Source: https://patents.google.com/patent/EP1291650B1/en
Timestamp: 2019-04-22 04:22:54
Document Index: 128698241

Matched Legal Cases: ['art 96', 'art 96', 'art 96', 'art 96', 'art 46', 'art 46', 'art 7', 'art 7', 'art 46', 'art 7', 'art 7', 'art 7', 'art 7', 'art 7', 'art 7', 'art 7', 'art 7', 'art 7', 'art 7', 'arts 7', 'arts 46', 'arts 46', 'art 7']

EP1291650B1 - Biosensor and method for manufacturing the same - Google Patents
EP1291650B1
EP1291650B1 EP20010930170 EP01930170A EP1291650B1 EP 1291650 B1 EP1291650 B1 EP 1291650B1 EP 20010930170 EP20010930170 EP 20010930170 EP 01930170 A EP01930170 A EP 01930170A EP 1291650 B1 EP1291650 B1 EP 1291650B1
EP20010930170
EP1291650A4 (en
EP1291650A1 (en
2000-05-16 Priority to JP2000143340 priority
2001-05-15 Application filed by Arkray Inc filed Critical Arkray Inc
2001-05-15 Priority to PCT/JP2001/004055 priority patent/WO2001088526A1/en
2003-03-12 Publication of EP1291650A1 publication Critical patent/EP1291650A1/en
2009-05-13 Publication of EP1291650A4 publication Critical patent/EP1291650A4/en
2014-04-02 Publication of EP1291650B1 publication Critical patent/EP1291650B1/en
As a disposable biosensor, a capillary type biosensor that is constituted such that a sample liquid is fed to a reaction part using a capillary phenomenon is known. Fig. 10 is an exploded perspective view of such a conventional biosensor 9, and Fig. 11 is a sectional view of the assembled biosensor 9. The biosensor 9 has a structure in which an insulating base 90, a spacer 91 and a cover 92 are built up on top of one another. As shown in Fig. 11, a capillary 93 having an inlet port 93a is defined by the spacer 91 between the base 90 and the cover 92. A working electrode 94, a counter electrode 95, and a reaction part 96, which is formed on top of the working electrode 94 and the counter electrode 95, are provided inside the capillary 93. The reaction part 96 contains any of various reagents necessary for a prescribed reaction system, for example a redox enzyme, in accordance with the subject of quantification.
With a biosensor 9 having such a structure, the sample liquid moves from the inlet port 93a through the capillary 93 due to a capillary phenomenon, and reaches the reaction part 96. The reagent contained in the reaction part 96 then dissolves in the sample liquid, and a redox reaction occurs. By measuring the oxidation current at this time, the concentration of a specific component in the sample liquid is quantified.
The present invention provides a biosensor comprising: a capillary channel with an inlet port for taking in a sample liquid; a membrane for promoting movement of said sample liquid through said channel, and a reaction part containing a reagent that reacts with a component to be tested in said sample liquid whose movement has been promoted by said membrane; characterized in that said membrane is an asymmetric membrane or composite membrane comprising, thicknesswise, a first layer having smaller pores, and a second layer having larger pores, and wherein said first layer is held in contact with said reaction part.
Preferably, the membrane has a plurality of pores each of which is no less than 0.25µm but no more than 45µm.
Preferably, the membrane is an asymmetric membrane made of a polysulfone.
Preferably, the pores of the first layer have a pore diameter of no less than 0.25µm but no more than 0.45µm, and the pores of the second layer have a pore diameter of no less than 25µm but no more than 45µm.
Preferably, a method for manufacturing a biosensor according to the present invention comprises forming a working electrode and a counter electrode in an elongate configuration on an insulating base; forming an elongate reaction part intersecting said working electrode and said counter electrode; laminating an elongate asymmetric or composite membrane on said reaction part; providing a pair of walls along longitudinal edges on both sides of said reaction part and said membrane; and forming a capillary channel by forming a cover bridging said pair of walls.
Other features and advantages of the present invention will become more apparent from the following detailed description while referring to the accompanying exemplary drawings.
Fig. 2 is a sectional view along line II-II of the biosensor of Fig. 1 in an assembled state.
A biosensor 1 according to the present invention will now be described with reference to Figs . 1 to 3. The biosensor 1 includes an insulating base 2 which is formed in a strip shape (e.g. 6×30×0.25mm) from a glass epoxy resin or the like. A capillary 3 that extends widthwise of the insulating base 2 (the X-direction in Fig. 1) is formed on top of the insulating base 2.
As is shown clearly in Fig. 1, the electrode system 4 comprises a counter electrode 40, a working electrode 41 and a reference electrode 42, each of which extends longitudinally of the insulating base 2 (the Y-direction in Fig. 1). An inter-electrode insulator 43 is provided between the counter electrode 40 and the working electrode 41. Similarly, an inter-electrode insulator 44 is provided between the working electrode 41 and the reference electrode 42. These inter-electrode insulators 43 and 44 are flush with the electrodes 40, 41 and 42. The electrodes 40, 41 and 42 are each formed to a thickness of approximately 40µm and a width of approximately 2mm using a technique such as screen printing, sputtering or vapor deposition. An insulating layer 45 of thickness approximately a few tens of µm is further formed on top of the electrodes 40, 41 and 42 and the inter-electrode insulators 43 and 44. The insulating layer 45 is divided by a groove part 46 that extends in the width direction of the insulating base 2. The groove part 46 has a width of approximately 0.5 to 1.5mm.
The reaction part 7 is a solid material that contains an enzyme that reacts with a specific component (substrate) contained in a biological sample liquid such as blood, and is constituted such as to dissolve when impregnated with the sample liquid. As is shown clearly in Figs. 2 and 3, the reaction part 7 is filled into the groove part 46, with the thickness of the reaction part 7 being made to be approximately a few tens of µm. In the case that a redox enzyme is used as the enzyme, an electron acceptor may be put into the reaction part 7 in advance.
As is shown clearly in Fig. 3, the membrane 8 is disposed on top of the reaction part 7 such as to extend in the width direction of the insulating base 2, is white, and has a thickness of approximately 130µm. The membrane 8 is a porous synthetic polymer membrane, and can form a porous membrane having a smaller pore diameter than a glass filter. In the present embodiment, the diameter of the plurality of pores in the membrane 8 is at least 0.25µm but not more than 45µm. As the membrane 8, for example one containing a polysulfone type raw material, an aromatic polyamide type raw material, cellulose acetate or the like can be adopted.
In the case of using an asymmetric membrane or composite membrane as the membrane 8, the membrane 8 is disposed such that the dense layer is in contact with the reaction part 7. Preferably, the diameter of the plurality of pores formed in the dense layer is at least 0.25µm but not more than 0.45µm. Preferably, the diameter of the plurality of pores formed in the porous layer is at least 25µm but not more than 45µm.
As is shown clearly in Fig. 3, each of the pair of spacers 5 are disposed extending in the width direction of the insulating base 2 along the two side edges of the reaction part 7 and the membrane 8 so as to sandwich the membrane 8 therebetween. The spacers 5 are thicker than the membrane 8, having for example a thickness of approximately 200µm.
The cover 6 bridges between the pair of spacers 5. The cover 6 is a transparent member or a semitransparent member formed from a resin such as polyethylene terephthalate (PET), and has a thickness of approximately 15 to 30µm
As is shown clearly in Fig. 2, a channel 30 is formed between the cover 6 and the membrane 8, with the height of the channel 30 being approximately 50µm. As is shown clearly in Fig. 3, the two ends of the channel 30 are open at openings 30a and 30b of the capillary 3. One opening 30a functions as an inlet port for introducing the sample liquid into the capillary 3. The other opening 30b functions as an escape route for air in the capillary 3 when the sample liquid moves through the capillary 3, and as a result a good capillary phenomenon is secured in the channel 30.
With the biosensor 1 constituted in this way, if the sample liquid such as blood is introduced from the opening 30a of the capillary 3, then the sample liquid moves through the channel 30 toward the opening 30b side due to a capillary phenomenon that acts in the longitudinal direction of the capillary 3. The amount of the sample liquid required in this case is for example 0.2 to 1.5µl. A part of the sample liquid introduced into the capillary 3 comes into contact with the membrane 8. A suction force acts on the sample liquid in contact with the surface of the membrane 8, and hence the sample liquid moves through the pores of the membrane toward the reaction part 7. As the sample liquid moves from pore to pore through the membrane 8, a suction force acts to also pull the following sample liquid in the channel 30 into the membrane 8. As a result, the movement of the sample liquid through the channel 30 is promoted, and hence it becomes easy to make the sample liquid reach every region of the reaction part 7.
With the present embodiment, the reaction part 7 and the membrane 8 on top thereof are formed up to the opening 30a, and hence the sample liquid introduced from the opening 30a comes into contact with the membrane 8 and some of the sample liquid moves into the pores of the membrane 8 immediately. The movement of the sample liquid through the channel 30 is then promoted by the movement of the sample liquid into the membrane 8. Consequently, even with a small amount of sample liquid, the sample liquid arrives at the reaction part 7 instantly and reliably.
With the present embodiment, the cover 6 is made to be transparent, and the membrane 8 is made to be white. According to this constitution, it can easily be checked from the outside via the cover 6 how far the sample liquid has filled into the channel 30. Such visual checking is possible even if the electrodes 40, 41 and 42 are formed from carbon black or the like so as to be black, and even if the sample liquid is red as in the case of blood. Moreover, the position that the sample liquid has reached can also be identified by detecting the state of electrical continuity between the electrodes 40, 41 and 42. This is because as the sample liquid moves through the capillary 3 from the opening 30a to the opening 30b while impregnating into the membrane 8 and the reaction part 7, the state of electrical continuity between the electrodes 40, 41 and 42 via the sample liquid changes.
First, as shown in Fig. 4, conductor layers 40A, 41A and 42A that will ultimately become counter electrodes 40, working electrodes 41 and reference electrodes 42 are formed in a state divided by grooves 43A on a mother base 2A made of a glass epoxy resin, a ceramic or the like. The conductor layers 40A, 41A and 42A can be formed individually by a technique such as screen printing, sputtering or vapor deposition. Alternatively, the conductor layers 40A, 41A and 42A can be formed simultaneously by forming a conductor layer over the whole surface of the mother base 2A and then providing a plurality of grooves 43A in the conductor layer. The conductor layers 40A, 41A and 42A are each formed from carbon black, copper, silver, gold or the like, and have a thickness of for example approximately 40µm.
Next, as shown in Fig. 7, reaction layers 7A that will become reaction parts 7 are formed so as to fill up the groove parts 46A. These reaction layers 7A are formed, for example, by filling the groove parts 46A with an aqueous solution of an enzyme that has been selected in accordance with the specific component to be quantified, or a mixed aqueous solution of such an aqueous solution and a hydrophilic polymer, and then drying. The reaction layers 7A have a thickness of for example approximately a few tens of µm.
The membrane layers 8A have a thickness of for example approximately 130µm, with the width and length matching those of the reaction layers 7A.
The adhesive layers 5A are formed for example by applying a hot melt adhesive, or by sticking on double-sided tape. The adhesive layers 5A in the present embodiment are provided either side of each membrane layer 8A, but may also be provided such that parts thereof are formed on top of side edge parts of the reaction layers 7A or the membrane layers 8A. The thickness (height) of the adhesive layers 5A is for example approximately 200µm.
The covers 6A are formed for example from PET so as to be transparent, and have a thickness of approximately 15 to 30µm. In the case where a hot melt adhesive or double-sided tape has been used as the adhesive layers 5A, the covers 6A can be fixed onto the spacers 5A through the adhesiveness of the adhesive layers 5A.
Table 1 shows the results of measuring the time required for a sample liquid to be filled more-or-less completely into the capillary for the biosensor 1 according to the present invention in which the membrane 8 is provided on top of the reaction part 7 and a biosensor having exactly the same structure except that the membrane is not provided. A membrane of product number SD450 made by Memtec was used as the membrane 8. The measurements were carried out using three sample liquids exhibiting different hematocrits to one another. The volume of the channel in the capillary of each of the biosensors was made to be 6mm×0.5mm×130µm. The thickness of the membrane of the biosensor 1 was made to be 130µm. The measurement values in Table 1 are the mean values over three samples. TABLE 1
a capillary channel (30) with an inlet port (30a) for taking in a sample liquid;
a membrane (8) for promoting movement of said sample liquid through said channel (30); and
a reaction part (7) containing a reagent that reacts with a component to be tested in said sample liquid whose movement has been promoted by said membrane (8);
characterized in that said membrane (8) is an asymmetric membrane (8) or composite membrane comprising, thicknesswise, a first layer having smaller pores, and a second layer having larger pores, and wherein said first layer is held in contact with said reaction part (7).
The biosensor according to claim 1, wherein said membrane (8) is provided in the vicinity of said inlet port (30a).
The biosensor according to claim 1, wherein said membrane (8) has a plurality of pores each of which is no less than 0.25µm but no more than 45µm in diameter.
The biosensor according to claim 1, wherein said membrane (8) is laminated on said reaction part (7).
The biosensor according to claim 4, wherein said reaction part (7) and said membrane (8) are formed to extend to said inlet port (30a).
The biosensor according to claim 1, wherein the pores of said first layer have a pore diameter of no less than 0.25µm but no more than 0.45µm, and the pores of said second layer have a pore diameter of no less than 25µm but no more than 45µm.
The biosensor according to claim 1, wherein said capillary channel (30) has an opening (30b) serving as an air escape hole.
The biosensor according to claim 1, wherein said capillarychannel (30) is provided with a transparent or semitransparent part (6) for internally observing said capillary channel (30).
The biosensor according to claim 9, wherein said membrane (8) is white.
The biosensor according to claim 1, further comprising an elongate working electrode (41) and an elongate counter electrode (40) provided on an insulating base (2), wherein said capillary channel (30) is formed to intersect said working electrode (41) and said counter electrode (40) on said insulating base (2).
The biosensor according to claim 10, wherein said counter electrode (40) is generally parallel to said working electrode (41).
The biosensor according to claim 10, wherein said capillary channel (30) is formed by a pair of upright walls (5) on said base(2), and a cover (6) bridging the pair of walls (5).
A method for manufacturing a biosensor according to claim 1, comprising the steps of:
forming a working electrode(41) and a counter electrode (40) in an elongate configuration on an insulating base (2) ;
forming an elongate reaction part (7) intersecting said working electrode (41) and said counter electrode (40);
laminating an elongate asymmetric or composite membrane (8) on said reaction part (7);
providing a pair of walls (5) along longitudinal edges on both sides of said reaction part (7) and said membrane (8) ; and
forming a capillary channel (30) by forming a cover (6) bridging said pair of walls (5).
The biosensor manufacturing method according to claim 13, wherein said pair of walls (5) is formed by applying a hot melt adhesive.
The biosensor manufacturing method according to claim 13, wherein said pair of walls (5) is formed by sticking double-sided tape onto said insulating base (2).
EP20010930170 2000-05-16 2001-05-15 Biosensor and method for manufacturing the same Active EP1291650B1 (en)
EP1291650A1 EP1291650A1 (en) 2003-03-12
EP1291650A4 EP1291650A4 (en) 2009-05-13
EP1291650B1 true EP1291650B1 (en) 2014-04-02
EP20010930170 Active EP1291650B1 (en) 2000-05-16 2001-05-15 Biosensor and method for manufacturing the same
WO2012018632A2 (en) 2010-07-26 2012-02-09 Senova Systems, Inc. Analyte sensor
CN1198137C (en) 2005-04-20
Ipc: G01N 27/327 20060101AFI20011126BHEP
Ipc: C12Q 1/00 20060101ALI20090408BHEP
Ipc: G01N 33/487 20060101ALI20090408BHEP
Inventor name: YAMAOKA, HIDEAKI
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