JP4343736B2 - Biosensor - Google Patents

Description

  The present invention relates to a small and disposable biosensor capable of analyzing a specific component in a specimen sample solution by a reaction between the specimen sample solution and a reagent, and more particularly to a biosensor in which the specimen sample solution is sufficiently aspirated.

  A biosensor is a sensor that utilizes the molecular recognition ability of biological materials such as microorganisms, enzymes, antibodies, DNA, and RNA, and applies the biological material as a molecular identification element. That is, a reaction that occurs when an immobilized biological material recognizes a target substrate, an enzyme consumption due to respiration of microorganisms, an enzyme reaction, luminescence, and the like are utilized. Among biosensors, enzyme sensors are being put into practical use. For example, enzyme sensors for glucose, lactic acid, urea, and amino acids are used in medical measurement and the food industry.

  For example, an enzyme sensor reduces an electron acceptor by electrons generated by a reaction between a substrate contained in a specimen sample solution and an enzyme, and the measurement device electrochemically measures the reduction amount of the electron acceptor. Quantitative analysis of the specimen is performed (for example, see Patent Document 1).

  Hereinafter, a conventional biosensor will be described with reference to FIGS. 4 and 5. FIG. 4A is an exploded perspective view showing the configuration of the biosensor, and FIG. 4B is a plan view of the biosensor shown in FIG.

  4A and 4B, the biosensor 400 includes a first insulating substrate (hereinafter referred to as “first substrate”) 41 made of polyethylene terephthalate or the like, a reagent layer 45, and a specimen supply. A spacer 46 having a notch for forming a passage 47, and a second insulating substrate (hereinafter referred to as a “second substrate”) 48 provided with an air hole 49. The spacer 46 and the reagent layer 45 are sandwiched between the substrate 41 and the second substrate 48 and are integrally arranged.

  On the surface of the first substrate 41, for example, a conductor layer 410 made of a noble metal such as gold or palladium or an electrically conductive material such as carbon is formed by a screen printing method or a sputtering deposition method. The upper conductor layer 410 is divided by a plurality of slits to form measurement electrodes (hereinafter also referred to as working electrodes) 42, counter electrodes 43, detection electrodes 44, and substantially arc-shaped slits 414 and 415. In addition, 411, 412 and 413 are terminals of the measurement electrode 42, the counter electrode 43, and the detection electrode 44, respectively.

  A reagent layer 45 is formed on the electrodes 42, 43, and 44 by applying a reagent containing an enzyme, an electron carrier and a water-soluble polymer that specifically reacts with a specific component in the sample solution. The spreading of the reagent applied on the electrodes 42, 43, 44 is restricted by the substantially arc-shaped slits 414, 415 formed in the counter electrode 43.

  Further, a spacer 46 is laminated thereon, and a specimen supply path 47 is formed by a rectangular cutout provided at the center of the front edge of the spacer 46.

  Then, the second substrate 48 is laminated and bonded on the spacer 46 so that one end of the notch portion of the spacer 46 communicates with the air hole 49 provided in the second substrate 48.

  A flow of measuring the content of the substrate in the sample liquid using the conventional biosensor 400 configured as described above will be described below.

First, a measurement device (not shown) not shown is connected to the biosensor 400, and a constant voltage is applied between the counter electrode 43 or the measurement electrode 42 of the biosensor 400 and the detection electrode 44 by the measurement device. Apply. Then, the sample solution is supplied to the inlet of the specimen supply path 47 with a voltage applied between the two electrodes of the biosensor 400 described above. The supplied sample liquid is sucked into the specimen supply path 47 by capillary action, passes through the counter electrode 43 and the measurement electrode 42, reaches the detection electrode 44, and the sample liquid that has reached the detection electrode 44 is The reagent layer 45 is dissolved. On the measurement device side, an electrical change occurring between the counter electrode 43 or the measurement electrode 42 and the detection electrode 44 is detected, and the measurement operation is started.
International Publication No. 01/36953 pamphlet

  By attaching at least two first and second substrates 41 and 48 as described above, a sample supply path 47 through which a sample sample is aspirated is formed between the substrates. In the biosensor in which a reagent for analyzing a component in the aspirated specimen sample is arranged and an air hole 49 communicating with the outside from the specimen supply path 47 is formed in at least one of the substrates, the air hole Conventionally, when forming 49, a part of the second substrate 48 is punched out by pressing to form a hole.

  FIG. 5A is a perspective view showing the shape of air holes formed by conventional pressing, and FIG. 5B is a cross-sectional view of the air holes formed by pressing.

  However, in recent years, there has been a demand for a reduction in the amount of blood that is a specimen sample supplied to the biosensor 400, and accordingly, the size of the specimen supply path 47 has also become smaller. The size of the air holes 49 provided in the substrate 48 must be reduced.

  However, when the air hole 49 formed by the press working is made smaller, the punched residue remains in the air hole 49 even if a hole is formed by pressing the second substrate 48. Therefore, if a biosensor in which the extracted residue remains in the air hole 49 is commercialized as it is, the biosensor cannot suck a sufficient amount of the supplied specimen sample, which causes a problem in terms of measurement accuracy. To do. And the minimum diameter of the air hole which can be formed by press work which does not cause such a problem is 0.3 mm in diameter. Further, considering the productivity, the air hole by press work is 0.35 mm in diameter. .

  As a method for solving the above-described problems, it is conceivable that the second substrate 48 is not punched by pressing, but the second substrate 48 is heated and melted with a laser or the like to form minute holes. According to this method, there is no left-over residue in the air holes 49 unlike the press work, and it is also possible to make finer air holes.

  However, if the air hole formed on the second substrate 48 is made too small by the laser processing, there is no problem when the specimen sample supplied to the biosensor is blood having a relatively high viscosity. In the case of a small control solution or the like, since the viscosity of the specimen sample is small, the speed of suction into the specimen supply path 7 is too high, and the air in the specimen supply path 47 cannot be extracted from the minute air holes. Bubbles remain in the sample supply path 47, and a new problem arises that a sufficient amount of sample sample cannot be aspirated.

  The present invention has been made to solve the above-described problem, and air is exhausted from the sample supply path well regardless of the viscosity of the sample supplied to the biosensor, and the sample supply path It is an object of the present invention to provide a biosensor including minute air holes that can sufficiently suck a specimen sample without bubbles remaining.

  In order to solve the above problems, the biosensor of the present invention forms a sample supply path through which a supplied sample sample is aspirated between the substrates by bonding at least the first and second substrates, A biosensor in which a reagent that reacts with a component in the specimen sample is disposed in a supply path, and an air hole communicating with the outside from the specimen supply path is provided in the second substrate, the air hole The diameter of the opening on one surface of the second substrate facing the sample supply path is larger than the diameter of the opening on the other surface of the second substrate.

  Thereby, regardless of the viscosity of the specimen sample supplied to the biosensor, a biosensor with minute air holes that can suck a sufficient amount of the specimen sample without bubbles remaining in the specimen supply path of the biosensor Can provide. Further, even if the user mistakenly deposits a sample sample in the air hole, the diameter of the opening on the side facing the outside of the second substrate is very small, so that the sample sample is placed in the sample supply path. There is also an effect that it is not sucked.

In the biosensor of the present invention, the air hole is formed by a laser.
As a result, a biosensor having finer air holes can be realized, and there is an effect that no waste is generated when the air holes are drilled.

In the biosensor of the present invention, the area of the opening on one surface of the second substrate facing the sample supply path is 1.76 × 10 ^{−2 to} 3.14 × 10 ⁻² mm ² . The area of the opening on the other surface of the second substrate is 1.96 × 10 ^{−3 to} 7.85 × 10 ⁻³ mm ² .

  Thereby, when a specimen sample is supplied to the biosensor, a biosensor having a finer air hole that can suck a sufficient amount of the specimen sample without bubbles remaining in the specimen supply path of the biosensor. Can provide.

  In the biosensor of the present invention, the shape of the opening of the air hole is substantially circular, and the diameter of the opening on one surface of the second substrate facing the sample supply path is 0.15 to 0. 20 mm, and the diameter of the opening on the other surface of the second substrate is 0.05 to 0.10 mm.

  Thereby, when a specimen sample is supplied to the biosensor, a biosensor having a finer air hole that can suck a sufficient amount of the specimen sample without bubbles remaining in the specimen supply path of the biosensor. Can provide.

  In the biosensor of the present invention, the shape of the opening of the air hole is any one of a circle, an ellipse, a linear shape having a very narrow width, a triangle, a square, a rectangle, and a polygon.

  According to the biosensor of the present invention, a specimen supply path through which the supplied specimen sample is aspirated between the substrates is formed by bonding at least the first and second substrates, and the specimen is provided in the specimen supply path. In the biosensor, in which a reagent that reacts with a component in the sample is disposed and an air hole that communicates with the second substrate through the sample supply path is formed, the air hole is formed on the second substrate. Since the diameter of the opening on one side facing the sample supply path is larger than the diameter of the opening on the other side of the second substrate, the supplied sample sample is sucked into the sample supply path In this case, air can be exhausted from the specimen supply path through the air hole, and bubbles can be prevented from being generated in the specimen supply path regardless of the viscosity of the specimen sample. Specimen sample can be aspirated

  In addition, according to the biosensor of the present invention, the smaller the diameter of the opening of the air hole drilled in the second substrate is on the outer surface side of the biosensor. Even if the specimen sample is spotted in the hole, there is an effect that the specimen sample is not sucked into the specimen supply path.

  Further, according to the biosensor of the present invention, since the air holes are drilled by a laser, it is possible to make a finer hole than the hole formed by press working, and further, the air hole is formed by press working. There is an effect that the punched residue generated at the time of forming is not generated.

(Embodiment 1)
Hereinafter, biosensor 100 according to Embodiment 1 of the present invention will be described in detail with reference to the drawings.
FIG. 1A is an exploded perspective view of the biosensor, and FIG. 1B is a plan view of the biosensor shown in FIG.

  1A and 1B, reference numeral 1 denotes a first insulating substrate (hereinafter referred to as a “first substrate”) made of polyethylene terephthalate or the like, and is formed on the surface of the first substrate 1. The conductive layer 10 made of a noble metal such as gold or palladium or an electrically conductive material such as carbon is formed by a screen printing method or a sputtering deposition method. The conductive layer 10 may be formed on the entire surface or at least a part of the first substrate 1.

  On the first substrate 1, the conductive layer 10 is divided by a plurality of slits, and the counter electrode 3, the measurement electrode 2, the detection electrode 4, and the reagent overflow prevention lines 14 and 15 are formed. .

Reference numeral 8 denotes a second insulating substrate (hereinafter referred to as a “second substrate”) in which a substantially circular air hole 9 is formed at the center by a CO ₂ laser marker LP-211 made by SUNX. The material is preferably a plastic film, and examples thereof include polyester, polyolefin, polyamide, polyether, polyamideimide, polystyrene, polycarbonate, poly-ρ-phenylene sulfide, and polyvinyl chloride. Further, for the second substrate 8, those copolymers, blends, and further crosslinked ones can be used, and those having a thickness of 0.01 mm to 0.5 mm can be used. The size of the air hole 9 can change the laser condition or the irradiation condition. For example, the diameter of the laser irradiation light is increased, the laser power is increased, or the laser irradiation time is lengthened. This can be achieved.

  The second substrate 8 includes a spacer 6 having a notch for forming a sample reagent supply path 7 for supplying a sample reagent into the biosensor 100, and a reagent layer 5 impregnated with the reagent. It is sandwiched between the first substrate 1 and disposed integrally with the first substrate 1.

  The spacer 6 is disposed so as to cover the counter electrode 3, the measurement electrode 2, and the detection electrode 4 on the first substrate 1, and is formed by a rectangular notch provided at the center of the front edge portion of the spacer 6. A sample supply path 7 is formed, and the reagent layer 5 is formed on the counter electrode 3, the measurement electrode 2, and the detection electrode 4 exposed from the notch portion of the spacer 6, with an enzyme, an electron acceptor, an amino acid, and It is formed by applying a reagent containing a sugar alcohol or the like. Reference numerals 11, 12, and 13 denote terminals of the measurement electrode 2, the counter electrode 3, and the detection electrode 4, respectively.

Here, the shape of the air holes 9 formed in the second substrate 8 will be described in detail with reference to FIGS. 2 and 3. FIG. 2 is a diagram showing the shape of air holes drilled in the second substrate by CO ₂ laser processing according to the first embodiment, and FIG. 2A is viewed from the CO ₂ laser irradiation surface side. FIG. 2B is a perspective view of the air hole as seen from the non-irradiated surface side of the CO ₂ laser, and FIG. 2C is a cross section of the air hole formed by CO ₂ laser processing. FIG.

An opening 9b in FIG. 2C indicates an opening on the CO ₂ laser irradiation surface 8a side, and the opening 9a indicates an opening on the CO ₂ laser non-irradiation surface 8a side.
As shown in FIG. 2, the air holes 9 in the first embodiment are substantially circular in the shape of the openings 9a and 9b, and the diameter y of the opening 9b on the laser irradiation surface 8b side of the second substrate 8 is. Accordingly, the aperture x of the opening 9a on the laser non-irradiation surface 8a side is small.

FIG. 3 is formed when air holes of various diameters are drilled in the second substrate 8 made of a polyethylene terephthalate material having a thickness of 0.1 mm using a SUNX CO ₂ laser marker LP-211. The transition of the apertures x and y of the openings 9a and 9b on both surfaces of the second substrate is shown. In the following description, “air hole diameter” refers to the diameter x of the opening 9 a formed on the laser non-irradiation surface 8 a side of the air hole 9 penetrating the second substrate 8.

As apparent from the graph of FIG. 3, the aperture y of the opening 9b on the CO ₂ laser irradiation surface 8b side is larger than the aperture x of the opening 9a on the laser non-irradiation surface 8a side at any air hole size. As shown in FIG. 2C, the cross-sectional shape is such that the aperture diameter decreases from the laser irradiation surface 8b side toward the laser non-irradiation surface 8a side.

The size of the air hole diameter that can be drilled using a CO ₂ laser is 0.05 to 0.30 mm in consideration of productivity, that is, the diameter of the opening 9a on the laser non-irradiation surface 8a side is 0. 0.05 to 0.30 mm, and the diameter of the opening 9b on the laser irradiation surface 8b side is 0.15 to 0.45 mm.

  Hereinafter, the sample supply when the sample sample is supplied to the biosensor 100 of the first embodiment having the second substrate 8 having such air holes 9 and sucked into the sample supply path 7. The verification results for verifying the remaining state of bubbles in the path 7 are shown in Table 1 below.

  Here, as described above, the air hole 9 of the first embodiment is different in the diameter of the opening between the laser irradiation surface 8b and the laser non-irradiation surface 8a. In order to verify the remaining state of bubbles in 7, it is necessary to verify two patterns: a case where the side facing the specimen supply path 7 is the laser irradiation surface 8 b and a case where the side facing the sample supply path 7 is the non-laser irradiation surface 8 a.

Table 1 shows that air holes of various diameters are drilled in a second substrate 8 made of a polyethylene terephthalate material having a thickness of 0.1 mm using a SUNX CO ₂ laser marker LP-211, which is 1.5 mm × 3.4 mm. Table 1 (a) shows the verification result of the remaining state of bubbles inside the sample supply path when a low viscosity control liquid of 25 mPas is supplied to the sample supply path 7 of 0.155 mm. Is a verification result when the laser irradiation surface 8b is used on the sample supply path 7 side, and Table 1 (b) is a verification result when the laser non-irradiation surface 8a is used on the sample supply path 7 side.

  As shown in Table 1 (a), when the laser irradiation surface 8b is used on the side of the specimen supply path 7, there is no remaining bubble even if the air hole diameter is as fine as 0.05 to 0.10 mm. Even if the viscosity of the specimen sample is small, a sufficient amount of specimen sample can be sucked into the specimen supply path 7, but as shown in Table 1 (b), when the laser non-irradiation surface 8a is used on the specimen supply path 7 side If the air hole diameter is as fine as 0.05 to 0.1 mm, bubbles remain, and if the viscosity of the specimen sample is small, a sufficient amount of specimen sample cannot be sucked into the specimen supply path 7.

From this, the air hole 9 drilled in the second substrate 8 is 0.05 mm to 0.30 mm, that is, the aperture 9a formed in the laser non-irradiated surface 8a has a diameter of 0.05 to 0.30 mm ( In terms of area, the diameter of the opening 9b formed in the laser irradiation surface 8b is 0.15 to 0.45 mm (in terms of area) (1.96 × 10 ^{−3 to} 7.07 × 10 ⁻² mm ² ). 1.76 × 10 ^{−2 to} 1.58 × 10 ⁻¹ mm ² ), more preferably, the CO ₂ laser irradiation surface 8b is disposed so as to face the specimen supply path 7 side, and the air hole diameter Is at least 0.05 mm to 0.1 mm, that is, the opening 9 a of the laser non-irradiated surface 8 a has a diameter of 0.05 to 0.10 mm (in terms of area, 1.96 × 10 ^{−3 to} 7.85 × 10 ⁻³ mm ^2), and diameter of the opening portion 9b of the laser irradiation surface 8b 0.15 It is desirable that the 0.20 mm (in terms of the area of ^{1.76 × 10 -2 ~3.14 × 10 -2} mm 2).

  If the air hole 9 is shaped as described above, air escape in the specimen supply path 7 of the biosensor 100 is promoted, and bubbles are generated in the specimen supply path 7 regardless of the viscosity of the specimen sample. Can be prevented.

As described above, according to the first embodiment, the second substrate 8 of the biosensor 100 is heated and melted from one surface side of the second substrate 8 toward the other surface by the CO ₂ laser. A fine air hole 9 having a small diameter is formed from the laser irradiation surface 8b toward the non-irradiation surface 8a, and the CO ₂ laser irradiation surface 8b of the _second substrate 8 on which the air hole 9 is formed is supplied with the sample. Since it is arranged so as to be on the path 7 side, even if the air hole 9 of the biosensor 100 is fine, the generation of bubbles regardless of the viscosity of the specimen sample supplied to the specimen supply path 7 It is possible to prevent the sample sample from being sucked into the sample supply path 7 with a fine air hole 9 to obtain a measurement result of the substrate contained in the sample sample.

Further, according to the first embodiment, since the air holes 9 are perforated by irradiating the _second substrate 8 of the biosensor 100 with the CO ₂ laser, the punching residue is different from the conventional press working. It is possible to reduce industrial waste.

  Furthermore, according to the first embodiment, the laser non-irradiation surface 8a side where the aperture of the air hole formed in the second substrate 8 is small faces the outer surface side of the biosensor 100. Since they are arranged, there is an effect that even if the user mistakenly deposits the sample sample in the air hole 9, the sample sample is not sucked into the sample supply path 7.

In the first embodiment, the case where the air holes 9 are perforated using a CO ₂ laser device has been described as an example. However, the air holes 9 may be perforated using another laser device or the like. Further, the air holes 9 of the first embodiment can be formed by laser processing if the shape can be formed so as to be fine holes whose diameter decreases from one surface side toward the other surface as shown in FIG. It is not limited to formation, and for example, it can be formed by perforating with a heated thin needle. However, fine processing with an air hole diameter of 0.05 mm is impossible except for laser irradiation.

  Furthermore, in the first embodiment, the case where the shapes of the openings 9a and 9b of the air hole 9 are substantially circular has been described as an example. However, the present invention is not limited to this. It may be linear, triangular, square, rectangular, polygonal, etc.

  Since the biosensor according to the present invention can obtain an accurate response value without depending on the viscosity of the specimen sample, it is useful for a biosensor for analyzing specimens having individual differences in viscosity such as blood. In addition, since the biosensor according to the present invention processes the air holes by melting using a laser, unlike conventional press processing, there is no occurrence of punching debris and the industrial waste is reduced. Is also useful.

1 is an exploded perspective view of a biosensor according to a first embodiment of the present invention. It is a top view of the biosensor concerning Embodiment 1 of the present invention. It is a perspective view which shows the shape of the opening part by the side of a laser irradiation surface of the air hole concerning Embodiment 1 of this invention. It is a perspective view which shows the shape of the opening part by the side of a laser non-irradiation surface of the air hole concerning Embodiment 1 of this invention. It is sectional drawing of the air hole concerning Embodiment 1 of this invention. In the biosensor according to the first embodiment of the present invention, the apertures of the openings on both surfaces of the second substrate formed when the second substrate is punched with air holes of various apertures using a CO ₂ laser. It is a graph which shows transition of. It is a disassembled perspective view of the conventional biosensor. It is a top view of the conventional biosensor. It is a perspective view which shows the shape of the air hole by the conventional press work. It is sectional drawing of the air hole by the conventional press work.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1,41 1st insulator board | substrate 2,42 Measuring electrode 3,43 Counter electrode 4,44 Detection electrode 5,45 Reagent layer 6,46 Spacer 7,47 Specimen supply path 8,48 2nd insulator board 8a Laser Non-irradiation surface 8b Laser irradiation surface 9,49 Air hole 9a, 9b Opening 10,410 Conductor layer 11, 12, 13, 411, 412, 413 Terminal 14, 15, 414, 415 Slit 100, 400 Biosensor

Claims (5)

At least the first and second substrates are bonded together to form a sample supply path through which the supplied sample sample is aspirated between the substrates, and a reagent that reacts with a component in the sample sample is formed in the sample supply path. A biosensor comprising an air hole that is disposed on the second substrate and communicates with the outside from the sample supply path,
In the air hole, the diameter of the opening on one surface of the second substrate facing the sample supply path is larger than the diameter of the opening on the other surface of the second substrate.
A biosensor characterized by that. The biosensor according to claim 1, wherein
The air holes are drilled by a laser;
A biosensor characterized by that. The biosensor according to claim 1, wherein
The area of the opening on one side of the second substrate facing the specimen supply path is 1.76 × 10 ^{−2 to} 3.14 × 10 ⁻² mm ² ;
The area of the opening on the other surface of the second substrate is 1.96 × 10 ^{−3 to} 7.85 × 10 ⁻³ mm ² .
A biosensor characterized by that. The biosensor according to claim 1, wherein
The shape of the opening of the air hole is substantially circular,
The diameter of the opening on one side of the second substrate facing the specimen supply path is 0.15 to 0.20 mm, and the diameter of the opening on the other side of the second substrate is 0.05 to 0.10 mm,
A biosensor characterized by that. The biosensor according to claim 1, wherein
The shape of the opening of the air hole is any one of a circle, an ellipse, a line having a very narrow width, a triangle, a square, a rectangle, and a polygon.
A biosensor characterized by that.

Images