to be detected is immobilized in a region having a diameter of several tens to several hundreds of µm, and the detection means is an electrode having a diameter of 1 mm or less.
22. A sensor device according to claim 21, wherein when the subject to be detected is measured, the electrode approaches the region in which the subject to be detected is immobilized.
23. A sensor device according to claim 1, wherein the electrode is provided on the support, and the subject to be detected is immobilized in a region having a diameter of several tens to several hundreds of µm.
24. A sensor device according to claim 4, wherein the subject to be detected is immobilized in a region having a diameter of several tens to several hundreds of µm, and the electrode surrounds the region.
25. A sensor device according to claim 1, wherein the support is a material selected from the group consisting of glass, ceramics, noble metals, and resins.
26. A sensor device according to claim 1, further comprising means for promoting the immobilizing of the subject to be detected.
27. A sensor device according to claim 25, wherein the promoting means is used to stir the solution in the cell.
28. A sensor device according to claim 26, wherein the promoting means is used to exchange the solution in the cell.
29. A sensor device according to claim 26, wherein the promoting means is used to supply the solution in the cell.
30. A sensor device according to claim 26, wherein the promoting means is used to allow the solution to flow through the cell.
Applications Claiming Priority (5)
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| JP2000138671 | 2000-05-11 | ||
| JP2000223406 | 2000-07-25 | ||
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| JP2000-138671 | 2000-11-05 | ||
| PCT/JP2001/003917 WO2001086275A2 (en) | 2000-05-11 | 2001-05-10 | Chemical sensor device |
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| CA2412302A1 true CA2412302A1 (en) | 2002-10-30 |
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| ATE367449T1 (en) * | 2002-09-20 | 2007-08-15 | Univ Kingston | DETECTION OF BIOLOGICAL MOLECULES USING DIFFERENTIAL SEPARATION OF ENZYME SUBSTRATES AND PRODUCTS |
| WO2011156915A2 (en) | 2010-06-18 | 2011-12-22 | Pathogen Detection Systems, Inc. | Method and system for detecting biological molecules in samples |
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| AU562373B2 (en) | 1982-11-15 | 1987-06-11 | Data General Corporation | Digital data processing system |
| US6846654B1 (en) * | 1983-11-29 | 2005-01-25 | Igen International, Inc. | Catalytic antibodies as chemical sensors |
| DK531185A (en) * | 1985-11-18 | 1987-05-19 | Radiometer As | SENSOR TO DETERMINE THE CONCENTRATION OF A BIOCHEMICAL SPECIES |
| EP0284518B1 (en) * | 1987-03-27 | 1992-10-07 | Isao Karube | Miniaturized oxygen electrode and miniaturized biosensor and production process thereof |
| US5063081A (en) * | 1988-11-14 | 1991-11-05 | I-Stat Corporation | Method of manufacturing a plurality of uniform microfabricated sensing devices having an immobilized ligand receptor |
| US5200051A (en) | 1988-11-14 | 1993-04-06 | I-Stat Corporation | Wholly microfabricated biosensors and process for the manufacture and use thereof |
| US5491097A (en) * | 1989-06-15 | 1996-02-13 | Biocircuits Corporation | Analyte detection with multilayered bioelectronic conductivity sensors |
| US5183740A (en) * | 1990-02-23 | 1993-02-02 | The United States Of America As Represented By The Secretary Of The Navy | Flow immunosensor method and apparatus |
| GB9325718D0 (en) * | 1993-12-16 | 1994-02-16 | Ars Holding 89 Nv | Sensor device for sandwich assay |
| DE4408352C2 (en) * | 1994-03-12 | 1996-02-08 | Meinhard Prof Dr Knoll | Miniaturized substance-recognizing flow sensor and method for its production |
| AU2365895A (en) * | 1994-04-26 | 1995-11-16 | Regents Of The University Of Michigan, The | Unitary sandwich enzyme immunoassay cassette, device and method of use |
| US5805048A (en) * | 1995-09-01 | 1998-09-08 | Sumitomo Wiring Systems, Ltd. | Plate fuse and method of producing the same |
| US6387707B1 (en) * | 1996-04-25 | 2002-05-14 | Bioarray Solutions | Array Cytometry |
| JP4663824B2 (en) * | 1996-12-31 | 2011-04-06 | ハイ スループット ジェノミクス インコーポレイテッド | Multiplexed molecular analyzer and method |
| US5759364A (en) * | 1997-05-02 | 1998-06-02 | Bayer Corporation | Electrochemical biosensor |
| DE19822123C2 (en) * | 1997-11-21 | 2003-02-06 | Meinhard Knoll | Method and device for the detection of analytes |
| US6388788B1 (en) * | 1998-03-16 | 2002-05-14 | Praelux, Inc. | Method and apparatus for screening chemical compounds |
| US6780591B2 (en) * | 1998-05-01 | 2004-08-24 | Arizona Board Of Regents | Method of determining the nucleotide sequence of oligonucleotides and DNA molecules |
| US6322963B1 (en) * | 1998-06-15 | 2001-11-27 | Biosensor Systems Design., Inc. | Sensor for analyte detection |
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| US6254830B1 (en) * | 1999-11-05 | 2001-07-03 | The Board Of Governors For Higher Education, State Of Rhode Island And Providence Plantations | Magnetic focusing immunosensor for the detection of pathogens |
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- 2001-05-10 EP EP01930043A patent/EP1335200A4/en not_active Ceased
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| Publication number | Publication date |
|---|---|
| EP1335200A4 (en) | 2004-03-24 |
| AU2001256695A1 (en) | 2001-11-20 |
| WO2001086275A2 (en) | 2001-11-15 |
| JP3759039B2 (en) | 2006-03-22 |
| EP1335200A1 (en) | 2003-08-13 |
| US7662612B2 (en) | 2010-02-16 |
| US20030180184A1 (en) | 2003-09-25 |
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