JP2006201080A - Biosensor chip - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a biosensor chip capable of detecting a substance to be inspected, with high sensitivity, by simple constitution. <P>SOLUTION: This biosensor chip is provided with a substrate 3 having a flow passage 1 for allowing a flow of a specimen, a micro-protruding group 5 provided in the flow passage 1 and formed of a material, having a biofunctional molecule coupling group, and a working electrode 7 for detecting an electrode active substance and a reference electrode 9. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a biosensor chip, and more particularly to a biosensor chip that can detect a test substance with high sensitivity by electrolysis of an electrode active material.

  Conventionally, as a biosensor chip for electrochemically measuring the concentration of a test substance, one described in Patent Document 1 is known. As shown in FIG. 8, the biosensor chip includes a pair of base lead electrodes 53 on a substrate 51. A working electrode 55 and a counter electrode 57 are provided on the lead electrode 53, respectively. On the working electrode 55, a charge transfer medium layer 61 and an enzyme reaction layer 63 are provided. The enzyme reaction layer 63 contains an oxidoreductase corresponding to the test substance, and the charge transfer medium layer 61 contains an electron acceptor that undergoes an oxidoreduction reaction coupled with the enzyme reaction of the oxidoreductase. The working electrode 55 and the counter electrode 57 are insulated by an insulating layer 59.

In this biosensor chip, when a sample liquid such as blood is dropped onto the enzyme reaction layer 63, the substrate in the sample reacts with the oxidoreductase, and the electron acceptor is reduced. After completion of the reaction, a voltage is applied to the electrode system to oxidize the reduced form of the electron acceptor, and the substrate concentration in the sample liquid is determined from the oxidation current value obtained at this time.
JP 2004-294231 A

  In the conventional biosensor chip, since the enzyme reaction layer 63 is formed on the lead electrode, it is difficult to sufficiently increase the contact surface area between the oxidoreductase contained in the enzyme reaction layer 63 and the substrate. Yes, it is difficult to increase the reaction speed and sensitivity.

  The present invention has been made in view of such circumstances, and provides a biosensor chip capable of detecting a test substance with high sensitivity with a simple configuration.

  The biosensor chip of the present invention includes a substrate having a channel for flowing a specimen, a microprojection group provided in the channel and formed of a material having a biofunctional molecular binding group, and a downstream of the microprojection group Provided with a working electrode and a reference electrode for detecting the electrode active material.

  Since the microprojection group of this biosensor chip has a very large surface area, a large amount of biofunctional molecules are bound thereto. For this reason, the test substance in the specimen reacts efficiently with the biofunctional molecule, and the detection sensitivity is improved. The microprojection group of this biosensor chip can be easily formed by an imprint method or the like, and the working electrode and the reference electrode can also be easily formed by a screen print method or the like. Therefore, this biosensor chip can be easily manufactured.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings. The drawings are used for convenience of explanation, and the scope of the present invention is not limited to the embodiments shown in the drawings.

1. First Embodiment FIG. 1 is a cross-sectional view of a biosensor chip according to a first embodiment of the present invention. (A) is a top view, (b) is II sectional drawing in (a). This biosensor chip is provided with a substrate 3 having a channel 1 for flowing a specimen, a microprojection group 5 provided in the channel 1, and a downstream of the microprojection group 5, and electrolyzes an electrode active material. And a working electrode 7 and a reference electrode 9 for detection. The microprojection group 5 is formed of a thermoplastic resin, and this resin has a carboxyl group that is a biofunctional molecule binding group. As shown in FIG. 2 (enlarged plan view of the microprojection group 5), the microprojection group 5 is configured by arranging a plurality of microprojections 5a adjacent to each other. Each microprotrusion 5a has a diameter of 2 μm and a height of 40 μm. The distance between adjacent microprotrusions (the width between adjacent microprotrusions) is 2 μm.

  The flow path 1 and the microprojection group 5 are applied with a thermoplastic resin on the substrate and pressed with a mold having a structure corresponding to the flow path and the microprojection group in a state where the resin is heated to the glass transition temperature or higher. Then, it is formed by cooling in this state and then releasing the mold (imprint method, refer to Japanese Patent Application Laid-Open No. 2002-184719 for details). The mold is prepared using a photolithography technique or the like. The thermoplastic resin used here is a copolymer of styrene and methacrylic acid, and methacrylic acid is contained in the 5-15% polymer chain. The melting temperature of this polymer is 150-190 ° C.

  The working electrode 7 is formed by screen printing carbon ink containing platinum or palladium particles on the substrate 3, and the reference electrode 9 is formed by screen printing carbon ink containing silver particles on the substrate 3.

  The working electrode 7 is provided adjacent to the microprojection group 5. The working electrode 7 and the reference electrode 9 are each electrically connected to a potentiostat 17. A voltage of 0.6 V is always applied between the working electrode 7 and the reference electrode 9.

  The flow path is covered with a lid 11. The lid 11 has an immobilization enzyme inlet 13a, an immobilization enzyme outlet 13b, a specimen inlet 15a, and a specimen outlet 15b. A solution containing the enzyme is introduced into the flow channel 1 from the immobilization enzyme inlet 13 a and brought into contact with the microprojection group 5. After this solution has passed through the microprojection group 5, this solution is sucked from the immobilizing enzyme outlet 13b, and again injected into the immobilizing enzyme inlet 13a. By repeating this step, the enzyme is fixed to the microprojection group 5. This immobilization is performed by an amide bond between the carboxyl group of the microprojection group 5 and the amino group contained in the lysine residue in the enzyme. The carboxy group is previously reacted with EDC {1-ethyl-3- (dimethilaminopropyl) carbodiimide} as a crosslinking agent.

  Here, the principle that enables highly sensitive measurement using the biosensor chip of the present embodiment will be described below. A case where glucose oxidase is used as an enzyme and rat serum is used as a specimen and the amount of glucose in the serum is detected is taken as an example. Glucose oxidase is previously bound to the microprojection group 5 by the method described above.

When serum is introduced into the flow channel 1 from the specimen inlet 15 a, the serum flows through the flow channel 1 and reaches the microprojection group 5. Glucose in serum is decomposed according to the reaction formula 1 by the action of glucose oxidase fixed to the microprojection group 5. In this case, H ₂ O ₂ becomes the electrode active material.
(Reaction Formula 1) Glucose + O ₂ → Gluconic acid + H ₂ O ₂

Gluconic acid and H ₂ O ₂ produced by the decomposition further flow along the flow path and reach the working electrode 7. Since a voltage of 0.6 V is applied between the working electrode 7 and the reference electrode 9, H ₂ O ₂ is decomposed according to the reaction formula 2, and at the time of this decomposition, the working electrode 7 and the reference electrode 9 A current corresponding to the H ₂ O ₂ concentration flows between them, and by measuring this current value, the glucose concentration in serum can be measured.
(Reaction Formula 2) H ₂ O ₂ → O ₂ + 2H ⁺ + 2e ⁻

  Up to this point, the specific embodiment has been described as an example, but various modifications are possible.

The diameter and height of the microprotrusions 5a are not particularly limited, and can be, for example, 0.5 to 10 μm or 5 to 200 μm. The aspect ratio (height / diameter) of the microprojections 5a is preferably about 10-20. This is because when the aspect ratio is small, the surface area of the microprojection group 5 is not sufficiently large, and when the aspect ratio is large, the flow path is also high, so that the generated H ₂ O ₂ and the working electrode are This is because it becomes difficult to contact and the sensitivity is lowered. The distance between the microprotrusions 5a is not particularly limited, and can be, for example, 0.5 to 10 μm.

The enzyme fixed to the microprotrusions 5a is not limited to glucose oxidase, and can be appropriately selected according to the test substance. For example, when the test substance is uric acid or total cholesterol, uricase, cholesterol esterase + cholesterol oxidase may be immobilized on the microprojections 5a, respectively. The enzyme immobilization method is not particularly limited, and may be an ester bond or the like in addition to the amide bond. Moreover, as long as it is sufficiently fixed, an electrostatic force or the like may be used. The electrode active material only needs to be oxidized or reduced by voltage application, and quinones can be used in addition to H ₂ O ₂ .

  Polylactic acid may be used as a material for forming the flow path 1 and the microprojections 5, and a mixed polymer of polyacrylic acid, PET, polycarbonate, various thermosetting resins, and polymethacrylic acid may be used. Further, the flow path 1 and the microprojection group 5 may be formed by using photolithography, etching technique, or the like in addition to the imprint method.

If the working electrode 7 is downstream of the microprojection group 5, the working electrode 7 can be arranged at a position away from the microprojection group 5. In this case, the produced H ₂ O ₂ is diluted before reaching the working electrode 7, but even in this case, the test substance can be measured.

  The method of forming the working electrode 7 and the reference electrode 9 is not limited. For example, the working electrode 7 and the reference electrode 9 are formed by forming a metal film on the entire surface of the substrate by sputtering or the like and removing unnecessary portions by photolithography and etching or lift-off. May be. The materials of the working electrode 7 and the reference electrode 9 are not particularly limited as long as the electrode active material can be detected by the above-described action.

  As a specimen, in addition to serum, a low molecular weight substrate in whole blood or brain cells may be used. In addition, dialysis is performed in advance as necessary.

2. Second embodiment.

  FIG. 3 is a cross-sectional view of the biosensor chip according to the second embodiment of the present invention. (A) is a top view, (b) is II sectional drawing in (a). In the present embodiment, three channels are provided, and the microprojection group 5 and the working electrode 7 are provided in each channel. Each of the working electrodes 7 is electrically connected to a potentiostat 17. Different enzymes are immobilized on the microprojection group 5 of each channel. For example, glucose oxidase, uricase, cholesterol esterase + cholesterol oxidase are immobilized. The specimen thrown into the flow path flows through the three flow paths at the same time and is measured simultaneously by the respective working electrodes 7. Therefore, according to the present embodiment, a plurality of test substances can be measured simultaneously. The number of flow paths is not limited to three, and may be two or four or more.

3. Third Embodiment The biosensor chip of the third embodiment is similar to the first embodiment, except that it is single-stranded DNA that is fixed to the microprojection group 5. In this embodiment, the test substance is complementary DNA.

  Here, the principle of detecting the amount of complementary DNA will be described with reference to FIG. The single-stranded DNA is modified with the amino group at the 5 'end and then fixed to the microprojection group 5 by the same method as in the first embodiment. As a sample, a solution containing complementary DNA is prepared.

  When the sample is introduced into the flow channel 1 from the sample inlet 15a, the sample flows through the flow channel 1 and reaches the microprojection group 5. The complementary DNA is efficiently combined with the single-stranded DNA fixed to the microprojection group 5 to form double-stranded DNA. Next, a solution containing an intercalator molecule (for example, Hoechst 33258 (Doujinsha)), which is an electrode active material, is similarly introduced into the flow path 1. When this solution reaches the microprojection group 5, it is trapped by double-stranded DNA, and the concentration of intercalator molecules in the solution decreases. Since the trap amount of the intercalator molecule depends on the amount of double-stranded DNA, the decrease in the concentration of the intercalator molecule also depends on the amount of double-stranded DNA. For this reason, the amount of complementary DNA contained in the specimen can be measured by measuring the concentration of intercalator molecules (the amount of decrease in concentration) with the working electrode 7 and the reference electrode 9.

  The type of intercalator molecule is not limited as long as it is an electrode active material. There may be two or more channels, and a plurality of complementary DNAs can be measured simultaneously by fixing another single-stranded DNA in each channel.

4). Fourth Embodiment The biosensor chip according to the fourth embodiment is similar to the first embodiment except that an antibody is fixed to the microprojection group 5. In this embodiment, the test substance is an antigen that undergoes an antigen-antibody reaction with the antibody.

  Here, the detection principle of the antigen will be described with reference to FIGS. The antibody is fixed to the microprojection group 5 by the same method as in the first embodiment (a state in which the antibody 19 is fixed to the microprotrusion 5a is schematically shown in FIG. 4A). A solution containing an antigen is prepared as a specimen.

When the sample is introduced into the flow channel 1 from the sample inlet 15a, the sample flows through the flow channel 1 and reaches the microprojection group 5. The antigen 21 contained in the specimen efficiently binds to the antibody 19 immobilized on the microprojection group 5 to form a complex (FIG. 4B). The amount of complex formation depends on the amount of antigen 21. Next, a solution containing the antibody 23 modified with the enzyme 25 is similarly introduced into the flow path 1. When this solution reaches the microprojection group 5, the antibody 23 binds to the complex (FIG. 4 (c)). FIG. 4C shows that the enzyme 25 is fixed to the microprotrusions 5a via the antigen 21 and the like. It can be seen that the amount of enzyme 25 immobilized depends on the amount of antigen 21. Thereafter, for example, a glucose solution having a known concentration is introduced into the flow path 1, and the oxidation current of the generated H ₂ O ₂ is measured by the same method as in the first embodiment. The amount of oxidation current depends on the amount of enzyme 25. Therefore, the amount of the antigen 21 can be determined by measuring the amount of oxidation current.

  Two or more channels may be used, and a plurality of antigens can be measured simultaneously by fixing different antibodies in each channel. It is possible to measure the amount of antibody in the specimen by fixing the microprojection group 5 with an antigen.

5. Fifth Embodiment A biosensor chip according to a fifth embodiment is similar to the fourth embodiment, but includes two microprojection groups 5 and 27 in the flow path as shown in FIGS. Is different. The upstream microprojection group (consisting of a plurality of microprojections 27a) 27 has a smaller cross-sectional area and a higher density than the downstream microprojection group (consisting of a plurality of microprojections 5a) 5. Is formed. Each microprotrusion 27a has a diameter of 100 nm and a height of 40 μm. The distance between adjacent microprotrusions (the width between adjacent microprotrusions) is 200 nm. The antibody is immobilized only on the microprojection group 5 on the downstream side.

  Here, the function of the upstream microprojection group 27 will be described with reference to FIG. As a sample, a sample containing a plurality of types of antigens is prepared. This embodiment is suitably used when a relatively small size of a plurality of types of antigens is a test substance.

  When the sample is introduced into the flow channel 1 from the sample inlet 15 a, the sample flows through the flow channel 1 and reaches the microprojection group 27. A relatively small antigen (small antigen) quickly passes through the microprojection group 27, whereas a relatively large antigen (large antigen) slowly passes through the microprojection group 27. For this reason, the microprojection group 27 functions as a filter that separates antigens for each size. When the microprojection group 27 is not provided, the large antigen and the small antigen may reach the microprojection group 5 at the same time, and the binding of the small antigen to the antibody fixed to the microprojection group 5 may be inhibited. In this embodiment, a small antigen efficiently binds to the antibody. Thereafter, the amount of antigen can be determined by the same method as in the fourth embodiment.

  In addition, the diameter and height of the microprotrusion 27a are not specifically limited, For example, it can be 50-200 nm and 5-200 micrometers. The distance between the minute protrusions 27a is not particularly limited, and can be set to, for example, 50 to 200 nm. Three or more microprojections may be provided to perform stepwise separation.

(Enzyme sensor chip)
A three-channel enzyme sensor chip as shown in FIG. 3 was prepared and used to measure glucose, uric acid, and total cholesterol in serum.

(1) Immobilization method of enzyme From the holes 13a and 13b before and after the microprojection group 5, a solution of a cross-linking agent EDC (10 mM, acetate buffer (0.05 M, pH 5.7)) was allowed to flow at a flow rate of 10 μl / min, EDC was allowed to react with 1, 2 and 3. Then, using the holes before and after channel 1, 0.5 mg / ml glucose oxidase (0.1 M phosphate buffer, pH 7.0) solution was added at 10 μl / ml. The glucose oxidase enzyme was immobilized by circulating for 40 minutes at a rate of minutes, and 0.5 mg / ml uricase solution was similarly applied to channel 2 to immobilize uricase. Both enzymes were immobilized by flowing a solution containing 5 mg cholesterol oxidase and 0.5 mg cholesterol esterase, and finally 0.1 M phosphate buffer (pH 7. ) And flowed to obtain a sensor chip is washed.

(2) Measurement method A 0.1 M phosphate buffer (pH 7.5) containing 0.5% of the surfactant Triton X-100 was used as the mobile phase (carrier solution). Sample injection was performed using an auto-injector, and 1 μl of serum serving as a sample was injected into the sample loop of the injector from the inlet 15a of the chip by automatic valve switching. An applied voltage of 0.6 V (relative to the Ag / AgCl reference electrode) was applied to the electrode part, and the oxidation current of hydrogen peroxide generated by the enzyme reaction occurring in the microprotrusion group 5 of each channel was detected.

(3) Results In each channel, there was a linear relationship between the substrate concentration and the response current in the concentration range of 5-500 mg / dl for glucose, uric acid, and total cholesterol, respectively. Table 1 shows the results of determining the accuracy and reproducibility (7-times repeatability) of the measurement by this method using control sera labeled with each of the four substrate concentrations.

  As can be seen from Table 1, the manufacturer indication values and analysis values were good. The reproducibility of the measurement was 1-2% in terms of variation coefficient. In addition, the stability of the sensor chip maintained 88% or more of the initial activity even after continuous use for 1 month (30 samples every day).

(Enzyme sensor chip for measurement of neurotransmitters in rat brain)
The microdialysis probe shown in FIG. 7 was fixed to the rat brain linear band by surgery, and using the same system as in Example 1, neurotransmitters (L-glutamic acid, dopamine, acetylcholine) were simultaneously detected. In FIG. 7, symbols A and B are fused silica tubes, and C is a dialysis tube (inner diameter 200 μm, outer diameter 220 μm).

(1) Immobilization of enzyme Glutamate oxidase is immobilized on microprojection group 5 of channel 1, tyrosinase is immobilized on channel 2, and acetylcholinesterase and choline oxidase are immobilized on channel 3 in the same manner as in Example 1. I got a chip.

(2) Measurement method An applied voltage of 0.6 V (relative to the Ag / AgCl reference electrode) was applied to the electrode portions of channel 1 and channel 3, and the oxidation current of hydrogen peroxide generated by the enzyme reaction was detected. On the other hand, an applied voltage of −0.15 V was applied to the electrode portion of channel 2, and the reduction current of dopaquinone produced by the enzyme reaction was measured. Using 0.1 M phosphate buffer as the mobile phase (carrier solution), the carrier solution was allowed to flow at a constant flow rate of 5 μl / min. Further, Ringer's solution was used as a dialysis solution, and it was allowed to flow at a flow rate of 2 μl / min with a syringe pump.

(3) Results In each channel, L-glutamic acid, dopamine, and acetylcholine were in a concentration range of 1 μM-100 μM, and the response current was proportional to each concentration. The lower limit of detection was 0.4 μM. In addition, when the stability of the sensor chip was used continuously for 1 month, the sensitivity of the L-glutamic acid sensor part and the acetylcholine sensor part maintained 90% or more of the initial sensitivity, but the sensitivity of the dopamine sensor part decreased to 72%. .

(DNA sensor chip)
A one-channel DNA sensor chip as shown in FIG. 1 was prepared, and an experiment for functional evaluation as a DNA sensor chip was performed.

In the same manner as in Example 1, one 5′-amino group-modified DNA (d (T) ₂₂ : DNA in which 22 thymines were linked) was bound to the microprojection group 5. As a specimen, d (A) ₂₂ , single base mismatch DNA, and double base mismatch DNA are injected, and hybridization is performed with d (T) ₂₂ on the microprojection group. Next, donomycin (1 μM solution) was injected as an intercalator molecule, and the current was measured at an applied voltage of 0.7V.

For d (T) ₂₂ , the current value decreased by 70%, for single-base mismatched DNA it decreased by 3.5%, and for double-base mismatched DNA it was 0%. That is, d (T) ₂₂ on the microprojection group 5 selectively hybridized with d (A) ₂₂ and showed selectivity for complementary DNA molecules.

(Protein sensor chip)
A 1-channel protein sensor chip as shown in FIG. 1 was prepared, and human immunoglobulin G (HIgG) was measured.

  A HIgG antibody (Anti HIgG) was immobilized on the microprojection group 5 in the same manner as in Example 1. Using a slide valve, HIgG was injected (1 μl, 30 seconds), then glucose oxidase-labeled antibody (GOD-Anti HIgG) was injected (60 seconds), glucose solution (0.1 mM) was injected (30 seconds), The labeled glucose oxidase detected the oxidation current of hydrogen peroxide generated from glucose at the downstream electrode. There was a good linear relationship between response current and HIgG concentration in the concentration range of 2 μmg / ml to 80 μg / ml.

It is (a) top view and (b) II sectional view of the biosensor chip of a 1st embodiment of the present invention. FIG. 2 is an enlarged plan view of a microprojection group in FIG. 1. It is (a) top view of the biosensor chip | tip of 2nd Embodiment of this invention, (b) It is II sectional drawing. It is a conceptual diagram which shows the principle of 4th Embodiment. It is (a) top view of the biosensor chip | tip of 5th Embodiment of this invention, (b) It is II sectional drawing. FIG. 6 is an enlarged plan view of the minute protrusion group in FIG. 5. The structure of a microdialysis probe is shown. It is sectional drawing of the conventional biosensor chip.

Explanation of symbols

1: Flow path 3: Substrate 5, 27: Microprotrusion group 5a, 27a: Microprotrusion 7: Working electrode 9: Reference electrode 11: Lid 13a: Immobilization enzyme inlet 13b: Immobilization enzyme outlet 15a: Sample inlet 15b : Sample outlet 17: Potentiostat 19, 23: Antibody 21: Antigen 25: Enzyme 51: Substrate 53: Base lead electrode 55: Working electrode 57: Counter electrode 61: Charge transfer medium layer 63: Enzyme reaction layer

Claims (9)

A substrate having a channel for flowing a specimen, a microprojection group formed of a material having a biofunctional molecular binding group provided in the channel, a downstream of the microprojection group, and an electrode active material A biosensor chip comprising a working electrode for detection and a reference electrode. The chip according to claim 1, wherein the binding group includes a carboxyl group. The chip according to claim 1, wherein the microprojection group has an aspect ratio of 10-20. The chip according to claim 1, wherein the electrode active material is made of hydrogen peroxide or quinone. The chip according to claim 1, wherein the working electrode is disposed adjacent to the microprojection group. The chip according to claim 1, comprising a plurality of flow paths, each of which is provided with a working electrode. The chip according to claim 1, wherein a biofunctional molecule is bound to the binding group. The chip according to claim 7, wherein the biofunctional molecule is composed of an enzyme, an antibody, or a single-stranded DNA. In the flow path, at least two microprojection groups are provided,
The microprojection group on the upstream side has a smaller cross-sectional area of the microprojection than the microprojection group on the downstream side, and is formed with a high density,
The chip according to claim 1, wherein a biofunctional molecule is bound to a group of microprojections on the downstream side.

Images