WO2019187574A1 - Biocapteur - Google Patents

Biocapteur Download PDF

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Publication number
WO2019187574A1
WO2019187574A1 PCT/JP2019/002472 JP2019002472W WO2019187574A1 WO 2019187574 A1 WO2019187574 A1 WO 2019187574A1 JP 2019002472 W JP2019002472 W JP 2019002472W WO 2019187574 A1 WO2019187574 A1 WO 2019187574A1
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WIPO (PCT)
Prior art keywords
reagent
conductive
reagent part
biosensor
area
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PCT/JP2019/002472
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English (en)
Japanese (ja)
Inventor
直 林野
圭吾 羽田
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Ｐｈｃホールディングス株式会社
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Publication of WO2019187574A1 publication Critical patent/WO2019187574A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements

Definitions

  • the present invention relates to a biosensor, and more particularly to a biosensor using an electrochemical measurement method.
  • Biosensors are used to measure a substance to be detected in a biological sample in various fields such as the medical field and clinical laboratory field.
  • a biosensor using an electrochemical measurement method is known (for example, Patent Document 1).
  • This biosensor forms a working electrode, a counter electrode, and a reference electrode on an insulating substrate, and is in contact with these electrodes and includes an enzyme reaction layer (also referred to as a reagent unit) including an enzyme and an electron acceptor (hereinafter referred to as a mediator). ) Is formed.
  • an enzyme reaction layer also referred to as a reagent unit
  • an enzyme and an electron acceptor hereinafter referred to as a mediator
  • Is formed Is formed.
  • various substances can be measured by selecting an enzyme that uses the substance to be measured as a substrate.
  • a glucose sensor that measures glucose concentration in a sample solution by selecting glucose oxidase as an enzyme has been put into practical use.
  • HbA1c hemoglobin A1c
  • HbA1c value is a test value indicating the proportion of hemoglobin bound to sugar in hemoglobin in erythrocytes. Since the HbA1c value reflects the average blood glucose level in the past 1 to 2 months, it is less affected by the diet before the test and is important as an index for managing diabetes.
  • the HbA1c value is measured by an HPLC method or an immunization method using an optical spectroscopic method.
  • the test sample is diluted by, for example, about 100 times by hemolysis (hereinafter, in this specification, a sample solution to be measured is subjected to a pretreatment such as hemolysis. Called liquid). Therefore, the HbA1c concentration in the test solution is significantly lower than the glucose concentration, and is usually a low concentration on the order of ⁇ M.
  • a method for measuring the HbA1c value using an electrochemical measurement method has not been put into practical use.
  • the HbA1c value can be measured with high accuracy by using the electrochemical measurement method.
  • proteins other than HbA1c such as hemoglobin, glycated albumin, glucose, cholesterol, lactic acid, ketone bodies (3-hydroxybutyric acid), antibodies, etc. can be measured with high accuracy if the blank current can be reduced. It becomes possible.
  • an object of the present invention is to provide a biosensor used in an electrochemical measurement method, which can measure with high accuracy by reducing blank current.
  • a biosensor of the present invention is a biosensor that analyzes a component in a test solution using a protein and a mediator, and a spacer is interposed between the insulating substrate and the cover. And having at least one formed space portion, and having at least one inner surface of the space portion having a conductive portion and a non-conductive portion, and including a first reagent portion containing the protein and a second mediator. Reagent parts are separately disposed at different locations on the inner surface, and at least one of the first reagent part and the second reagent part is disposed in the non-conductive part.
  • a biosensor capable of highly accurate measurement can be provided by reducing the blank current.
  • FIG. 2 is a perspective view showing an example of a structure of biosensor A according to Embodiment 1.
  • FIG. It is a disassembled perspective view of the biosensor A of FIG. It is a top view of the insulating base material which comprises the biosensor A of FIG. It is a longitudinal cross-sectional view along the longitudinal direction of the biosensor A of FIG. 6 is an exploded perspective view showing an example of a structure of a biosensor B according to Embodiment 2.
  • FIG. It is a longitudinal cross-sectional view along the longitudinal direction of the biosensor B of FIG.
  • FIG. 6 is a plan view of an insulating base material constituting a biosensor C according to a third embodiment.
  • FIG. It is a longitudinal cross-sectional view along the longitudinal direction of the biosensor C which concerns on Embodiment 3.
  • FIG. It is a graph which shows the blank electric current value in a reference example. It is a graph which shows the blank electric current value in a reference example. It is a graph which shows the blank electric current value in a reference example. It is an example of the graph which shows the relationship between the detection electric current value in this invention, and the density
  • the biosensor of the present invention is a biosensor that analyzes a component in a test solution using a protein and a mediator, and is one or more formed through a spacer between an insulating substrate and a cover.
  • the inner surface of the at least one space portion has a conductive portion and a non-conductive portion, and the first reagent portion containing the protein and the second reagent portion containing the mediator are different in the inner surface. It is arrange
  • FIG. 1 is a perspective view showing an example of the structure of the biosensor A according to the present embodiment
  • FIG. 2 is an exploded perspective view.
  • an insulating base material 1 having a conductive portion 4 and a cover 2 are laminated via a spacer 3.
  • FIG. 1 shows an example having a long rectangular piece shape in which the X direction is the longitudinal direction and the Y direction is the width direction.
  • a conductive portion 4 is formed on one main surface of the insulating substrate 1 having a pair of opposing main surfaces.
  • the conductive portion 4 is a conductive portion, and includes a first electrode pair 41 and a second electrode pair 42, a terminal portion 43 formed at one end of the insulating base material 1, a first electrode pair 41, and a second electrode pair 42. And a lead part 44 for connecting the terminal part 43 to each other.
  • FIG. 3 is a plan view of the insulating substrate 1, and the first electrode pair 41 is disposed in the longitudinal direction and sandwiches the pair of working electrodes 41 a and 41 a that are electrically connected to each other and the working electrodes 41 a in plan view.
  • the second electrode pair 42 acts via a working electrode 42a disposed in the longitudinal direction so as to be separated from the first electrode pair 41, and a minute gap (described later) so as to sandwich the working electrode 42a in plan view. It has a pair of counter electrodes 42b and 42b facing the pole 42a.
  • the pair of working electrodes 41a and 41a is connected to the terminal 43a through the lead 44a
  • the two pairs of counter electrodes 41b and 41b are connected to the terminal 43d through the lead 44d
  • the working electrode 42a is connected to the terminal 43b through the lead 44b.
  • the pair of counter electrodes 42b and 42b are connected to the terminal 43c via a lead 44c. 2 and FIG. 3, the structure excluding the first reagent part and the second reagent part described later is shown. Further, the two electrode pairs of the first electrode pair 41 and the second electrode pair 42 can be used when measuring two different types of substances to be detected in the test liquid.
  • the case of measuring two kinds of substances to be detected is, for example, the case of measuring the HbA1c value, measuring the concentration of HbA1c with the first electrode pair 41 and measuring the concentration of Hb with the second electrode pair 42. Can do.
  • the example which used two working electrodes for the 1st electrode pair 41 was shown, it can also be set as the structure using one working electrode similarly to the case of the 2nd electrode pair 42.
  • the spacer 3 only needs to have at least one opening, but in the present embodiment, the spacer 3 has two openings 3a and 3b formed to be separated from each other along the longitudinal direction.
  • the spacer 3 is shorter than the insulating base 1 so that the terminal portion 43 is exposed.
  • the cover 2 has an opening 2a at one end in the longitudinal direction, an opening 2c at the other end, and an opening 2b at an intermediate portion between the opening 2a and the opening 2c.
  • the cover 2 is also made shorter than the insulating base material 1 so that the terminal part 43 is exposed similarly to the case of the spacer 3.
  • the opening of the spacer 3 and the opening of the cover 2 are such that when the spacer 3 and the cover 2 are stacked, the opening 2 b at the center of the cover 2 is the end on the center side of the opening 3 a of the spacer 3. And the opening 2a at one end of the cover 2 is at least partially overlapped with the side end of the opening 3a of the spacer 3, so that the cover 2
  • the opening 2c at the other end of the spacer 3 is disposed so as to at least partially overlap the side end of the opening 3b of the spacer 3.
  • FIG. 4 is a longitudinal sectional view along the longitudinal direction of the biosensor A shown in FIG.
  • the openings 3 a and 3 b of the spacer 3 form two first space portions 6 and second space portions 7 that are independent from each other when the spacer 3 is sandwiched between the insulating base material 1 and the cover 2. ing.
  • one main surface of the insulating base material 1 exposed to a space part comprises the bottom face of a space part.
  • the cover 2 has a pair of opposing main surfaces, and one main surface exposed to the space portion constitutes the top surface of the space portion.
  • the inner surface of the opening part of the spacer 3 comprises the side surface of a space part.
  • the biosensor of the present invention has a conductive portion and a non-conductive portion on the inner surface of the space portion.
  • the conductive portion is a conductive portion, and includes an electrode including a working electrode and a counter electrode, a lead portion, and a terminal portion.
  • the conductive portion in the first space portion 6 is the first electrode pair 41 and the lead portion.
  • the non-conductive portion in the first space portion 6 is a portion that is not conductive, specifically, a portion where no electrode or lead is formed on the insulating base material 1, or the opening 3 a of the spacer 3.
  • a main surface 5 that is not in contact with the cover 2 (hereinafter also referred to as a back surface) can be exemplified.
  • the conductive part in the second space 7 is the second electrode pair 42 and the lead part.
  • the non-conductive portion of the second space portion 7 is a portion of the insulating base material 1 where no electrodes or leads are formed, the inner peripheral surface of the opening 3b of the spacer 3, and a pair of spacers 3 facing each other.
  • the back surface 5 that is not in contact with the cover 2 can be cited.
  • the first reagent part containing protein and the second reagent part containing mediator are separately arranged at different locations on the inner surface of at least one space part, and at least one of the first reagent part and the second reagent part is Arranged in the non-conductive portion.
  • the first reagent portion 8 is disposed on the surfaces of the first electrode pair 41 and the second electrode pair 42 that are conductive portions, and the second reagent portion 9 is non-conductive. It is arranged on the back surface 5 of the cover 2 which is a part.
  • the second reagent portion 9 is disposed on the surface of the second electrode pair 42.
  • the opening 2b in the center of the cover 2 can be used as an introduction opening for the test liquid, and the introduction opening communicates with the space.
  • the space part in the present invention provides a holding area for holding the test liquid and a reaction area for advancing the reaction between the test liquid and the first reagent part and the second reagent part.
  • the reaction region refers to a region occupied by the conductive part and the reagent part arranged in the conductive part.
  • the openings 2 a and 2 c at both ends of the cover 2 provide air to the inside of the first space portion 6 and the second space portion 7, respectively, and the test liquid is supplied from the opening portion 2 b at the center portion of the cover 2. When it is dropped, it works to release the internal air as the test solution is drawn.
  • the area of one reagent part of the first reagent part and the second reagent part may be larger than the area of the other reagent part.
  • one reagent part of the first reagent part and the second reagent part is arranged in the non-conductive part
  • the other reagent part is arranged in the conductive part
  • one of the reagent parts arranged in the non-conductive part The area of the reagent part may be larger than the area of the other reagent part arranged in the conductive part.
  • the area of the second reagent portion 9 disposed on the back surface 5 of the cover 2 that is a non-conductive portion is the first space portion disposed on the surface of the first electrode pair 41 that is a conductive portion.
  • An example larger than the area of one reagent part 8 is shown.
  • the area of the reagent part is an area of the reagent part in plan view.
  • the reagent part is formed by a coating method, it is the area of the coating film in plan view (hereinafter, the area of the coating film in plan view may be referred to as the coating film area).
  • the two 1st reagent parts 8 exist in the same space part (1st space part 6).
  • the area of the reagent part is a total area of two or more reagent parts.
  • the test liquid flowing in from the introduction opening moves from the upstream to the downstream in the space, and at this time, a part of the reagent in the first reagent part and / or the second reagent part is dissolved in the test liquid. May move out of the reaction zone.
  • a sufficient amount of reagent can be supplied to the reaction region.
  • the area of one reagent part arranged in the non-conductive part is larger than the area of the other reagent part arranged in the conductive part, the reagent contained in one reagent part arranged in the non-conductive part Even if a part flows out of the reaction region together with the test solution, a sufficient amount of reagent can be supplied to the working electrode of the conductive portion. As a result, the response value is increased and the S / N ratio is improved, so that highly accurate measurement is possible. Furthermore, it is preferable to arrange the second reagent part in the non-conductive part and make the area of the second reagent part larger than the area of the first reagent part.
  • the area of the reagent part is, for example, in the range of 0.5 mm 2 to 200 mm 2
  • the area of one reagent part is equal to or larger than the area of the other reagent part, preferably 1.05 to 10 times. Times, more preferably 1.5 times to 4 times.
  • the area of one reagent part of the first reagent part and the second reagent part arranged in the non-conductive part may be larger than the area of the working electrode included in the conductive part.
  • the test solution flowing in from the introduction opening moves from the upstream to the downstream in the space portion.
  • a part of the reagent may be dissolved in the test solution and move out of the reaction region.
  • a part of the reagent contained in the reagent part arranged in the non-conductive part flows out of the reaction region and is not supplied to the working electrode.
  • the area of the working electrode is the area of the working electrode in plan view. Specifically, for example, when the area of the reagent part is in the range of 0.5 to 200 mm 2 , the area of one reagent part is equal to or more than the area of the working electrode, preferably 1.5 to 30 times, More preferably, it is 2 to 20 times.
  • the area of one reagent part of the first reagent part and the second reagent part arranged in the non-conductive part is based on the total area of the working electrode area and the counter electrode area of the conductive part. May be larger.
  • the test solution flowing in from the introduction opening moves from the upstream to the downstream in the space portion.
  • a part of the reagent may be dissolved in the test solution and move out of the reaction region.
  • a part of the reagent contained in the reagent part arranged in the non-conductive part flows out of the reaction region and is not supplied to the working electrode.
  • the area of the reagent part arranged in the non-conductive part is larger than the total area of the working electrode area and the counter electrode area, even if a part of the reagent flows out of the reaction region together with the test solution, A sufficient amount of reagent can be supplied to the working electrode and the counter electrode.
  • a sufficient amount of reagent for the reduction reaction reacts on the counter electrode within a fixed time, and a sufficient amount of reagent for the oxidation reaction reacts on the working electrode, increasing the response value and improving the S / N ratio. Therefore, measurement with high accuracy is possible.
  • the area of the working electrode is the area of the working electrode in plan view
  • the area of the counter electrode is the area of the counter electrode in plan view.
  • the area of the reagent part is in the range of 0.5 to 200 mm 2
  • the area of one reagent part is equal to or larger than the total area of the working electrode area and the counter electrode area, preferably It is 1.05 times to 10 times, more preferably 1.5 times to 4 times.
  • one reagent part of the first reagent part and the second reagent part arranged in the non-conductive part is closer to the introduction opening for the test solution than the other reagent part. It may be arranged. As described above, the test solution flowing in from the introduction opening moves from the upstream to the downstream in the space portion. At this time, a part of the reagent may be dissolved in the test solution and move out of the reaction region. At this time, a part of the reagent contained in the reagent part arranged in the non-conductive part flows out of the reaction region and is not supplied to the working electrode.
  • one reagent part arranged in the non-conductive part is arranged closer to the introduction opening for the test solution than the other reagent part, that is, upstream of the other reagent part.
  • the response value increases and the S / N ratio is improved, so that more accurate measurement is possible.
  • at least a part of one reagent part may be arranged so as to be closer to the introduction opening for the test solution than the other reagent part.
  • one reagent part of the first reagent part and the second reagent part arranged in the non-conductive part may be arranged closer to the introduction opening than the working electrode.
  • the test solution flowing in from the introduction opening moves from the upstream to the downstream in the space portion.
  • a part of the reagent may be dissolved in the test solution and move out of the reaction region.
  • a part of the reagent contained in the reagent part arranged in the non-conductive part flows out of the reaction region and is not supplied to the working electrode.
  • one of the reagent parts arranged in the non-conductive part is arranged so as to be closer to the introduction opening for the test solution than the working electrode, that is, by arranging it upstream of the working electrode. Even if a part of the reagent flows out of the reaction region, a sufficient amount of the reagent can be supplied to the working electrode. As a result, the response value increases and the S / N ratio is improved, so that more accurate measurement is possible. It should be noted that at least a part of one of the reagent parts may be arranged so as to be closer to the introduction opening for the test solution than the working electrode.
  • the material used for the insulating substrate is not particularly limited.
  • resin materials or glass materials can be used.
  • Preferred are polyethylene terephthalate, polycarbonate, and polyimide, and more preferred is polyethylene terephthalate.
  • the size of the insulating substrate is not particularly limited. For example, the total length is 5 to 100 mm, the width is 2 to 50 mm, and the thickness is 0.05 to 2 mm.
  • the total length is 7 to 50 mm, the width is 3 to 20 mm, and the thickness is 0.1 to 1 mm. More preferably, the total length is 10 to 30 mm, the width is 3 to 10 mm, and the thickness is 0.1 to 0.6 mm.
  • the conductive part on the insulating substrate is formed of a conductive layer by sputtering or vapor deposition using, for example, carbon, gold, platinum, palladium or the like, and then formed into a predetermined electrode pattern by laser trimming. It can be formed by processing.
  • the laser trimming method the minute gap between the working electrode and the counter electrode, the minute gap between the leads, and the minute gap between the terminals are formed, so that the electrical insulation between the electrodes, between the leads, and between the terminals. Is secured.
  • the material of the spacer is not particularly limited.
  • the same material as the insulating base material can be used.
  • the size of the spacer is not particularly limited.
  • the total length is 5 to 100 mm
  • the width is 2 to 50 mm
  • the thickness is 0.01 to 1 mm
  • the total length is 7 to 50 mm
  • the width is 3 to 20 mm
  • the thickness is 0.05 to 0.5 mm.
  • the total length is 10 to 30 mm
  • the width is 3 to 10 mm
  • the thickness is 0.05 to 0.25 mm.
  • the cover material is not particularly limited.
  • the same material as the insulating base material can be used.
  • the size of the cover is not particularly limited.
  • the total length is 5 to 100 mm
  • the width is 3 to 50 mm
  • the thickness is 0.01 to 0.5 mm
  • the total length is 10 to 50 mm
  • the width is 3 to 20 mm
  • the thickness is 0.05 to 0.25 mm.
  • the total length is 15 to 30 mm
  • the width is 5 to 10 mm
  • the thickness is 0.05 to 0.1 mm.
  • the cover is preferably formed with a plurality of openings used as air holes or test liquid inlets. As the shape of the opening, for example, a circle, an ellipse, a polygon or the like can be used.
  • the maximum diameter is 0.01 to 10 mm, preferably 0.05 to 5 mm, more preferably 0.1 to 2 mm.
  • the opening may be formed by drilling with a laser or a drill, or may be formed by molding using a mold.
  • a biosensor can be manufactured by laminating an insulating base material, a spacer, and a cover in this order, and bonding and integrating them with an adhesive or heat fusion.
  • An epoxy adhesive, an acrylic adhesive, a polyurethane adhesive, a hot melt adhesive, a UV curable adhesive, or the like can be used as the adhesive.
  • the first reagent part includes a protein
  • the second reagent part includes a mediator.
  • the protein include enzymes, antibodies, immunoglobulins, bovine serum albumin, human serum albumin and the like.
  • the enzyme include oxidoreductases such as glucose oxidase, lactate oxidase, cholesterol oxidase, bilirubin oxidase, glucose dehydrogenase, lactate dehydrogenase, fructosyl amino acid oxidase, fructosyl peptide oxidase, and 3-hydroxybutyrate dehydrogenase.
  • oxidoreductases are oxidases or dehydrogenases that act on glucose, lactic acid, cholesterol, bilirubin, glycated amino acids, or glycated peptides, ketone bodies (3-hydroxybutyric acid).
  • the amount of oxidoreductase is, for example, 0.01 to 100 U, preferably 0.05 to 10 U, more preferably 0.1 to 100 U per sensor or per measurement. 5U.
  • Mediators include, but are not limited to, metal complexes (eg, osmium complexes, ruthenium complexes, iron complexes, etc.), quinone compounds (eg, benzoquinone, naphthoquinone, phenanthrenequinone, phenanthrolinequinone, anthraquinone, and derivatives thereof).
  • metal complexes eg, osmium complexes, ruthenium complexes, iron complexes, etc.
  • quinone compounds eg, benzoquinone, naphthoquinone, phenanthrenequinone, phenanthrolinequinone, anthraquinone, and derivatives thereof.
  • a phenazine compound, a viologen compound, a phenothiazine compound, and a phenol compound eg., a viologen compound, a phenothiazine compound, and a phenol compound.
  • the salt examples include, but are not limited to, sodium salt, potassium salt, calcium salt, magnesium salt, lithium salt and the like.
  • the blending amount of the mediator is not particularly limited, and is, for example, 0.1 pmol to 100 ⁇ mol, preferably 10 pmol to 10 ⁇ mol, more preferably 50 pmol to 1 ⁇ mol per measurement or per biosensor. is there.
  • the first reagent part containing the protein and the second reagent part containing the mediator are separately arranged at different locations on the inner surface of the space part. Therefore, the first reagent part containing the protein and the second reagent part containing the mediator Are prepared separately. If necessary, additives such as a buffer and a hydrophilic polymer can be added to the first reagent part and the second reagent part, respectively. An enzyme stabilizer can also be added to the first reagent part. Each of these substances is dissolved in water, applied to the application part, and dried to form a reagent part.
  • the portion to be coated is the first electrode pair that is a conductive portion in the case of the first reagent portion, and the back surface of the cover in the case of the second reagent portion.
  • the terminal of the terminal portion 43 is connected to a measuring device (not shown).
  • test liquid is sucked up with a dropper, and the test liquid is dropped into the opening 2b for introducing the test liquid in the cover 2.
  • the first electrode pair 41 measures the concentration of HbA1c
  • the second electrode pair 42 measures the concentration of Hb.
  • the 1st reagent part 8 of the 1st space part 6 contains fructosyl peptide oxidase, for example, and a 2nd reagent part contains potassium ferricyanide, for example.
  • the fructosyl valyl histidine in the test solution dropped into the test solution introduction opening 2 b of the cover 2 reacts with the fructosyl peptide oxidase of the first reagent unit 8 arranged in the first space 6.
  • the potassium ferricyanide contained in the second reagent unit 9 is reduced to potassium ferrocyanide, but is oxidized to potassium ferricyanide with the application of voltage. Since the oxidation current changes according to the concentration of HbA1c in the test solution, the concentration of HbA1c in the test solution can be measured by measuring the oxidation current.
  • the concentration of Hb can be measured.
  • the HbA1c value can be calculated.
  • a coating film obtained by applying a coating liquid containing both an enzyme and a mediator to an electrode and drying it is used as a reagent part.
  • the first reagent part containing the protein and the second reagent part containing the mediator are arranged separately, and at least one of the first reagent part and the second reagent part is made non-conductive.
  • a crack may occur in the conductive part, which may greatly affect the performance, but at least one of the first reagent part and the second reagent part Can be prevented from being generated on the conductive portion.
  • the first reagent unit is arranged on the surface of the first electrode pair and the second reagent unit is arranged on the back surface of the cover.
  • the first reagent unit is arranged on the back surface of the cover. The same effect can be obtained even if the second reagent part is disposed on the back surface of the first electrode pair.
  • the second reagent part is provided in the second space part and used for measurement of two types of detected substances.
  • the reagent part in the second space part Only one electrode pair can be used for measurement of one kind of two kinds of detected substances, and a plurality of electrode pairs can be provided on the insulating substrate in accordance with the number of detected substances.
  • Embodiment 2 In the first embodiment, an example using two electrode pairs of the first electrode pair and the second electrode pair has been described, but in this embodiment, an example using one electrode pair will be described. In the following description, the description overlapping with the description of Embodiment 1 is omitted.
  • FIG. 5 is an exploded perspective view showing an example of the structure of the biosensor B according to the present embodiment.
  • an insulating base material 21 having a conductive portion 24 and a cover 22 are laminated via a spacer 23.
  • a conductive portion 24 is formed on one main surface of the insulating base material 21 having a pair of opposing main surfaces.
  • the conductive portion 24 is a conductive portion, and includes a first electrode pair 241, a terminal portion 243 formed at one end of the insulating base material 21, and a lead portion 242 that connects the first electrode pair 241 and the terminal portion 43.
  • the first electrode pair 241 has a working electrode 241a and a pair of counter electrodes 241b and 241b facing the working electrode 241a through a minute gap so as to sandwich the working electrode 241a in plan view.
  • the working electrode 241 is connected to the terminal 243a via the lead 242a, and the pair of counter electrodes 241b and 241b is connected to the terminal 243b via the lead 242b.
  • FIG. 6 is a longitudinal sectional view of the biosensor B along the longitudinal direction.
  • the opening 23 a of the spacer 23 forms a first space 26 as one space by sandwiching the spacer 23 between the insulating base material 21 and the cover 22.
  • the conductive portion in the first space portion 26 is the first electrode pair 241 and the lead portion.
  • the non-conductive portion in the first space portion 26 is a portion that is not conductive, specifically, a portion where no electrode or lead is formed on the insulating base material 21, or the opening 23 a of the spacer 23.
  • a main surface 25 hereinafter also referred to as a back surface
  • the first reagent portion 28 is disposed on the surface of the first electrode pair 241 that is a conductive portion, and the second reagent portion 29 is disposed on the back surface 25 of the cover 22 that is a nonconductive portion.
  • the opening 22a at the end in the longitudinal direction of the cover 22 can be used as an opening for introducing the test liquid.
  • the test liquid flows into the first space 26.
  • the first reagent portion 28 and the second reagent portion 29 are in contact with each other.
  • the opening 22b at the center of the cover 22 provides air to the inside of the first space 26, and when the test liquid is dropped from the opening 22a at the end of the cover 22, Along with the pull-in, it works to release the air inside.
  • Biosensor B can be used when measuring one type of substance to be detected.
  • the 1st reagent part 28 of the 1st space part 26 may contain glucose dehydrogenase, for example
  • the 2nd reagent part may contain potassium ferricyanide, for example.
  • the glucose in the test liquid dropped into the test liquid introduction opening 22a of the cover 22 reacts with the glucose dehydrogenase of the first reagent part 28 disposed in the first space part 26, and the second reagent part.
  • the potassium ferricyanide contained in 29 is reduced to potassium ferrocyanide, but is oxidized to potassium ferricyanide with the application of voltage. Since the oxidation current changes according to the glucose concentration in the test solution, the glucose concentration in the test solution can be measured by measuring the oxidation current.
  • the area of one reagent part of the first reagent part and the second reagent part may be larger than the area of the other reagent part.
  • positioned at a nonelectroconductive part may be larger than the area of the working electrode contained in a conductive part.
  • the area of one reagent part of the first reagent part and the second reagent part arranged in the non-conductive part may be larger than the total area of the area of the working electrode and the counter electrode of the conductive part.
  • one reagent part of the first reagent part and the second reagent part arranged in the non-conductive part may be arranged so as to be closer to the introduction opening for the test solution than the other reagent part.
  • one reagent part of the first reagent part and the second reagent part arranged in the non-conductive part may be arranged so as to be closer to the introduction opening than the working electrode.
  • the biosensor according to the present embodiment also has the same effect as the biosensor according to Embodiment 1, and can be suitably used when measuring one kind of substance to be detected.
  • Embodiment 3 In the first embodiment, the example in which the second reagent part is disposed on the back surface of the cover has been described. In the present embodiment, an example in which the second reagent part is disposed in the non-conductive part on the insulating substrate will be described. . In the following description, the description overlapping with the description of Embodiment 1 is omitted.
  • FIG. 7 is a plan view of the insulating substrate 31 used in the biosensor C according to the present embodiment
  • FIG. 8 is a longitudinal sectional view along the longitudinal direction of the biosensor C.
  • a conductive portion 34 is formed on one main surface of the insulating substrate 31 having a pair of opposing main surfaces.
  • the conductive portion 34 includes a first electrode pair 341 and a second electrode pair 342, a terminal portion 343 formed at one end of the insulating base material 31, a first electrode pair 341 or a second electrode pair 342 and a terminal portion 343.
  • a lead portion 344 to be connected is provided.
  • the first electrode pair 341 has a working electrode 341a and a pair of counter electrodes 341b and 341b facing the working electrode 341a through a minute gap so as to sandwich the working electrode 341a in plan view.
  • the second electrode pair 342 is disposed in the longitudinal direction apart from the first electrode pair 341, and faces the working electrode 342a through a minute gap so as to sandwich the working electrode 342a in plan view.
  • a pair of counter electrodes 342b and 342b is provided.
  • the pair of working electrodes 341a and 341a is connected to the terminal 343a via the lead 344a, and the two pairs of counter electrodes 341b and 341b are connected to the terminal 343d via the lead 344d.
  • the working electrode 342a is connected to the terminal 343b via a lead 344b, and the pair of counter electrodes 342b and 342b is connected to the terminal 343c via a lead 344c.
  • Reference numeral 351 denotes a region where a conductive portion is not formed and corresponds to a non-conductive portion. In the case of FIG. 7, a structure excluding a first reagent part and a second reagent part described later is shown.
  • the openings 33 a and 33 b of the spacer 33 form two first space portions 36 and second space portions 37 that are independent from each other when the spacer 33 is sandwiched between the insulating base material 31 and the cover 32. ing.
  • the conductive portion in the first space portion 36 is the first electrode pair 341 and the lead portion.
  • the non-conductive portion in the first space portion 36 is an insulating base material surface 352 as a portion where no electrode or lead is formed in the insulating base material 31 and the inner peripheral surface of the opening 33a of the spacer 33.
  • a main surface 351 that is not in contact with the cover 32 hereinafter also referred to as a back surface
  • the conductive portion in the second space portion 37 is the second electrode pair 42 and the lead portion.
  • the non-conductive portion of the second space portion 37 is a portion of the insulating base 31 where no electrode or lead is formed, an inner peripheral surface of the opening 33b of the spacer 33, and a pair of opposed spacers 33.
  • a back surface 351 that is not in contact with the cover 32 can be cited.
  • the first reagent portion 38 is disposed on the first electrode pair 341 that is a conductive portion, and the second reagent portion 39 is a non-conductive portion. 352.
  • the second reagent portion 39 is disposed on the surface of the second electrode pair 342.
  • the opening 32b at the center of the cover 32 can be used as an opening for introducing the test liquid.
  • the test liquid When the test liquid is dropped into the opening 32b, the test liquid is the first. It flows to the space part 36 and the second space part 37, and contacts the first reagent part 38 and the second reagent part 39 in the first space part 36.
  • the openings 32 a and 32 c at both ends of the cover 32 provide air to the inside of the first space 36 and the second space 37, respectively, and the test liquid is supplied from the opening 32 b at the center of the cover 32. When it is dropped, it works to release the internal air as the test solution is drawn.
  • one reagent part of the first reagent part and the second reagent part arranged in the non-conductive part is arranged so as to be closer to the introduction opening for the test solution than the other reagent part.
  • the second reagent part 39 disposed on the insulating base material surface 352 that is a non-conductive part is more open than the first reagent part 38 as an opening 32 b that is an introduction opening for the test liquid. It is arranged to be close to.
  • the test liquid flowing in from the introduction opening contacts the second reagent part 39 and moves from the upstream to the downstream in the first space 36 while dissolving the reagent contained in the second reagent part 39.
  • the second reagent part 39 arranged on the insulating base surface 352 is arranged so as to be closer to the introduction opening than the first reagent part 38, that is, upstream from the first reagent part 38. Therefore, even if a part of the reagent flows out of the reaction region, a sufficient amount of the reagent can be supplied to the working electrode 341a. As a result, the response value increases and the S / N ratio is improved, so that more accurate measurement is possible.
  • one reagent part of the first reagent part and the second reagent part arranged in the non-conductive part is arranged so as to be closer to the introduction opening than the working electrode.
  • the second reagent part 39 disposed on the insulating base material surface 352 that is a non-conductive part is closer to the opening part 32b that is an opening for introducing the test solution than the working electrode 341a. It is arranged to be.
  • the test liquid flowing in from the introduction opening contacts the second reagent part 39 and moves from the upstream to the downstream in the first space 36 while dissolving the reagent contained in the second reagent part 39.
  • the second reagent part 39 disposed on the insulating base material surface 352 is disposed closer to the introduction opening than the working electrode 341a, that is, disposed upstream of the working electrode 341a. Therefore, even if a part of the reagent flows out of the reaction region, a sufficient amount of reagent can be supplied to the working electrode 341a. As a result, the response value increases and the S / N ratio is improved, so that more accurate measurement is possible.
  • the area of one reagent part of the first reagent part and the second reagent part may be larger than the area of the other reagent part.
  • positioned at a nonelectroconductive part may be larger than the area of the working electrode contained in a conductive part.
  • the area of one reagent part of the first reagent part and the second reagent part arranged in the non-conductive part may be larger than the total area of the area of the working electrode and the counter electrode of the conductive part.
  • the first reagent part 38 is disposed on the first electrode pair 341 which is a conductive part, and the second reagent part 39 is provided with an insulating group which is a non-conductive part.
  • the same effect as in the first embodiment can be obtained.
  • the two electrode pairs of the first electrode pair 341 and the second electrode pair 342 are provided, it can be used when two different kinds of substances to be detected in the test liquid are measured.
  • a coating film obtained by applying a coating solution containing both a protein such as an enzyme and a mediator to an electrode and drying it is used as a reagent part.
  • a coating film using a coating liquid containing both protein and mediator hereinafter referred to as a mixed support coating film
  • a coating film containing protein and a mediator is used as a reagent part.
  • the blank current value was compared for the case (hereinafter referred to as a separate supported coating film).
  • the mixed supported coating film In the mixed supported coating film, a predetermined amount of a reagent solution having the following composition is spotted on a palladium sheet prepared by vapor deposition on an insulating substrate constituting the biosensor A in FIG. % And dried for about 3 hours.
  • This coating film was redissolved in the same amount of sodium phosphate buffer (pH 7.0, 50 mM) as the reagent solution, and the redissolved solution was aspirated and collected.
  • the separate supported coating film was prepared using the same method as that for the mixed supported coating film except that a reagent solution containing no mediator was prepared and the reagent solution was spotted on the supporting sheet.
  • ⁇ Reagent solution Sodium phosphate buffer (pH 7.0) 10 mM Mediator 6mM Protein solution 10 mg / mL or 20 mg / mL
  • Mediator 1-methoxy-5-methylphenazinium methyl sulfate (PMS) 9,10-phenanthrenequinone-2-sulfonic acid sodium salt (PQSA)
  • Fructosyl peptide oxidase FPOX
  • GDH Glucose dehydrogenase
  • IgG Immunoglobulin G
  • BSA Bovine serum albumin
  • the redissolved solution was spotted on the biosensor A shown in FIG. 1 and subjected to electrochemical measurement.
  • the biosensor A was connected to a potentiostat, a voltage of 0.2 V was applied between the working electrode and the counter electrode for 30 seconds, and the current value when 10 seconds had elapsed after application was defined as a blank current.
  • FIG. 9 shows the result of another supported coating film containing only protein
  • FIG. 10 shows the result of the mixed supported coating film using 1-methoxy-5-methylphenazinium methyl sulfate (PMS) as a mediator
  • FIG. 11 shows the results of a mixed supported coating film using sodium 9,10-phenanthrenequinone-2-sulfonate (PQSA) as a mediator.
  • the blank current was 25 nA or less.
  • the blank currents of PMS and PQSA were 235 nA and 18 nA, respectively.
  • the blank current increased as compared with the separate supported coating film of protein and the separately supported coating film of mediator, and particularly increased when PMS was used as the mediator.
  • PMS the blank current
  • the blank current in the case of PQSA, it increased from 75 to 189 nA depending on the type and concentration of protein.
  • PMS it increased from 513 to 2707 nA depending on the type and concentration of protein. From this result, it can be expected that the blank current can be suppressed by using another supported coating film in the reagent part instead of the mixed supported coating film.
  • the reason why the blank current is large is not always clear, but the high concentration state of the enzyme and the mediator is maintained by drying the coating liquid, and the amino group having an electron donating property in the enzyme is the mediator. It is conceivable that the blank current is increased by partially reducing.
  • Example 1 ⁇ Production of biosensor> 1 ⁇ l of the following reagent solution 1 is applied to the surface of the first electrode pair on the insulating base material constituting the biosensor A of FIG. 1 and dried at 25 ° C. and 50% humidity for about 3 hours. Formed. On the other hand, 24 ⁇ L of the following reagent solution 2 was applied to the back surface of the cover constituting the biosensor A of FIG. 1 and dried at 25 ° C. and 50% humidity for about 3 hours to form a second reagent part. And the biosensor shown in FIG. 1 was produced.
  • ⁇ Reagent liquid 1 Sodium phosphate buffer (pH 7.0) 25 mM Glucose dehydrogenase (GDH) 2000 U / mL Carboxymethylcellulose (CMC) 0.2% Dodecyl maltoside 0.0065% ⁇ Reagent liquid 2> 9,10-phenanthrenequinone-2-sulfonic acid sodium salt (PQSA) 10 mM
  • the biosensor A was spotted with 50 ⁇ L of a test solution having the following composition and subjected to electrochemical measurement.
  • the biosensor A was connected to a potentiostat, a voltage of 0.2 V was applied between the working electrode and the counter electrode for 30 seconds, and the current value when 10 seconds had elapsed after the application was measured.
  • ⁇ Test solution Sodium phosphate buffer (pH 7.0) 25 mM Glucose 0, 20, 50, 100 ⁇ M
  • FIG. 12 shows the relationship between the glucose concentration and the detected current value ( ⁇ mark). A linear relationship was obtained in the range of 0-100 ⁇ M.
  • Comparative Example 1 ⁇ Production of biosensor> 4 ⁇ L of the following reagent solution 3 is applied to the surface of the first electrode pair on the insulating substrate constituting the biosensor A of FIG. 1 and dried at 25 ° C. and 50% humidity for about 3 hours to form a reagent part.
  • a biosensor was produced by the same method as in Example 1 except that.
  • the prepared biosensor was spotted, and electrochemical measurement was performed in the same manner as in Example 1.
  • FIG. 12 shows the relationship between the glucose concentration and the detected current value ( ⁇ mark). A linear relationship was obtained in the range of 0-100 ⁇ M.
  • a biosensor capable of highly accurate measurement can be provided.

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Abstract

L'invention concerne un biocapteur utilisé dans un procédé de mesure électrochimique, le biocapteur étant capable d'effectuer une mesure avec une précision élevée en réduisant le courant muet. Ce biocapteur analyse un constituant d'un liquide à tester en utilisant une protéine et un médiateur, le biocapteur comportant au moins une partie d'espace formée à travers un espaceur situé entre un substrat isolant et un couvercle, au moins une surface des parties d'espace comportant une partie électroconductrice et une partie non électroconductrice, une première partie de réactif contenant la protéine et une seconde partie de réactif contenant le médiateur étant disposées individuellement en différents emplacements sur la surface interne et la première partie de réactif et/ou la seconde partie de réactif étant disposée(s) dans la partie non électroconductrice.
PCT/JP2019/002472 2018-03-26 2019-01-25 Biocapteur WO2019187574A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001249103A (ja) * 1999-12-27 2001-09-14 Matsushita Electric Ind Co Ltd バイオセンサ
JP2001343349A (ja) * 2000-05-31 2001-12-14 Techno Medica Co Ltd 中性脂肪測定用センサ
WO2008007499A1 (fr) * 2006-07-13 2008-01-17 Panasonic Corporation Puce a dosage immunologique electrochimique
WO2015002184A1 (fr) * 2013-07-05 2015-01-08 株式会社村田製作所 Biocapteur
JP2016520844A (ja) * 2013-06-07 2016-07-14 ライフスキャン・スコットランド・リミテッド 裸電極に相対する可溶性電気化学的活性コーティングを備える電気化学式分析試験ストリップ

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2001249103A (ja) * 1999-12-27 2001-09-14 Matsushita Electric Ind Co Ltd バイオセンサ
JP2001343349A (ja) * 2000-05-31 2001-12-14 Techno Medica Co Ltd 中性脂肪測定用センサ
WO2008007499A1 (fr) * 2006-07-13 2008-01-17 Panasonic Corporation Puce a dosage immunologique electrochimique
JP2016520844A (ja) * 2013-06-07 2016-07-14 ライフスキャン・スコットランド・リミテッド 裸電極に相対する可溶性電気化学的活性コーティングを備える電気化学式分析試験ストリップ
WO2015002184A1 (fr) * 2013-07-05 2015-01-08 株式会社村田製作所 Biocapteur

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