JP5204590B2 - Glucose sensor and manufacturing method thereof - Google Patents

Description

The present invention relates to a glucose sensor and its manufacturing how.

  As a conventional biosensor, there is one described in Patent Document 1, for example. This biosensor uses ferricium ions derived from a ferrocene electrolyte as an electron acceptor. Ferricium ions react directly with the electrons generated from the oxidation reaction of glucose and are reduced to ferrocene. Therefore, it is not necessary to convert the electron acceptor into an oxidant on the electrode in advance. Therefore, the enzyme reaction can be allowed to proceed in a short time without reducing the detection sensitivity.

By using ferricium ion as an electron acceptor, glucose in a sample such as a biological sample or food can be rapidly quantified.
JP-A-9-304329

  However, the prior art described in the above literature has room for improvement in the following points.

  First, in order to obtain the oxidation current of ferrocene, there was a problem that the applied voltage had to be increased.

  In the biosensor described in Patent Document 1, the magnitude of the ferrocene oxidation current is measured as a response current value. The magnitude of the response current value thus obtained is proportional to the glucose concentration present in the sample. Therefore, glucose in the sample can be quantified by measuring the response current value.

  However, a high applied voltage is required for the oxidation of ferrocene. If it does so, it will become easy to receive to the influence of the interference substance contained in a sample, for example, uric acid, bilirubin, ascorbic acid, acetaminophen, etc.). As a result, the measurement accuracy is reduced.

  Secondly, ferricium ion is an expensive reagent, and there is a problem that a disposable biosensor is too expensive.

  From the above, an electron acceptor having a lower applied voltage for obtaining an oxidation current at low cost has been desired.

  The present invention has been made in view of the above circumstances, and provides a biosensor capable of quickly and accurately quantifying a base mass such as glucose without reducing detection sensitivity.

According to the present invention,
A substrate,
An electrode provided on the substrate;
A reaction layer including an oxidoreductase, an oxidized tetrathiafulvalene or an oxidized tetrathiafulvalene derivative that is in an oxidized state before the start of measurement, and a surfactant;
With
The oxidoreductase is glucose oxidase or glucose reductase ;
Prior Symbol reaction layer, said oxidized tetrathiafulvalene or oxidized tetrathiafulvalene derivatives are inclusion in cyclodextrins,
The electrode detects an oxidation current value of tetrathiafulvalene or tetrathiafulvalene derivative obtained by reducing the oxidized tetrathiafulvalene or the oxidized tetrathiafulvalene derivative with an oxidoreductase that has reacted with glucose in the sample. A glucose sensor is provided.

Moreover, according to the present invention,
Preparing a substrate;
Providing an electrode on the substrate;
Providing a reaction layer containing an oxidoreductase, oxidized tetrathiafulvalene or oxidized tetrathiafulvalene derivative in an oxidized state, and a surfactant so as to be in contact with the electrode;
Including
The oxidoreductase is glucose oxidase or glucose reductase ;
Prior Symbol reaction layer, said oxidized tetrathiafulvalene or the oxidized tetrathiafulvalene derivatives are inclusion in cyclodextrins,
The electrode detects an oxidation current value of tetrathiafulvalene or tetrathiafulvalene derivative obtained by reducing the oxidized tetrathiafulvalene or the oxidized tetrathiafulvalene derivative with an oxidoreductase that has reacted with glucose in the sample. A method for manufacturing a glucose sensor is provided.

Further, Oite the present invention, the use of the glucose sensor can alternatively be as follows:
Adding a sample to a reaction layer containing an oxidoreductase, an oxidized tetrathiafulvalene or an oxidized tetrathiafulvalene derivative that is in an oxidized state before the start of measurement , and a surfactant;
Reacting glucose in the added sample with the oxidoreductase;
Reacting the reacted oxidoreductase with the oxidized tetrathiafulvalene or the oxidized tetrathiafulvalene derivative;
Applying a potential to tetrathiafulvalene or a tetrathiafulvalene derivative produced by a reaction between the oxidoreductase and the oxidized tetrathiafulvalene or the oxidized tetrathiafulvalene derivative; and
Measuring an oxidation current value of an oxidation current generated from the tetrathiafulvalene or the tetrathiafulvalene derivative to which the potential is applied;
Including
In the reaction layer, the oxidized tetrathiafulvalene or the oxidized tetrathiafulvalene derivative that is inclusion in cyclodextrins.

According to the present invention, oxidized tetrathiafulvalene or oxidized tetrathiafulvalene derivative is used as the electron acceptor. Thereby, oxidized tetrathiafulvalene or oxidized tetrathiafulvalene derivative can react directly with the oxidoreductase reduced by reacting with the substrate. Therefore, the current value detected by applying a voltage to the electrode is only the oxidation current value of the reductant produced by the reaction between the oxidoreductase and oxidized tetrathiafulvalene or oxidized tetrathiafulvalene derivative. Thus, the base current can be made lower than when a reduced electron acceptor is charged. Therefore, it is possible to quickly and accurately measure the base mass in the sample without reducing the detection sensitivity.

The applied voltage for obtaining the oxidation current is lower for tetrathiafulvalene or tetrathiafulvalene derivatives than for ferrocyan. Therefore, the applied voltage for reoxidizing tetrathiafulvalene or tetrathiafulvalene derivatives converted from oxidized to reduced by reaction with oxidoreductase can be lowered, making it less susceptible to interference substances. it can. Therefore, the base mass in the sample can be measured quickly and with high accuracy without reducing the detection sensitivity.

  Furthermore, an oxidant such as tetrathiafulvalene can be easily prepared by reacting tetrathiafulvalene or the like with an oxidizing agent such as peroxide. Therefore, it is more advantageous in terms of cost than ferricium ion. Therefore, the configuration of the present invention is suitable for a disposable biosensor.

In the present invention, that employs a configuration in which oxidized tetrathiafulvalene or oxidized tetrathiafulvalene derivative is inclusion in cyclodextrins. Thereby, the hydrophilicity of oxidized tetrathiafulvalene or oxidized tetrathiafulvalene derivative can be improved. Therefore, oxidized tetrathiafulvalene or oxidized tetrathiafulvalene derivative can be easily applied to the electrode.

In the present invention, that employs a configuration that contains a surfactant in the reaction layer. Thereby, the solubility with respect to the water of the tetrathiafulvalene obtained by the oxidation type tetrathiafulvalene reacting with an oxidoreductase can be improved. Similarly, the reduced tetrathiafulvalene derivative obtained by reacting an oxidized tetrathiafulvalene derivative with an oxidoreductase can also improve the solubility in water.

In the present invention, the reaction layer includes an enzyme layer containing an oxidoreductase and a mediator layer containing an oxidized tetrathiafulvalene or an oxidized tetrathiafulvalene derivative , and the enzyme layer is in contact with the mediator layer, A configuration in which a mediator layer is provided on the surface of the electrode can be employed. By doing so, the enzyme that has reacted with the substrate can immediately act on the oxidized tetrathiafulvalene or oxidized tetrathiafulvalene derivative at the interface between the enzyme layer and the mediator layer. Therefore, electrons can be efficiently transmitted and received between the enzyme and oxidized tetrathiafulvalene or oxidized tetrathiafulvalene derivative, and the base mass in the sample can be measured more quickly.

Further, the present invention, it is possible to adopt a configuration in which oxidized Te tiger tetrathiafulvalene or oxidized tetrathiafulvalene derivative is contained in the reaction layer is distributed throughout the reaction layer. Thereby, the enzyme which reacted with the substrate can immediately act on the oxidized tetrathiafulvalene or tetrathiafulvalene derivative . Therefore, electrons can be efficiently transmitted and received between the enzyme and oxidized tetrathiafulvalene or oxidized tetrathiafulvalene derivative, and the base mass in the sample can be measured more quickly.

In addition, the present invention can employ a configuration in which oxidized tetrathiafulvalene or oxidized tetrathiafulvalene derivative contained in the mediator layer is distributed throughout the mediator layer. Thus, the enzyme that has reacted with the substrate can immediately act on the oxidized tetrathiafulvalene or tetrathiafulvalene derivative at the interface between the enzyme layer and the mediator layer. The reduced form of tetrathiafulvalene or tetrathiafulvalene derivative can quickly move toward the surface of the electrode using the concentration gradient difference. Therefore, the base mass in the sample can be measured more rapidly.

In the present invention, that adopts the configuration oxidoreductase is glucose oxidase or glucose reductase. Specifically, glucose dehydrogenase or glucose oxygenase can be used. Thereby, the present invention can be used as a glucose sensor.

Furthermore, the structure of this invention can employ | adopt the structure whose oxidized tetrathiafulvalene or oxidized tetrathiafulvalene derivative is a two-electron oxidant. Thereby, the enzyme which reacted with the substrate in the sample can react with oxidized tetrathiafulvalene or oxidized tetrathiafulvalene derivative more rapidly. Therefore, the substrate concentration contained in the sample can be measured more rapidly.

  According to the present invention, a base mass such as glucose can be measured quickly and with high accuracy without reducing the detection sensitivity.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings. In all the drawings, the same reference numerals are given to the same components, and the description will be omitted as appropriate.

  FIG. 2 is a schematic plan view showing the biosensor 1 of the present embodiment. 1 is a cross-sectional view taken along line AA ′ of FIG. The biosensor 1 includes an insulating substrate 101, an electrode 106 provided on the insulating substrate 101, and a reaction layer 104 provided in contact with the electrode 106 and containing an oxidoreductase and an electron acceptor. In addition, the biosensor 1 contains oxidized tetrathiafulvalene or an oxidized tetrathiafulvalene derivative in the reaction layer 104 as an electron acceptor. The term “tetrathiafulvalene (TTF)” or “tetrathiafulvalene derivative” is also collectively referred to as “TTF compound” hereinafter. The oxidized TTF compound contained in the reaction layer 104 is distributed throughout the reaction layer 104.

  For the insulating substrate 101, ceramic, plastic, silicon, an alumina glass plate, or a polymer material can be used. Preferably, synthetic resin plates such as polyethylene terephthalate, polyvinyl chloride, and polycarbonate can be used.

  As the electrode 106, a bipolar electrode composed of a working electrode 102 and a counter electrode 103 can be employed. The working electrode 102 and the counter electrode 103 can be formed using a conductive material. The conductive material is carbon or a metal material such as palladium, silver, platinum, gold, copper, nickel, or an alloy thereof. Further, as the electrode 106, a tripolar electrode composed of the working electrode 102, the counter electrode 103, and the reference electrode 105 as shown in FIGS. 1 and 2 can be adopted. By employing a triode electrode, a response current can be stably obtained, and measurement accuracy can be stabilized. Similarly to the working electrode 102 and the counter electrode 103, the reference electrode 105 can also be formed using a conductive material. The electrode may be formed of one type of conductive material, or may be formed of two or more types of conductive material. As a result, a stable response current is obtained, and the accuracy is further stabilized. The working electrode 102, the counter electrode 103, and the reference electrode 105 can be formed by applying to the insulating substrate 101 by screen printing, sputtering, or vapor deposition. The electrode 106 is provided with an insulating region whose surface is covered with the insulating film 30, a measurement region covered with the reaction layer 104, and an application region for applying a voltage. In the application region, the surface of the electrode 106 is exposed.

  The reaction layer 104 contains at least one oxidoreductase and at least one electron acceptor.

As the electron acceptor, at least one oxidized TTF compound is used, but two or more oxidized TTF compounds may be used. The oxidized TTF compound (oxidized TTF and oxidized TTF derivative) refers to a one-electron oxidant or a two-electron oxidant of the TTF compound. When the TTF compound emits one electron, it becomes a one-electron oxidant, and when two electrons are emitted from the TTF compound, it becomes a two-electron oxidant. Examples of the TTF derivative include 4-ethoxycarbonyl-4 ′, 5,5′-trimethyltetrathiafulvalene (Me ₃ TTF-CO ₂ Et), 8-mercaptooctyltetrathiafulvalenecarboxylate, and the like.

  The oxidized TTF compound can be prepared by dissolving the TTF compound in a suitable solvent and allowing it to act with an oxidizing agent. Any solvent can be used as long as it is a solvent in which the TTF compound and its oxidant are dissolved. For example, acetonitrile can be exemplified. As the oxidizing agent, a peroxide can be exemplified. As the peroxide, for example, iron (III) perchlorate can be used.

Reaction formula 1 shows the redox reaction of TTF. Compound 1 is TTF, and when TTF emits one electron, tetrathiafulvalene-cation radical (TTF. ⁺ ) (Compound 2), which is a one-electron oxidant of TTF ^, is obtained. Further, by emitting one electron from compound 2, tetrathiafulvalene cation radical (TTF.2 ⁺ ) (compound 3), which is a two-electron oxidant of TTF ^, is obtained. In other words, Compound 2 and Compound 3 are oxidized TTF. Each reaction is a reversible reaction, and compound 2 and compound 3 can be reduced to compound 1 by acting with a reducing agent. As the reducing agent, for example, ascorbic acid, dithiothreitol (DTT) and the like can be used.

(Reaction Formula 1)

  The content of the oxidized TTF compound in the reaction layer 104 is not particularly limited, and an appropriate amount can be selected as necessary. Preferably, the 1-electron oxidized form of the TTF compound is 0.05 to 10 mM. including.

  The oxidoreductase is oxidized by reacting with various substrates to be measured. The base mass can be quantified by the oxidation of the oxidized enzyme with the oxidized form of the TTF compound.

  For example, glucose oxidase (GOD) or glucose dehydrogenase can be used as the oxidoreductase. In addition, lactate oxidase, lactate dehydrogenase, cholesterol oxidase (ChOD), and combinations thereof can be used.

  When the biosensor 1 is a glucose sensor, glucose oxidase (GOD) or glucose dehydrogenase can be used as the oxidoreductase.

  The measurement mechanism when GOD is used as the oxidoreductase is based on reaction formula 2.

(Reaction Formula 2)
Glucose + GOD (oxidized substance) → Gluconic acid + GOD (reduced form)
GOD (reduced form) + oxidized TTF compound → GOD (oxidized form) + TTF compound (reduced form)

  As shown in Reaction Formula 2, glucose in the measurement sample is oxidized to gluconic acid by reducing GOD by the catalytic reaction of GOD. The GOD transfers electrons to the oxidized TTF compound to return to the original state, and the oxidized TTF compound is reduced to become a TTF compound (reduced form). The TTF compound is oxidized electrochemically to become an oxidized TTF compound. At this time, the obtained oxidation current is measured as a response current value. This response current value is proportional to the glucose concentration present in the measurement sample.

  The content of the oxidoreductase in the reaction layer 104 is not particularly limited, and an appropriate amount can be selected as necessary. For example, when glucose oxidase is used, its content is preferably 0.1 to 40 units / μL. Here, “1 unit” refers to the amount of oxidoreductase required to oxidize 1 μmol of substrate in 1 minute.

  The biosensor 1 can employ a configuration in which an oxidized TTF compound is included in cyclodextrin. As a result, the hydrophilicity is improved, so that it is easy to apply to the electrode.

  Although it does not restrict | limit especially as a cyclodextrin, For example, (alpha) -cyclodextrin, (beta) -cyclodextrin, (gamma) -cyclodextrin etc. can be used. Further, it may be a chemically modified cyclodextrin, such as hydroxycyclodextrin, hydroxypropyl-β-cyclodextrin (hp-β-CD), dimethyl-β-cyclodextrin, carboxymethyl-β-cyclodextrin, etc. Of these, hydroxypropyl-β-cyclodextrin is preferred. One kind of these may be used, or two or more kinds may be used in combination.

  The oxidized TTF compound is preferably included by, for example, 40% by weight or less of cyclodextrin per mole, more preferably 0.1 to 20% by weight, particularly preferably 1 to 10% by weight. is there. If the concentration of cyclodextrin is too high, there is a problem in that the viscosity increases, and if it is too low, there is a problem in that solubilization becomes difficult.

  The oxidized TTF compound can be included by a known inclusion method using cyclodextrin. For example, a cyclodextrin solution in which cyclodextrin is dissolved in an aqueous solvent and an oxidant suspension in which an oxidized TTF compound is suspended are prepared, and the oxidized TTF compound is included by mixing both. Can do.

  The ratio of the oxidized TTF compound to the cyclodextrin solution is not particularly limited as long as the oxidized TTF compound can be dissolved. For example, the oxidized TTF compound is dissolved to a concentration of 5 mM in a solution of cyclodextrin of 40% by weight or less. Can do. The solution of cyclodextrin is more preferably 1 to 10% by weight.

  As an aqueous solvent, water, a buffer solution, etc. can be used, for example. As the buffer solution, TES (N-Tris (hydroxymethyl) methyl-2-aminoethanesulfide acid) buffer solution, HEPES (2- [4- (2-hydroxyethylyl) -1-piperazinyl] ethanesulfide acid) buffer solution, etc. may be used. it can. The buffer solution may contain sodium chloride.

  The pH of the solvent is preferably 4-8. If the pH is too large or too small, the enzyme is deactivated, which is not preferable.

  In addition, the reaction layer 104 can include a surfactant.

  By using an oxidized TTF compound as an electron acceptor, as shown in Reaction Formula 2, the reduced form of the oxidoreductase reacts with the oxidized TTF compound to oxidize the oxidoreductase and oxidize the TTF compound. Produces a TTF compound (reduced form). TTF compounds have low water solubility. For this reason, a reductant produced by water contained in the measurement sample may be deposited on the reaction layer. Therefore, by adding a surfactant to the reaction layer 104, the solubility of the generated TTF compound (reduced form) in water can be improved.

  Examples of the surfactant include lecithin, octylthioglucoside, sodium cholate, dodecyl-β-maltoside, sodium deoxycholate, sodium taurodeoxycholate, Tritron-X (registered trademark), Lubrol PX (registered trademark), DK-ester®, BIGCHAP®, DeoxCHAP®, sodium dodecyl sulfate (SDS), and Tween 20 (polyoxyethylene sorbitan monolaurate) can be used. It is particularly preferable to use Tritron-X (registered trademark), SDS, and Tween20.

  Examples of the measurement sample include blood, urine, body fluid such as saliva, and food such as fruit juice.

  It continues and demonstrates an example of the manufacturing method of the biosensor 1. FIG.

  First, a substrate is prepared, and the working electrode 102 is formed on the substrate by a known method such as screen printing, sputtering, or vapor deposition. Next, the insulating film 30 is formed by screen printing an insulating paste on the substrate to obtain the insulating substrate 101. The insulating layer 30 is formed so as to cover the outer periphery of the working electrode 102. A counter electrode 103 and a reference electrode 105 are provided so as to be parallel to the working electrode 102.

  Thereafter, a reaction layer 104 containing an oxidoreductase and an electron acceptor is provided on the insulating substrate 101. Specifically, the reaction layer 104 can be formed on the insulating substrate 101 as follows.

  An oxidized TTF compound, cyclodextrin, surfactant and oxidoreductase are mixed and adjusted in an aqueous solvent, applied to the surface of the insulating substrate 101, and then dried to complete the biosensor 1. Drying is preferably performed at a temperature at which the oxidoreductase is not inactivated and at an optimum temperature or lower.

  Next, a method for measuring the substrate concentration contained in the sample solution using the biosensor 1 will be described.

  First, a measurement sample is added to the reaction layer 104. The reaction layer 104 is dissolved by the addition of the measurement sample, and the reaction between the substrate in the sample and the oxidoreductase, and the reaction between the oxidoreductase and the electron acceptor following this reaction proceed. In order to advance these reactions, after the biosensor 1 is left for a predetermined time, a pulse voltage (0 to +0.3 V) is applied to the working electrode 102 in the anode direction with respect to the counter electrode 103. A response current is generated, and the response current value is measured by a known method. The substrate concentration is determined from the obtained response current value using a calibration curve between the substrate concentration and the response current value prepared in advance using a substrate having a known concentration.

  It continues and demonstrates the effect | action and effect of the biosensor 1 using FIG.

  According to the biosensor 1, an oxidized TTF compound is used as an electron acceptor. Thus, the oxidized TTF compound can directly react with the oxidoreductase reduced by reacting with the substrate. Therefore, only the oxidation current of the TTF compound produced by the reaction with the oxidoreductase is detected as the response current value, and the base current can be lowered even when the reduced electron acceptor is charged. Therefore, it is possible to quickly and accurately measure the base mass in the sample without reducing the detection sensitivity.

  Traditionally, TTF compounds have been used as electron acceptors. However, oxidoreductases react only with oxidized TTF compounds. For this reason, when the TTF compound is charged into the reaction layer as it is, it is necessary to convert the TTF compound into an oxidized TTF compound once. As a result, there is a problem that the base current is increased due to the current generated during the oxidation reaction. That is, when the base current is increased, the response current to the substrate is relatively decreased, and as a result, there is a problem that the detection sensitivity is lowered.

  In addition, when an oxidized TTF compound is generated at the time of use, there is a variation in the oxidation reaction, which causes a decrease in the performance of the biosensor. In addition, it was impossible to predict how much the TTF compound was oxidized, and it was impossible to predict how much the electron acceptor should be charged.

  However, in the biosensor 1, an oxidized TTF compound is charged in advance. Thereby, the quantity of the electron acceptor required for preparation can be estimated, and a biosensor can be designed. Thereby, the performance of each biosensor can be made uniform, and variation among chips can be reduced. Therefore, a stable measurement result can be obtained even with a disposable biochip. It is also possible to prepare a biosensor having desired performance.

  The applied voltage for oxidizing the TTF compound again to the oxidized TTF compound is 0 to +0.3 V, which is lower than the applied voltage (+0.4 to +0.7 V) for oxidizing ferrocyan. Therefore, the TTF compound is not easily affected by the interfering substance contained in the sample, and the substrate concentration in the sample can be quantified with high accuracy.

  Furthermore, the oxidized TTF compound can be easily prepared by reacting the TTF compound with a peroxide. Therefore, it is more advantageous in terms of cost than ferricium ion. Therefore, the biosensor 1 can be used as a disposable biosensor.

  In addition, the biosensor 1 can employ a two-electron oxidant of a TTF compound as the oxidized TTF compound contained in the reaction layer 104. By using a two-electron oxidant, it is possible to react more rapidly with electrons generated by an enzyme reaction. Therefore, the substrate concentration such as glucose contained in the sample can be measured more rapidly.

  In addition, the biosensor 1 can employ a configuration in which the oxidized TTF compound contained in the reaction layer 104 is distributed throughout the reaction layer 104. Thereby, the reaction layer 104 can be reacted rapidly with the oxidoreductase acting on the substrate throughout the reaction layer 104. Therefore, the substrate concentration in the sample can be measured more rapidly.

  Furthermore, the biosensor 1 can employ a configuration in which an electron acceptor is included in cyclodextrin. Thereby, the hydrophilicity of an electron acceptor can be improved. Therefore, the electron acceptor can be easily applied to the electrode.

  As described above, according to the biosensor 1, a substrate such as glucose can be quickly and accurately quantified without reducing the detection sensitivity.

  As mentioned above, although embodiment of this invention was described with reference to drawings, these are the illustrations of this invention, Various structures other than the above are also employable.

  For example, the configuration of the biosensor 2 shown in FIG. 3 can be adopted. In the biosensor 2, the reaction layer 204 includes an enzyme layer 204b containing an oxidoreductase and a mediator layer 204a containing an electron acceptor. The enzyme layer 204 b is in contact with the mediator layer 204 a, and the mediator layer 204 a is provided on the surface of the electrode 106. The biosensor 2 and the biosensor 1 are different in that a reaction layer 204 is provided instead of the reaction layer 104. The oxidized TTF compound contained in the mediator layer 204a is distributed throughout the mediator layer 204a.

  Since the oxidoreductase and the electron acceptor diffuse in the reaction layer, the mediator layer 204a may contain an oxidoreductase, or the enzyme layer 204b may contain an electron acceptor. However, the mediator layer 204a contains the oxidized TTF compound more uniformly and at a higher concentration than the enzyme layer 204b. The enzyme layer 204b contains the oxidoreductase at a higher concentration and more uniformly than the mediator layer 204a.

  The biosensor 2 can employ a configuration in which an oxidized TTF compound is included in a cyclodextrin, Nafion (registered trademark), or an organic solvent. As with the biosensor 1, it is particularly preferable to include it with cyclodextrin. As a result, the hydrophilicity is improved, so that it is easy to apply to the electrode.

  Further, the biosensor 2 may form a surfactant layer containing a surfactant between the mediator layer 204a and the enzyme layer 204b. As the surfactant, the same surfactant as that used in the biosensor 1 can be employed.

  In the biosensor 2, for example, the reaction layer 204 can be formed on the insulating substrate 101 as follows.

  The oxidized TTF compound is dissolved in a solvent, and the obtained oxidized TTF compound solution is applied to the surface of the insulating substrate 101 and dried. Thereafter, the oxidized TTF compound solution is applied to each of the working electrode 102, the counter electrode 103, and the reference electrode 105, and dried again. After drying, the mediator layer 204a can be uniformly formed by repeatedly applying the oxidized TTF compound solution to the surface of the formed mediator layer 204a. After coating, the mediator layer 204a is carefully dried by putting it in a desiccator or the like.

  Here, as the solvent for dissolving the oxidized TTF compound, it is preferable to use a solvent that dissolves the oxidized TTF compound and easily volatilizes. For example, water or a volatile organic solvent can be used, and it is more preferable to use acetonitrile.

  Thereafter, a solution of the surfactant is applied, and further dried by putting it in a desiccator. Thereby, the layer of surfactant can be formed. Presence of the surfactant can prevent precipitation of the reduced TTF compound generated at the interface between the mediator layer 204a and the enzyme layer 204b.

  Next, an oxidoreductase solution is applied to form the enzyme layer 204b. By using GOD or glucose dehydrogenase as the oxidoreductase, the biosensor 2 can be a glucose sensor. Finally, the biosensor 2 is completed by drying at a low temperature. The drying temperature is preferably a temperature at which the oxidoreductase is not inactivated and is not more than the optimum temperature.

  Hereinafter, examples of the biochip of the present invention will be described, but the present invention is not limited thereto.

(Method for adjusting oxidized TTF and measuring method thereof)
1. Oxidation of TTF in acetonitrile Tetrathiafulvalene (TTF) was adjusted to 1 mM with acetonitrile (hereinafter referred to as “TTF solution 1”). Iron (III) perchlorate (Fe (ClO ₄ ) ₃ ) was adjusted with acetonitrile so as to be 500 mM (hereinafter referred to as “iron (III) perchlorate solution 1”). Acetonitrile is further added to the iron (III) perchlorate solution 1, and the concentration of iron (III) perchlorate is 0, 0.5, 1, 2.5, 5, 10, 25, and 50 mM. (Hereinafter referred to as “iron (III) perchlorate dilution 1”). Next, the TTF solution 1 and the iron (III) perchlorate solution 1 are mixed, the TTF is 0.1 mM, the iron (III) perchlorate is 0, 0.5, 1, 2.5, 5, 10, It diluted with acetonitrile so that it might become each density | concentration of 25 and 50 mM (henceforth "the liquid mixture 1"). The liquid mixture 1 was diluted 10 times with ultrapure water, and the spectrum was measured with a spectrophotometer.

  The results are shown in FIG. FIG. 4 (a) shows the absorption spectrum for each concentration of the iron (III) perchlorate diluent 1. FIG. 4 (b) shows the absorption spectrum of the mixed liquid 1 for each iron (III) perchlorate concentration.

  Here, it has been reported that the one-electron oxidant of TTF (compound 2) has absorption at 450 nm and 600 nm, respectively, and the two-electron oxidant of TTF (compound 3) has absorption at 375 nm. (M. Asakawa, PR Ashton, V. Balzani, et. Al, Angew. Chem. Int. Ed., 1998, 37, No. 3, 333-337). Moreover, it is known that TTF has absorption in 300 nm vicinity (S.Hnig, G.Kiessrich, H.Quast, D.Steutzow, Liebigs Ann.Chem., 1973,310-323). Therefore, when FIG. 4B is viewed, absorption is observed at 435 nm, 580 nm, and 360 nm, respectively. Further, as the concentration of iron (III) perchlorate increases, the absorption at 435 nm and 580 nm becomes stronger, and the absorption at 360 nm is also observed with iron (III) perchlorate at 50 mM. On the other hand, the absorption near 300 nm decreases with an increase in iron (III) perchlorate concentration.

  From the above, it was found that TTF was oxidized by iron (III) perchlorate in the mixed solution 1. Therefore, oxidation of TTF was possible in acetonitrile.

2. Oxidation of TTF in 90% acetonitrile + 10% ultrapure water TTF solution 1 and iron (III) perchlorate solution 1 are mixed, TTF is 0.1 mM, iron (III) perchlorate is 0, It adjusted with acetonitrile and ultrapure water (final concentration 10%) so that it might become each density | concentration of 0.5, 1, 2.5, 5, 10, 25, and 50 mM (henceforth "the liquid mixture 2"). . The liquid mixture 2 was diluted 10 times with ultrapure water, and the spectrum was measured with a spectrophotometer. Subsequently, re-reduction with ascorbic acid (AsA) was also performed. AsA was prepared to 5 mM with ultrapure water (hereinafter referred to as “AsA solution 1”). The AsA solution was further adjusted to concentrations of 0, 0.01, 0.05, 0.1, 0.2, and 0.5 mM (hereinafter referred to as “AsA diluted solution”. The iron (III) chlorate solution 1 and the AsA solution are mixed, the TTF is 0.1 mM, the iron (III) perchlorate concentration is 50 mM, the AsA concentration is 0, 0.01, 0.05, 0.1, The concentration was adjusted to 0.2 and 0.5 mM with acetonitrile and ultrapure water (final concentration 10%) (hereinafter referred to as “mixed solution 3”), and iron (III) perchlorate solution 1 and AsA solution 1 are mixed and mixed with acetonitrile and ultrapure water (final concentration 10%) so that Fe (ClO ₄ ) ₃ has a concentration of 50 mM and AsA has a concentration of 0.1, 0.2, and 0.5 mM. (Hereinafter referred to as “mixed liquid 4”) and TTF solution 1 sA solution 1 was mixed, and acetonitrile and ultrapure water (final) were adjusted so that TTF was 0.1 mM, and AsA was 0, 0.5, 1, 2.5, 5, 10, 25, and 50 mM. (Hereinafter referred to as “mixed liquid 5”) The mixed liquids 3 to 5 were diluted 10 times with ultrapure water, and the spectrum was measured with a spectrophotometer.

  The results are shown in FIG. FIG. 5A shows the absorption spectrum of the mixed liquid 2 according to the iron (III) perchlorate concentration. FIG.5 (b) shows the absorption spectrum of the solution according to the density | concentration of AsA dilution liquid. FIG. 5C shows an absorption spectrum of the mixed solution 4 according to AsA concentration. FIG. 5D shows an absorption spectrum of the mixed solution 5 by AsA concentration. FIG. 5 (e) shows an absorption spectrum of the mixed solution 3 by AsA concentration.

  As shown in FIG. 5 (a), the absorption at 435 nm and 580 nm increased with the increase of iron (III) perchlorate concentration. Absorption was observed at 360 nm at iron (III) perchlorate concentrations of 25 mM and 50 mM, respectively. On the other hand, the absorption near 300 nm decreased with the increase of iron (III) perchlorate concentration. Further, as shown in FIG. 5E, the absorption at 435 nm and 580 nm decreased with the increase of AsA concentration. On the other hand, the absorption near 300 nm increased.

  From the above results, it was found that the one-electron oxidant of TTF oxidized by iron (III) perchlorate was reduced to TTF by AsA and reversibly oxidized and reduced.

3. Oxidation of TTF in ultrapure water system TTF is 1 mM (hereinafter referred to as “TTF solution 2”), Fe (ClO ₄ ) in ultrapure water containing 10% hydroxypropyl-β-cyclodextrin (hp-β-CD). ₃ was adjusted to 500 mM (hereinafter referred to as “iron (III) perchlorate solution 2”). The iron (III) perchlorate solution 2 was further adjusted to have concentrations of 0, 1, 2.5, 5, 10, 25, 50, and 100 mM (hereinafter referred to as “iron (III) perchlorate dilution). Liquid 2 ". TTF solution 2 and iron (III) perchlorate solution 2 are mixed, and 10% hp-β-CD-containing ultrapure water has a TTF of 0.1 mM and iron (III) perchlorate of 0. , 0.5, 1, 2.5, 5, 10, 25, 50, and 100 mM (hereinafter, referred to as “mixed solution 6”). After dilution, the spectrum was measured with a spectrophotometer, then re-reduced with ascorbic acid, and an AsA solution was prepared with ultrapure water containing 10% hp-β-CD (hereinafter referred to as “AsA solution 2”). ) Mix TTF solution 2, iron (III) perchlorate solution 2 and AsA solution 2; The mixture was adjusted with ultrapure water containing 10% hp-β-CD (hereinafter, referred to as “mixed solution 7”) so that TF was 0.1 mM, iron (III) perchlorate was 10 mM, and AsA was at each concentration. The iron (III) perchlorate solution 2 and the AsA solution 2 are mixed, and the iron (III) perchlorate solution 2 has a concentration of 10 mM, and AsA has concentrations of 0, 0.01, 0.02, and 0.05 mM. (Hereinafter referred to as “mixed liquid 8”) The mixed liquids 6 to 8 were diluted 10 times with ultrapure water, and the spectrum was measured with a spectrophotometer.

  The results are shown in FIG. FIG. 6A shows an absorption spectrum according to concentration of the iron (III) perchlorate dilution 2. FIG. 6 (b) shows the absorption spectrum of the mixed solution 6 for each iron (III) perchlorate concentration. FIG. 6C shows an absorption spectrum according to concentration of the AsA solution 2. FIG. 6D shows an absorption spectrum of the mixed solution 8 by AsA concentration. FIG. 6 (e) shows the AsA concentration-specific absorption spectrum of the liquid mixture 7.

As shown in FIG. 6 (b), the absorption at 435 nm, 580 nm, and 360 nm increases as the iron (III) perchlorate concentration increases. On the other hand, the absorption in the vicinity of 300 nm decreases with the increase of the Fe (ClO ₄ ) ₃ concentration. Further, as shown in FIG. 6E, the absorption at 435 nm and 580 nm decreases with the increase of AsA concentration. On the other hand, the absorption in the vicinity of 300 nm increases as the AsA concentration increases. Therefore, it was possible to reversibly oxidize TTF with iron (III) perchlorate even in an aqueous system. It was also found that the one-electron oxidant of TTF oxidized by iron (III) perchlorate (compound 2) was reduced to TTF (compound 1) by AsA and reversibly oxidized and reduced.

(Purification method of oxidized TTF)
FIG. 7 is a diagram illustrating a method for purifying oxidized TTF using a solid phase extraction method. C18 (300 mg) was used as the solid phase carrier, acetonitrile (2 mL) was passed through (FIG. 7 (a)), and then ultrapure water (3mL) was passed through (FIG. 7 (b)). Thereafter, 300 μL of a mixed solution of TTF (10 mM) dissolved in 40% acetonitrile and iron (III) perchlorate (1M) was added (FIG. 7 (c)). After passing through, it was washed with ultrapure water (2 to 3 mL), and 500 μL of the extract was fractionated (FIG. 7D). Finally, elution was performed with acetonitrile (1-1.5 mL), and 500 μL of the eluate was collected (FIG. 7 (e)). Each fraction collected was subjected to spectrum measurement with a spectrophotometer. Moreover, each fraction extracted with acetonitrile was combined, concentrated and dried to obtain a residue. Assuming that the TTF used was 100% oxidized TTF, the amount of oxidized TTF in the obtained residue was converted, and an appropriate amount of acetonitrile was added to the residue to adjust the acetonitrile solution of oxidized TTF to a desired concentration. . The absorbance of the acetonitrile solution adjusted to each concentration was measured at 435 nm, and the correlation between the converted concentration of oxidized TTF and the absorbance was examined.

  The results are shown in FIG. Fig.8 (a) shows the absorption spectrum of each fraction extracted with the ultrapure water in the step shown in FIG.7 (d). FIG.8 (b) shows the absorption spectrum of each fraction extracted with acetonitrile in the step shown in FIG.7 (e). FIG. 8C is a graph showing the relationship between the absorbance at 435 nm and the converted concentration of oxidized TTF.

  FIG. 8A shows that oxidized TTF is not easily eluted with ultrapure water. Further, FIG. 8 (b) shows that oxidized TTF can be eluted with acetonitrile. It was also found that TTF existed in an oxidized state even after solid phase extraction. Furthermore, sufficient linearity was obtained in the relationship between the absorbance at 435 nm and the converted oxidized TTF concentration. This was used as a calibration curve and used as an index of the concentration of one-electron oxidant of TTF.

(Production method of biosensor)
1. Mediator / enzyme mixed system Glucose sensors A1 and A2 having the same configuration as that of the biosensor 1 were prepared by the following method. First, a PET (Polyethylene Terephthalate) substrate provided with a carbon working electrode, a silver / silver chloride reference electrode, and a carbon counter electrode was prepared. Next, TTF or TTF1 electron oxidant was dissolved in ultrapure water, and hp-β-CD (5%), tritonX-100 (1%), and GOD (10 U / μL) were added thereto. Thereafter, each solution was applied onto a 5 μL chip to form a reaction layer and dried in a refrigerator for 24 hours or more. What applied the TTF containing solution was made into glucose sensor A1, and what applied the TTF1 electronic oxidant containing solution was made into glucose sensor A2.

  The TTF1 electron oxidant solution was adjusted to a predetermined concentration by the following method. The absorbance of the acetonitrile eluate obtained in the step shown in FIG. Using the calibration curve shown in FIG. 8C, the absorbance measurement value is converted into the concentration. Weigh accurately the volume of the eluate and dry the eluate. Thus, a predetermined amount of one-electron oxidant powder is obtained. This powder is dissolved in a solvent and adjusted to a predetermined concentration.

2. Mediator, enzyme two-layer system Glucose sensors B1 and B2 having the same configuration as that of the biosensor 2 were produced by the following method. First, a PET substrate provided with a carbon working electrode, a silver / silver chloride reference electrode, and a carbon counter electrode was prepared. Subsequently, each 4 mM acetonitrile solution of TTF or TTF1 electron oxidant was applied to a 5 μL chip in 5 μl portions. Specifically, 1 μL was applied, and after drying, 1 μL was applied again on the electrode system, and this was repeated 5 times. After drying in a desiccator, 1 μL of a 5% triton X-100 aqueous solution was applied. Then, 2.5 μL of 20 U / μL GOD solution which had been dried and desalted in a desiccator was applied, and dried in a refrigerator for 24 hours or more. What applied the TTF containing solution was made into glucose sensor B1, and what applied the TTF1 electronic oxidant containing solution was made into glucose sensor B2.

(Electrochemical measurement)
As a standard sample, a glucose solution prepared with 0.1 M TES buffer (pH 7.5) containing 150 mM NaCl was prepared. In addition, human urine collected by volunteers and urine samples prepared by adding glucose to human urine were prepared. 5 μL of a sample was added to the reaction layer, and 10 seconds after the sample was added, a voltage (0.3 mV / vs) was applied to the working electrode in the anode direction with respect to the counter electrode, and measurement of the response current was started. The current value for 1 minute after the start was taken as the measured value. The glucose sensors A1, A2, B1, and B2 used for the measurement were exchanged for each measurement.

  FIG. 9 shows the results of glucose sensors A1 and A2. FIG. 9A shows the results of the standard sample. FIG. 9B is an enlarged view of FIG. FIG. 9C shows the result of the urine sample. FIG. 9 (d) is an enlarged view of FIG. 9 (c). ○ indicates the result of the glucose sensor A1, and Δ indicates the result of the glucose sensor A2. The horizontal axis of each graph indicates the glucose concentration (mM), and the vertical axis indicates the current value (μA). As shown in FIG. 9, the base current of the chip in which the TTF1 electronic oxide was prepared with ultrapure water decreased.

  FIG. 10 shows the results of glucose sensors B1 and B2. FIG. 10A shows the results of the standard sample. FIG. 10B is an enlarged view of FIG. FIG.10 (c) is a figure which shows the result of a urine sample. FIG. 10 (d) is an enlarged view of FIG. 10 (c). ◯ indicates the result of the glucose sensor B1, and □ indicates the result of the glucose sensor B2. The horizontal axis of each graph indicates the glucose concentration (mM), and the vertical axis indicates the current value (μA). When the standard sample was used for the glucose sensor B2, good linearity was obtained in the range of 4 to 10 mM. Therefore, it was suggested that the glucose sensor B2 can quantitatively measure low concentration glucose.

It is sectional drawing which showed typically the biosensor which concerns on embodiment. It is the top view which showed typically the biosensor which concerns on embodiment. It is sectional drawing which showed typically the biosensor which concerns on a modification. It is a result of the ultraviolet visible near-infrared light absorption spectrum of the oxidation type electron acceptor which concerns on an Example. It is a result of the ultraviolet visible near-infrared light absorption spectrum of the oxidation type electron acceptor which concerns on an Example. It is a result of the ultraviolet visible near-infrared light absorption spectrum of the oxidation type electron acceptor which concerns on an Example. It is a figure explaining the method to refine | purify the oxidation type electron acceptor which concerns on an Example. It is a result of the ultraviolet visible near-infrared light absorption spectrum after refine | purifying the oxidized electron acceptor which concerns on an Example. It is a figure which shows the measurement result using the biosensor which concerns on an Example. It is a figure which shows the measurement result using the biosensor which concerns on an Example.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Biosensor 2 Biosensor 101 Insulating substrate 102 Working electrode 103 Counter electrode 104 Reaction layer 105 Reference electrode 106 Electrode 204 Reaction layer 204a Mediator layer 204b Enzyme layer

Claims (6)

A substrate,
An electrode provided on the substrate;
A reaction layer including an oxidoreductase, an oxidized tetrathiafulvalene or an oxidized tetrathiafulvalene derivative that is in an oxidized state before the start of measurement, and a surfactant;
With
The oxidoreductase is glucose oxidase or glucose reductase ;
Prior Symbol reaction layer, said oxidized tetrathiafulvalene or the oxidized tetrathiafulvalene derivatives are inclusion in cyclodextrins,
The electrode detects an oxidation current value of tetrathiafulvalene or tetrathiafulvalene derivative obtained by reducing the oxidized tetrathiafulvalene or the oxidized tetrathiafulvalene derivative with an oxidoreductase that has reacted with glucose in the sample. Glucose sensor used for   The glucose sensor according to claim 1, wherein the oxidized tetrathiafulvalene or the oxidized tetrathiafulvalene derivative included in cyclodextrin contained in the reaction layer is distributed throughout the reaction layer. . The reaction layer includes an enzyme layer containing the oxidoreductase, and a mediator layer containing the oxidized tetrathiafulvalene or the oxidized tetrathiafulvalene derivative ,
The enzyme layer is in contact with the mediator layer;
The glucose sensor according to claim 1, wherein the mediator layer is provided on a surface of the electrode.   The glucose sensor according to claim 3, wherein the oxidized tetrathiafulvalene or the oxidized tetrathiafulvalene derivative included in cyclodextrin contained in the mediator layer is distributed throughout the mediator layer. The glucose sensor according to any one of claims 1 to 4, wherein the oxidized tetrathiafulvalene or the oxidized tetrathiafulvalene derivative is a two-electron oxidant. Preparing a substrate;
Providing an electrode on the substrate;
Providing a reaction layer containing an oxidoreductase, oxidized tetrathiafulvalene or oxidized tetrathiafulvalene derivative in an oxidized state, and a surfactant so as to be in contact with the electrode;
Including
The oxidoreductase is glucose oxidase or glucose reductase ;
Prior Symbol reaction layer, said oxidized tetrathiafulvalene or the oxidized tetrathiafulvalene derivatives are inclusion in cyclodextrins,
The electrode detects an oxidation current value of tetrathiafulvalene or tetrathiafulvalene derivative obtained by reducing the oxidized tetrathiafulvalene or the oxidized tetrathiafulvalene derivative with an oxidoreductase that has reacted with glucose in the sample. A method for manufacturing a glucose sensor used for the purpose.

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