JP6557090B2 - Electrode tip and electrode tip manufacturing method - Google Patents

Description

  The present invention relates to an electrode chip that can accurately and easily detect the concentration of endotoxin.

  Endotoxin is a substance present on the cell wall of Gram-negative bacteria and has various biological activities such as pyrogenicity. Therefore, for example, if pharmaceuticals and medical devices such as dialysate, injection, transplanted tissue, artificial insemination fertilized egg culture solution, etc. are contaminated with endotoxin, even a trace amount may cause serious results. The amount of contamination of microbial contaminants must be strictly controlled. However, endotoxins are ubiquitous in the environment and have high heat resistance, so that they are difficult to remove by heating and management of contamination prevention is very difficult.

  The endotoxin detection test includes a qualitative test and a quantitative test. As a qualitative test, a gelation method, a turbidimetric method as a quantitative test, a colorimetric method described in Patent Document 1, and a fluorescence method are generally known. In either method, a complicated procedure and a long time are required to perform highly accurate detection. Therefore, a rapid detection method is required with a simple operation.

  In order to solve such a problem, Patent Document 2 uses a measurement reagent including a peptide compound to which a dye is bound and a lysate reagent that liberates the dye from the peptide compound by reacting with endotoxin. An electrochemical endotoxin concentration detection method using voltammetry is described. In this method, by applying differential pulse voltammetry, the current peak derived from the reduction of the dye released from the peptide compound and the current peak derived from the reduction of the dye bound to the peptide compound are obtained separately. And endotoxin concentration can be detected with high accuracy.

Japanese Patent No. 51188849 Japanese Patent No. 5561613

However, the electrochemical endotoxin concentration detection method described in Patent Document 2 requires a step of adding a mixture formed by adding the analyte to the measurement reagent and mixing it to the measurement electrode chip. There is a problem that is complicated.
In addition, there is a problem in that endotoxin in the air is mixed in the step of adding the mixture to the electrode chip, and the accuracy of concentration detection may be reduced.

  The main object of the present invention is to provide an electrode chip that can accurately and easily detect the concentration of endotoxin.

To achieve the above object, the present invention provides a substrate, an electrode including at least a working electrode and a counter electrode formed on one surface of the substrate, and formed on one surface of the substrate. A connected terminal, a reagent immobilized on one surface of the substrate, a height formed on one surface of the substrate, the electrode and the reagent being arranged, and a height capable of accommodating a predetermined capacity And the reagent includes a factor C and a peptide compound to which a redox substance is bound, and the electrode is arranged to measure a current value by an electrochemical reaction. Provided is an electrode tip which is fixed in a region without being in contact with the electrode.

According to the present invention, an analyte containing endotoxin is introduced into the electrode chip by immobilizing a reagent containing a factor C and a peptide compound in which a redox substance is bound in the measurement region of the electrode chip. Only by this, it becomes possible to obtain a post-reaction mixture that is a target for measuring an electric current value by an electrochemical reaction, that is, a mixture of the analyte and the reagent components after the completion of the free reaction.
Therefore, the electrode tip can detect the endotoxin concentration accurately and easily.

In the present invention, the reagent preferably contains a buffer component. When the reagent contains a buffer component, measurement sensitivity can be improved. As a result, the electrode chip can detect the endotoxin concentration of the subject with high sensitivity.
Moreover, it is because the process which adds a buffer solution separately can be made unnecessary, As a result, the said electrode chip can detect an endotoxin density | concentration easily. In addition, endotoxin in the air can be prevented from being mixed when a buffer solution is separately added, and the electrode tip can accurately detect the concentration of endotoxin.

  In the present invention, it is preferable that the reagent has a porous structure. This is because when the reagent has a porous structure, the reagent quickly dissolves in the analyte. As a result, the electrode chip can detect the endotoxin concentration of the subject in a short time.

In this invention, it is preferable that the said measurement area | region is prescribed | regulated by the said accommodating part .

In this invention, it is preferable that the said accommodating part is provided with the dam part holding the said reagent of the height lower than the said accommodating part .

  In this invention, it is preferable to have a cover part which covers the said board | substrate upper surface. This is because contamination of endotoxin and the like in the air can be prevented, and the electrode tip can accurately detect the concentration of endotoxin. Further, it is possible to prevent drying of the specimen and the like, and the electrode chip can detect the endotoxin concentration with high accuracy.

The present invention includes a substrate, an electrode including at least a working electrode and a counter electrode formed on one surface of the substrate, a terminal formed on one surface of the substrate and connected to the electrode, and the substrate A layered body preparing step for preparing a layered body having a height that can accommodate the predetermined capacity and the electrode and the reagent are formed on one surface of the layer, and the layered body preparation After the step, there are an application step of applying a reagent solution containing a peptide compound in which a factor C and a redox substance are bonded on one surface of the substrate, and a drying step of drying the reagent solution to form a reagent. In the application step, the reagent solution is applied without being in contact with the electrode in a measurement region where the electrode is arranged and a current value is measured by an electrochemical reaction. How to make an electrode tip Subjected to.

  According to the present invention, by having the coating step and the drying step, it is possible to easily manufacture an electrode chip in which a reagent containing a factor C and a peptide compound in which a redox substance is bound is immobilized in the measurement region. .

  The present invention has an effect that it is possible to provide an electrode chip capable of easily and accurately detecting the endotoxin concentration.

It is a schematic plan view which shows an example of the electrode tip of this invention. It is the sectional view on the AA line of FIG. It is a schematic diagram which shows an example of the free reaction in this invention. It is a schematic diagram which shows an example of the free reaction in this invention. It is explanatory drawing explaining the immobilization position of the reagent in this invention. It is a schematic plan view which shows the other example of the electrode tip of this invention. It is the BB sectional view taken on the line of FIG. It is a schematic plan view which shows the other example of the electrode tip of this invention. It is CC sectional view taken on the line of FIG. It is a schematic sectional drawing which shows the other example of the electrode tip of this invention. It is process drawing which shows an example of the manufacturing method of the electrode chip of this invention. It is a schematic sectional drawing which shows an example of the endotoxin concentration measuring apparatus of this invention. It is process drawing which shows an example of the usage method of the endotoxin concentration measuring apparatus of this invention. 2 is a cyclic voltammogram in Example 1. FIG. 2 is a cyclic voltammogram in Comparative Example 1. 2 is a cyclic voltammogram at a reaction time of 30 minutes in Example 1 and Comparative Example 1. FIG. 2 is a cyclic voltammogram at a reaction time of 60 minutes in Example 1 and Comparative Example 1. FIG. 4 is a graph showing a peak current with respect to reaction time in Example 1 and Comparative Example 1. 6 is a graph showing a peak current with respect to reaction time in Example 3 and Comparative Example 2. It is a graph which shows the peak current with respect to the reaction time in Example 4 and Comparative Example 3. It is a graph which shows the peak current with respect to reaction time in Example 5 and Comparative Example 4.

The present invention relates to an electrode chip, a method for producing the same, and an endotoxin concentration measuring apparatus for detecting an endotoxin concentration using the electrode chip.
Hereinafter, the electrode chip, the electrode chip manufacturing method, and the endotoxin concentration measuring apparatus of the present invention will be described in detail.

A. Electrode Chip First, the electrode chip of the present invention will be described.
The electrode chip of the present invention includes a substrate, an electrode formed on one surface of the substrate, a terminal formed on one surface of the substrate and connected to the electrode, and one surface of the substrate A reagent immobilized thereon, wherein the reagent includes a factor C and a peptide compound to which a redox substance is bound, and further, the electrode is arranged to measure a current value by an electrochemical reaction. It is characterized in that it is fixed in the measurement area to be performed.

Such an electrode tip of the present invention will be described with reference to the drawings. FIG. 1 is a schematic plan view showing an example of the electrode tip of the present invention. 2 is a cross-sectional view taken along line AA in FIG. As illustrated in FIGS. 1 and 2, an electrode chip 10 of the present invention is formed on a substrate 1, an electrode 2 formed on one surface of the substrate 1, and one surface of the substrate 1. A terminal 3 connected to the electrode 2 and a reagent 4 immobilized on one surface of the substrate 1, wherein the reagent 4 is a peptide compound in which a factor C and a redox substance are bound. In addition, the electrode 2 is disposed and is fixed in a measurement region 5 where a current value is measured by an electrochemical reaction.
In this example, the electrode chip 10 has a conductive wire 6 that electrically connects the electrode 2 and the terminal 3, and an insulating layer 7 formed so as to cover the conductive wire 6. The electrode 2 is a three-electrode electrode having a working electrode 2a, a counter electrode 2b, and a reference electrode 2c.
In FIG. 1, the measurement region 5 is a region surrounded by a dotted line, and the reagent 4 is immobilized so as to cover a part of the electrode 2 in the measurement region 5.

According to the present invention, an analyte containing endotoxin is introduced into the electrode chip by immobilizing a reagent containing a factor C and a peptide compound in which a redox substance is bound in the measurement region of the electrode chip. This makes it possible to obtain the post-reaction mixture, which is a current value measurement target by an electrochemical reaction.
For this reason, the step of forming the post-reaction mixture using a container prepared separately from the electrode chip, and then removing the post-reaction mixture from the container and introducing it into the measurement electrode chip is unnecessary. be able to.
In addition, the step of taking out the post-reaction mixture from the container and introducing it into the measurement electrode chip can be eliminated. As a result, the post-reaction mixture is introduced into the measurement electrode chip when taking out the post-reaction mixture from the container. At the same time, mixing of endotoxin in the air can be prevented, and a decrease in the accuracy of endotoxin concentration detection can be suppressed.
Therefore, the electrode tip can detect the endotoxin concentration accurately and easily.

  Moreover, the measurement of an electric current value can be started immediately after formation of the said post-reaction mixture by arrange | positioning the said reagent in the said measurement area | region. For this reason, even when the reaction rate of the free reaction is high or the redox substance released from the peptide compound contained in the post-reaction mixture is deactivated in a short time, the electrode tip stably stabilizes the concentration of endotoxin. Can be detected.

Here, in the release reaction, an active factor C is generated from the factor C by causing an analyte containing endotoxin to act on the factor C, and then the peptide in which the redox substance is bound by the active factor C. The redox material can be liberated from the compound.
The release reaction may be a multistage reaction using the factor C together with factor B and a coagulase precursor.
FIG. 3 and FIG. 4 are schematic diagrams showing an example of the case where the liberation reaction is a multi-stage reaction, in which the redox substance is paramethoxyaniline (hereinafter sometimes simply referred to as pMA). An example of paraaminophenol (hereinafter sometimes simply referred to as pAP) is shown. As illustrated in FIG. 3 and FIG. 4, in a multi-step release reaction, an active factor C is converted from factor C, and an active factor B is converted from factor B by allowing an analyte containing endotoxin to act on factor C. Then, activated coagulase is generated one after another from the coagulase precursor, and pMA and pAP are released from the peptide compound to which pMA is bound and the peptide compound to which pAP is bound by the activated coagulase, respectively.
Endotoxin is quantified with respect to the said mixture after the said free reaction based on the electric current value measured by an electrochemical reaction. That is, pMA or pAP released from the peptide compound exists in the mixture after the release reaction, and pMA or pAP undergoes an oxidation reaction at a specific potential. There is a correlation between the current value derived from the oxidation reaction of pMA or pAP and the concentration of pMA or pAP, that is, the concentration of endotoxin, and the concentration of endotoxin is quantified using this correlation.

  Specifically, when the electrode potential is equal to or higher than the oxidation potential, for example, in the case of pMA, electrons are emitted according to the scheme of the following formula (1) and oxidized, and in the case of pAP, the following formula (2) According to the scheme, electrons are emitted and oxidized.

  At this time, electrons emitted from pMA or pAP and received by the working electrode are observed as current. The current value measured by the electrochemical reaction is proportional to the concentration of pMA or pAP. Since there is a correlation between the concentration of endotoxin and the progress of the multistep reaction, there is also a correlation between the concentration of endotoxin and the concentration of pMA or pAP generated, that is, the current value. Therefore, the endotoxin concentration can be measured from the current value by preparing in advance a calibration curve showing the correlation between the endotoxin concentration and the current value.

Further, in a method of detecting the endotoxin concentration by the absorbance of a sample such as a colorimetric method, it is necessary to transmit light for measurement through a predetermined optical path length, and thus a sample with a certain amount of solution or more is required.
On the other hand, endotoxin concentration detection using an electrochemical reaction makes it possible to detect the reaction on the electrode surface, and the endotoxin concentration can be detected even with a small amount of analyte. Therefore, the electrode chip can detect endotoxin concentration even with a very small amount of analyte. Furthermore, the use of so-called lysate reagents containing factor B and a coagulation enzyme precursor in factor C, and expensive enzymes such as factor C can be greatly reduced, and the electrode tip can detect endotoxin concentration at low cost. .

  The endotoxin concentration detection method using an electrochemical reaction is not a detection method using light unlike the colorimetric method, so low-transparency analytes and multi-component analytes such as tissue fluids are also subject to measurement. It is thought that it can be done and is extremely practical.

  As described above, according to the present invention, endotoxin can be quantified easily, quickly and with high accuracy at a medical site, and strict mixing prevention management of endotoxin can be performed on pharmaceuticals and medical instruments that are in direct contact with blood. .

The electrode chip of the present invention has a substrate, electrodes, terminals, and a reagent.
Hereafter, each structure of the electrode tip of this invention is demonstrated in detail.

1. Reagent The reagent used in the present invention is immobilized on one surface of the substrate.
The reagent is immobilized in the measurement region.
The reagent contains factor C and a peptide compound to which a redox substance is bound.
Note that “immobilized” means that the mixture of the components of the reagent is fixed in a dry state.

The above-mentioned factor C is particularly limited as long as it can generate an active factor C by endotoxin and further cause a free reaction to liberate the redox substance from the peptide compound by the active factor C. is not.
For example, as the above-mentioned factor C, factor C isolated and purified from a biological component such as a blood cell extract component of horseshoe crab, a recombinant factor C produced by a gene recombination technique, and the like can be used as appropriate.
In the present invention, among them, the above-mentioned factor C is used together with factor B and a clotting enzyme precursor, that is, the reagent comprises a multistage reaction composition containing factor C, factor B and a clotting enzyme precursor. It is preferable to include. This is because the multistage release reaction can efficiently release the redox substance from the peptide compound.
The composition for multistage reaction is not particularly limited as long as it contains factor C, factor B and a coagulation enzyme precursor. For example, it is based on a blood cell extract component of horseshoe crab called Limulus Ambocyte Lysate (LAL). The prepared lysate reagent can be used. In addition, at least one of C-factor, B-factor and coagulase precursor, such as those prepared by combining factor C, factor B and coagulase precursor isolated and purified from biological components such as blood cell extract components of horseshoe crab In addition, lysate reagents also include those prepared by combining those isolated and purified from biologically derived components such as blood cell extract components of horseshoe crab.
Furthermore, as the above-mentioned multistage reaction composition, a composition prepared by appropriately using recombinant factor C, recombinant factor B, or a recombinant coagulase precursor prepared by a gene recombination technique is used as an “LAL equivalent”. Can also be used.

The content of the factor C or the multistage reaction composition in the reagent is not particularly limited as long as it can stably detect the endotoxin concentration, and the type and size of the electrode tip of the present invention. It is appropriately set according to the amount of sample introduced.
As content in the said reagent of the said multistage reaction composition, it can be set as the same content as the lysate reagent for 1 test marketed for the endotoxin measurement used for an electrochemical method, for example.

The peptide compound to which the redox substance is bound is only required to have a redox substance and an oligopeptide to which the redox substance is bound, and the other redox substance is bound to one end of the oligopeptide. A peptide having a peptide protecting group bonded to the end can be used. Examples of such peptide compounds include those represented by X-A-Y. Here, X represents a protecting group, A represents an oligopeptide, and Y represents the redox substance.
Examples of the protecting group X include peptide protecting groups such as a t-butoxycarbonyl group (Boc), a benzoyl group, and an acetate group.

  The oligopeptide is not particularly limited as long as it can release the redox substance by the action of factor C or the multistage reaction composition. Of these, oligopeptides having 2 to 10 amino acids, particularly 2 to 5, and more preferably 3 to 4 amino acids are preferred.

  Examples of oligopeptides for factor C that can release redox substances by the action of factor C include, for example, Val-Pro-Arg, Asp-Pro-Arg, Leu-Gly-Arg, etc. as tripeptides for factor C. Can be mentioned.

Examples of the tripeptide for a multistage reaction composition capable of releasing a redox substance by the action of the multistage reaction composition include Leu-Gly-Arg and Thr-Gly-Arg. Examples of the tripeptide for the multistage reaction composition include a tripeptide having an L-amino acid in the general formula: R ¹ -Gly-Arg-Y. Here, R ¹ represents an N-blocked amino acid.
In addition, examples of the oligopeptide for the multistage reaction composition include tetrapeptides represented by the general formula: R ² -A ¹ -A ² -A ³ -A ⁴ -Y. Wherein R ² represents hydrogen, a blocked aromatic hydrocarbon or an acyl group, A ¹ represents an L-amino acid or D-amino acid selected from Ile, Val or Leu, and A ² represents Glu or Asp A ³ represents Ala or Cys, and A ⁴ represents Arg.
Peptide compounds for a multistage reaction composition in which a redox substance is bonded to one end of an oligopeptide and a protecting group for the peptide is bonded to the other end are specifically Boc-Leu-Gly-Arg-Y, acetate -Ile-Glu-Ala-Arg-Y etc. are mentioned.

The redox substance is not particularly limited as long as it can be released from the peptide compound by activated factor C or the activated coagulase and causes an oxidation reaction or a reduction reaction at a specific potential.
Specific examples of the redox substance include compounds represented by the following general formula (3) including pMA and the like, dyes, paraaminophenol (pAP), and the like.
Specific examples of the dye include 2,4-dinitroaniline (DNP), p-nitroaniline (pNA), 7-methoxycoumarin-4-acetic acid (MCA), Dansyl dye, and the like. .

(In the formula (3), R is —O—C _n H _{2n + 1} or —S—C _n H _{2n + 1} , and n is an integer from 1 to 4.)

  In the present invention, the compound represented by the general formula (3) is preferably pMA. The shorter the alkyl chain of the compound represented by the general formula (3), the higher the water solubility, and the activated factor C or the activated coagulase has the peptide sequence of the oligopeptide contained in the peptide compound. It becomes easy to recognize. For this reason, since the compound represented by the general formula (3) bonded to the oligopeptide as a redox substance is pMA, the peptide compound has increased reactivity with the activated factor C and the like. is there.

  In the present invention, the redox substance is preferably pMA or pAP. PMA and pAP released from the peptide compound and pMA and pAP bound to the peptide compound have different redox potentials, and the difference is relatively large. Therefore, the current derived from the oxidation reaction of pMA and pAP released from the peptide compound and the current derived from the oxidation reaction of pMA and pAP bound to the peptide compound can be easily separated and obtained. Therefore, by using a peptide compound to which pMA or pAP is bound, even if the concentration of pMA or pAP released from the peptide compound is extremely small, the concentration of endotoxin can be detected with high accuracy.

The content of the peptide compound in the reagent is not particularly limited as long as it can stably detect the endotoxin concentration, and is appropriately set according to the type of the electrode chip of the present invention. It is.
For example, the content can be an amount that falls within the range of 0.1 mM to 5.0 mM when the post-reaction mixture is formed.
The volume of the post-reaction mixture can be almost the same as the volume of the analyte, and can be in the range of several μL to several tens of μL.

Further, the total content of the factor C or the multistage reaction composition and the peptide compound to which the redox substance is bound is not particularly limited as long as it can stably detect the endotoxin concentration. Rather, it is appropriately set according to the type and size of the electrode tip of the present invention.
The total content of the multistage reaction composition and the peptide compound can be, for example, in the range of 1 mg to 10 mg, and more preferably in the range of 1 mg to 4 mg. This is because the content of expensive enzymes and the like can be reduced, and the price of the electrode tip of the present invention can be reduced.

The reagent preferably contains a buffer component. When the reagent contains a buffer component, measurement sensitivity can be improved. As a result, the electrode chip can detect the endotoxin concentration of the subject with high sensitivity.
Moreover, it is because the process which adds a buffer solution separately can be made unnecessary, As a result, the said electrode chip can detect an endotoxin density | concentration easily. In addition, endotoxin in the air can be prevented from being mixed when a buffer solution is separately added, and the electrode tip can accurately detect the concentration of endotoxin.
In addition, when the reagent contains the buffer component, it contains a peptide compound to which factor C and a redox substance are bound, and a buffer solution is added separately using a conventional reagent that does not contain the buffer component. Although it is not clear why the measurement sensitivity can be improved as compared with the case where a free reaction is performed, the factor C, the peptide compound, and the peptide compound that contact the analyte when the analyte contacts the reagent. It is surmised that the concentration of the buffer component can be increased.

The buffer component is a solid content of the buffer solution remaining when water is removed from the buffer solution by drying, and any buffer solution having a desired buffer capacity can be generated by adding pure water.
The buffer component is not particularly limited as long as it can generate a buffer solution capable of stably releasing the redox substance. For example, pH 6.0 to 9.0, and pH 7.0 to 8 are particularly preferable. Preferably, it is a component of the buffer solution of .5. This is because the release amount of the redox substance can be increased.
Examples of the buffer component include components such as Tris-Ac buffer, Tris-HCl buffer, phosphate buffer, HEPES buffer, and PIPES buffer.

The content ratio of the buffer component in the reagent is not particularly limited as long as it can generate a buffer solution having a desired pH range, and is appropriately set according to the type of the electrode chip of the present invention. It is what is done.
For example, the content ratio can be set to an amount capable of generating each buffer solution when the post-reaction mixture is formed.

The reagent preferably contains at least a factor C and a peptide compound, and further contains a factor B, a clotting enzyme precursor, and a buffer solution component, and further contains other components as necessary. Also good.
As said other component, the 2nd oxidation-reduction substance which can react with the oxidation-reduction substance liberated from the said peptide compound can be mentioned. This is because the endotoxin concentration can be detected with higher accuracy by causing the electrode to cause a redox reaction of the second reducing substance after reacting with the redox substance.
The second redox material can be appropriately selected according to the redox material.

The reagent may have a porous structure having a large number of pores and a surface area larger than a structure having no pores (non-porous structure) having the same volume, or a non-porous structure. good.
This is because when the reagent has a porous structure, the reagent quickly dissolves in the analyte. Moreover, when it is the said non-porous structure, it is because the drying method by normal temperature normal pressure which is mentioned later can be used as a drying method of the said reagent, and the said reagent can be formed with simple equipment.
In addition, as a method for determining whether the reagent has a porous structure or a non-porous structure, the reagent has a porous structure when it is clouded and has a transparency. Examples thereof include a method for visually judging that the structure is non-porous, and a method for confirming by observation with a scanning electron microscope (SEM).

The reagent is immobilized in the measurement region.
Here, being fixed in the measurement region is not limited to being formed so as to cover the entire surface of the measurement region in plan view, but may be fixed to a part of the measurement region in plan view. It includes what is formed.
The measurement region is a region where the electrode is arranged and a current value is measured by an electrochemical reaction.
Such a measurement region may be a region where a current value by an electrochemical reaction is stably measured using the electrode when the post-reaction mixture is formed. When dropped onto the reagent, the post-reaction mixture can be a region that can cover all the electrodes. More specifically, the area of the measurement area in plan view can be within a range where the distance from the end of the electrode is within 30 mm, depending on the volume of the subject to be dropped. In particular, it is preferably within a range of 20 mm or less, particularly preferably within a range of 10 mm or less. This is because, when the range of the measurement region is within the above range, the electrode tip can be easily reduced in size, and the endotoxin concentration can be detected stably.
The end of the electrode refers to the smallest rectangular outer periphery that can surround the electrode. For example, when the electrode includes a plurality of measurement electrodes (working electrode, counter electrode, reference electrode, etc.) as shown in FIG. 1, the end of the electrode is the minimum that can include all of the measurement electrodes. The outer periphery of the rectangle.
Further, the distance is specifically indicated by h in FIG. 1 already described.

The reagent immobilization position may be a position within the measurement region where the reagent can be brought into contact with the analyte introduced into the electrode chip to form the post-reaction mixture.
Such an immobilization position may be a location not in contact with the electrode, but is usually a location in contact with the electrode because the area in plan view of the electrode in the measurement region is relatively large. .
Specifically, the fixing position may include on the upper surface of the electrode, on the side surface of the electrode, on the surface of the substrate, on the upper surface of the insulating layer, on the side surface of the insulating layer, and the like.
When the electrode tip includes the housing part, the fixing position can include a side surface of the housing part and the like.
When the electrode chip has a lid that covers the upper surface of the substrate, the fixing position may include the surface of the lid on the substrate side, the side of the lid, and the like.
In addition, when the electrode chip has an adhesive layer formed on the substrate side surface of the lid portion in order to fix the lid portion to the substrate side, the immobilization position is the adhesion position. On the side of the layer and the like.
FIG. 5 is an explanatory view for explaining the immobilization position of the reagent in the present invention, and is a cross-sectional view showing the peripheral part of the measurement region when the electrode chip has the storage part. 5 shows that the fixing position is on the side surface of the electrode 2 and on the substrate 1 (FIG. 5A), on the upper surface of the electrode 2 (FIG. 5B), and on the upper surface of the electrode 2. And on the side surface, on the substrate 1 and on the side surface of the housing portion 12 (FIG. 5C), on the side surface of the housing portion 12 and on the substrate 1 (FIG. 5D). is there.

The method for forming the reagent is not particularly limited as long as the reagent can be immobilized in the measurement region, and after preparing a reagent solution containing reagent components such as the factor C and the peptide compound. A method can be used in which the reagent solution is applied and dried at a position where the reagent is immobilized in the measurement region. Moreover, when the said reagent contains the said buffer component, what contains the said buffer component as said reagent solution can be used.
The application method is not particularly limited as long as the reagent solution can be stably applied in the measurement region, and examples thereof include a dispenser method and an ink jet method.
In addition, when the electrode chip has a reagent fixing part described later, the application method may be a method of applying the reagent solution in the reagent fixing part. This is because it is easy to immobilize the reagent in the measurement region by using a method of applying the reagent in the reagent fixing part.

The drying method is not particularly limited as long as it can remove water from the reagent solution, and a method of drying at normal temperature and atmospheric pressure, a vacuum drying method of drying under reduced pressure, and the like can be used. . In the present invention, the drying method is preferably the vacuum drying method. This is because the reagent solution can be dried in a state in which contamination of endotoxin in the air is prevented by the drying method being the vacuum drying method. In addition, since drying at room temperature or less is possible in a short time, the cost is reduced, the risk of endotoxin contamination is reduced, and the factor C, factor B and coagulase precursor contained in the reagent are denatured and not activated. This is because the suppression of the structure can be achieved. Further, when the drying method is the vacuum drying method or the like, the reagent can have a porous structure.
In the present invention, the vacuum drying method is particularly preferably a freeze drying method. The reagent can have a lower density porous structure, that is, a larger void volume and a larger surface area, and the reagent has an excellent dissolution rate in the analyte. is there.
On the other hand, when the drying method is a method of drying under atmospheric pressure, the cost required to dry the reagent solution to obtain a reagent can be reduced.

2. Electrode The electrode in the present invention is formed on one surface of the substrate.
The electrode is not particularly limited, and a general electrode used for electrochemical measurement can be used. Examples of the electrode include carbon electrodes such as glassy carbon, carbon paste, graphite, and diamond-like carbon, and electrochemically stable metal electrodes such as Au, Pt, Pd, and Ni.

  Examples of the electrode include a two-electrode electrode including a working electrode and a counter electrode, a three-electrode electrode including a working electrode, a counter electrode, and a reference electrode, and a four-electrode electrode including two working electrodes, a counter electrode, and a reference electrode In general, the above three-electrode type electrode is used.

  The method for forming the electrode is not particularly limited as long as the electrode can be stably formed in the measurement region. However, after the substrate is prepared and the conductive film is formed in the measurement region, the electrode is formed. Examples include a method of patterning the conductive film by a photolithography method, and a method of forming an electrode having a desired pattern using a mask vapor deposition method, a screen printing method, a gravure printing method, a flexographic printing method, an ink jet method, or the like.

3. Terminal The terminal in the present invention is formed on one surface of the substrate and connected to the electrode.
The terminal is connected to a measuring device that quantifies the endotoxin concentration based on a current value measured by an electrochemical reaction.
The material of the terminal can be the same as the material of the electrode. The material of the terminal is not limited as long as the conductivity that does not affect the detected current value is ensured, and a general conductive material can be used. Among them, from the viewpoint of conductivity, Au, Pt, Ag It is preferably a metal such as Pd, Ni.
The method for forming the terminal can be the same as the method for forming the electrode.
The terminal may be formed simultaneously with the electrode or may be formed separately from the electrode.

  As the measurement device, a device generally used for electrochemical measurement can be used, and examples thereof include a potentiostat, a current amplifier, and a device having the same function as these.

4). Substrate The substrate in the present invention supports the electrode, the terminal, and the reagent.
The substrate used in the present invention is not particularly limited as long as an electrode can be formed and the surface has an insulating property.
Examples of the constituent material of the substrate include inorganic materials such as silicon and glass, acrylic resins, silicone resins such as polydimethylsiloxane (PDMS), epoxy resins, polyethylene terephthalate (PET), polyethylene naphthalate (PEN), and polystyrene sulfonic acid. Examples thereof include resin materials such as (PSS).
The shape of the substrate is not particularly limited, and may be any shape such as a square, a rectangle, a circle, and an ellipse.

5). Other Configurations The electrode chip of the present invention includes the substrate, the electrode, the terminal, and the reagent, but may include other configurations as necessary.
For example, as illustrated in FIG. 6 and FIG. 7, the post-reaction mixture can be accommodated, the electrode 2 is disposed therein, and the reagent 4 is immobilized, and the container 12. And a reagent fixing part 13 for fixing the reagent 4 and a cover part 15 covering the upper surface of the substrate 1.
6 and 7, the electrode chip 10 includes an electrode 2 formed on the substrate 1, a conducting wire 6 and a terminal 3, an insulating layer 7 formed on the conducting wire 6, and the insulating layer 7. A second substrate 8 formed thereon; a dam portion 23 formed on the substrate 1; and a lid portion 15 formed on the second substrate 8. The second substrate 8 includes: The dam portion 23 has an opening portion 13a formed at the same location as the reagent 4 in plan view, and has an opening portion 12a formed at a location corresponding to the storage portion 12. The lid portion 15 has an opening 15a having the same shape as the opening 12a of the second substrate 8 in plan view. Further, FIG. 6 omits the description of the lid for ease of explanation.
By having the housing portion, the electrode tip can stably hold the post-reaction mixture in the measurement region, and can stably measure an electric current value by an electrochemical reaction with respect to the post-reaction mixture. This is because it becomes possible.
In addition, by having the reagent fixing part, the electrode chip can easily form a reagent fixed in the measurement region simply by applying the reagent solution in the reagent fixing part and drying it. is there.
Further, by having the lid portion, it is possible to prevent endotoxin and the like in the air from being mixed, and the electrode chip is excellent in the accuracy of the concentration detection.

Further, as illustrated in FIGS. 8 and 9, the electrode chip may have a flow path 14 having one end connected to the housing portion 12 and the other end connected to the introduction port 11.
This is because the introduction port can be set at an arbitrary place by having the flow path. As a result, endotoxin and the like in the air can be prevented from being mixed, and the electrode tip is excellent in the accuracy of concentration detection.
Further, FIG. 8 omits the description of the lid for ease of explanation.

(1) Accommodating portion The electrode chip can accommodate a post-reaction mixture that is a mixture of the analyte and the reagent components after the end of the free reaction, the electrode is disposed therein, and the reagent is fixed It is possible to have an accommodating portion that is made into a single piece.
By having such a housing portion, the electrode tip can stably hold the post-reaction mixture in the measurement region, and a current value due to an electrochemical reaction stably with respect to the post-reaction mixture. This is because it becomes possible to measure.

The said accommodating part can accommodate the said mixture after reaction.
Such a housing portion may be formed by a groove of the substrate, and is formed by laminating the substrate and a second substrate in which a groove or opening corresponding to the housing portion is formed. Also good.
The constituent material of the second substrate can be the same as that of the first substrate. Further, the substrate and the second substrate may be made of different materials or the same material.
6 to 9 described above are examples in which the accommodating portion 12 is formed by laminating the substrate 1 and the second substrate 8 in which the opening 12a corresponding to the accommodating portion 12 is formed. Is shown.
FIG. 10 is a schematic cross-sectional view showing another example of the electrode chip of the present invention, and shows an example in which the accommodating portion 12 is formed by the groove 12b of the substrate 1.

The shape of the accommodating portion is not particularly limited, and examples of the shape of the accommodating portion in plan view include a circle, an ellipse, and a rectangle.
The cross-sectional shape of the housing portion can be, for example, a semicircular shape, a rectangular shape, or the like.
In the electrochemical endotoxin concentration detection method using a differential pulse voltammetry or the like, since a sample can be measured by introducing several μL to several tens of μL, the capacity of the container is 0.1 mm. preferably ³ in the range of ～2500Mm ^3, among others within the ^{5 mm} 3 500 mm ^3, and particularly preferably in the range of 10 mm ³ 100 mm ^3. As for the size of the accommodating portion, for example, when the shape of the accommodating portion in a plan view is circular, the diameter can be about 1 mm to 5 mm and the depth can be about 1 mm to 10 mm.
6 to 10 described above show examples in which the shape of the accommodating portion 12 is rectangular.

There are no particular limitations on the location where the accommodating portion is formed as long as it can accommodate the post-reaction mixture, can arrange the electrode inside, and can immobilize the reagent. A location where the inner circumference of the housing portion is the same as or inside the outer circumference of the measurement region, that is, the formation location of the housing portion is the same as the measurement region in plan view, and the measurement region is defined by the housing portion. The part where the inner periphery of the storage part includes the outside of the outer periphery of the measurement area, that is, the part where the storage part is formed in a plan view includes a wider range than the measurement area. . In the present invention, in particular, it is preferable that the formation portion of the housing portion is the same as the measurement region. This is because the mixture after the reaction can be stably brought into contact with the electrode. For example, when the method of introducing the analyte is a method of dropping from above the container, the analyte can be easily introduced into the measurement region to form the post-reaction mixture, and the post-reaction mixture This is because can be stably brought into contact with the electrode.
6 to 10 described above, the housing portion 12 includes all the electrodes 2 (the working electrode 2a, the counter electrode 2b, and the reference electrode 2c) in plan view, and further includes an insulating layer 7 that covers the conductive wire 6. An example including a part thereof is shown, and an example in which the formation portion of the housing portion 12 is the same as the measurement region 5 in a plan view is shown.

When the housing portion is formed using a groove of the substrate, or the substrate and a second substrate in which a groove or opening corresponding to the housing portion is formed, the substrate and the substrate The groove forming method for forming the groove or opening in the second substrate can be appropriately selected depending on the constituent material of the substrate and the like.
For example, when the constituent material is silicon, glass, or the like, as the groove forming method, a method of forming the groove using an anisotropic dry etching, wet etching using hydrofluoric acid, or the like. Etc.
Further, when the constituent material is a silicone resin or the like, as the groove portion forming method, a photoresist formed in an inverted shape corresponding to the accommodating portion on a silicon wafer using a normal photolithography method is used as a mold. Forming and pouring a mixture of unpolymerized silicone resin and polymerization initiator into this mold, heating and curing it, and then removing the cured silicone resin from the mold to remove the groove and the like. The method of forming etc. can be mentioned.
When the constituent material is a resin material such as an acrylic resin or an epoxy resin, as the groove portion forming method, a mold in which an uneven shape having an inverted shape corresponding to the housing portion is pressed against the resin material is pressed. Examples include a method of forming the groove and the like by curing the resin material by irradiation or heating with ultraviolet rays or the like, and then peeling the cured product of the resin material from the mold.
The groove forming method may be a method of forming a groove or the like in the constituent material using a laser or the like.
In the case where the housing portion is formed using the substrate and the second substrate, the second substrate can be attached to the substrate using an adhesive or an adhesive.

(2) Reagent fixing part In this invention, it is preferable to have a reagent fixing part which fixes the said reagent.
This is because, by having the reagent fixing part, the electrode chip can easily fix the reagent in the measurement region.
Specifically, as a method for forming the reagent, the reagent fixing part is formed at a position where the reagent is fixed in the measurement region, and then the reagent solution is applied onto the reagent fixing part, It is possible to use a method of drying in a state where the solution is fixed to the reagent fixing part, and the electrode chip can stably fix the reagent in the measurement region.

Here, immobilizing a reagent means that there is a function of retaining a reagent solution containing the components of the reagent in the reagent fixing part, for example, compared with the case where the reagent fixing part is not present, The range in which the reagent solution spreads out can be reduced.
Such a reagent fixing part is formed so as to overlap with the reagent fixing position in plan view, and is formed so as to overlap with the reagent storing part capable of storing the reagent solution, and the reagent fixing position in plan view. Examples thereof include a hydrophilic layer having high wettability with the reagent solution and a hydrophobic layer formed so as to surround the immobilization position of the reagent and having low wettability with the reagent solution.
As said reagent fixing | fixed part, these may be used independently and may be used in combination of multiple. As an example of using a plurality in combination, for example, the above-described reagent fixing part may be one in which the hydrophilic layer is formed on the bottom surface of the reagent containing part.

(A) Reagent storage part The reagent storage part is formed so as to overlap the immobilization position of the reagent in plan view, and can store the reagent solution. In the reagent container that can store the reagent solution, the reagent is formed by drying the reagent solution while being held in the reagent container, and the immobilization position of the reagent is determined by the reagent container. It is what has been.
In addition, since the reagent solution is formed by drying while being held in the reagent container, all of the outer periphery of the reagent is normally in contact with the inner surface of the reagent container in plan view. Is.
As such a reagent storage unit, as shown in FIGS. 6 and 7 already described, the reagent storage unit as the reagent fixing unit 13 has an opening corresponding to the reagent 4 on the substrate 1 and the substrate 1. As shown in FIGS. 8 to 10, a groove portion 13 b formed in the same position as the reagent 4 of the substrate 1 in a plan view is formed. it can.
6 to 10 already described show an example in which a reagent storage unit is formed as the reagent fixing unit 13 in the storage unit 12, and in this case, the volume of the reagent storage unit is as described above. It can be smaller than the volume of the mixture after the reaction.
Further, FIG. 5C already described shows an example in which the storage unit 12 is also used as the reagent storage unit.
The height of the dam portion is not particularly limited as long as it can hold the reagent. When the reagent storage portion is formed in the storage portion, the height of the dam portion is lower than the height of the storage portion. can do. In addition, the height of the dam part and the height of the housing part can be specifically shown by H2 and H1 in FIG.

The plan view shape and the cross-sectional view shape of the reagent storage unit are not limited as long as the reagent solution can be stored, and can be the same as the above “(1) storage unit”.
The capacity of the reagent storage section may be a can accommodate capacity the reagent ^solution, be in the range of 0.1mm ³ ~2500mm 3 is preferable, in the range of ^{5 mm} 3 500 mm ³ it is preferred, particularly preferably in the range of 10 mm ³ 100 mm ^3. Moreover, the capacity | capacitance of the said reagent accommodating part is normally smaller than the capacity | capacitance of the said post-reaction mixture, when the said electrode chip has the said accommodating part.
As the size of the reagent container, for example, when the planar shape of the reagent container is circular, the diameter is in the range of 0.1 mm to 25 mm and the depth is in the range of 0.025 mm to 5 mm. It can be.

The formation part of the reagent storage part may be in the measurement region, and differs depending on the immobilization position of the reagent, but is usually on the surface of the substrate.
For example, as illustrated in FIGS. 6 to 10 described above, the formation portion includes a part of the electrode 2 (a part of the working electrode 2a, the counter electrode 2b, and the reference electrode 2c) in plan view. be able to.

When the reagent container is the dam part, the material for forming the reagent container is not particularly limited as long as the reagent container having a desired shape can be formed and a physical shape can be formed. For example, a thermosetting resin, a photocurable resin, a metal material, or the like can be used alone or in combination.
As a method for forming the dam portion as the reagent storage portion, any method can be used as long as it can form a dam portion capable of storing the reagent. When the formation material is a resin material such as a photocurable resin, etc. Examples thereof include a photolithography method, a screen printing method, a gravure printing method, a flexographic printing method, and an ink jet method.
In addition, when the dam portion is formed using the same material as an insulating layer, an electrode, or a second substrate described later, the forming method is formed simultaneously with the insulating layer, the electrode, or the second substrate. be able to. For example, the reagent container may be formed using the same material as the insulating layer and in the same process as the insulating layer.

  When the reagent container is the groove formed in the substrate, the method for forming the reagent container is not particularly limited as long as the reagent container having a desired shape can be stably formed. Instead, it can be the same as the forming method described in the section “(1) Housing”.

(B) Hydrophilic layer The hydrophilic layer is formed so as to overlap the immobilization position of the reagent in plan view, and has high wettability for the reagent solution.

The degree of hydrophilicity of the hydrophilic layer may be any as long as it has higher hydrophilicity than the periphery of the hydrophilic layer. The reagent solution is usually an aqueous solution in which the components of the reagent are dispersed or dissolved. For this reason, it is because the said reagent solution can be stably fixed on the surface of the said hydrophilic layer because the said hydrophilic layer is a thing whose hydrophilicity is higher than the circumference | surroundings of the said hydrophilic layer.
The static contact angle of pure water on the surface of the hydrophilic layer is, for example, preferably 50 ° or less by the θ / 2 method, particularly preferably 25 ° or less, particularly 1 ° to A range of 20 ° is preferable. This is because, when the static contact angle is within the above range, the hydrophilic layer can stably fix the reagent solution.
The static contact angle is determined by dropping a 1.0 μL pure water droplet on the surface of the object to be measured, and 10 seconds after landing, a straight line connecting the left and right end points and the apex of the dropped droplet to the solid surface. The contact angle is measured according to the θ / 2 method for calculating the contact angle from the angle. As the measuring device, for example, a contact angle meter DM 500 manufactured by Kyowa Interface Science Co., Ltd. can be used. When the size of the measurement target portion is small, a measurement sample can be formed using the same material as the measurement target, and the static contact angle can be measured using this measurement sample.

The hydrophilic layer is not particularly limited as long as it has a desired hydrophilicity. Examples of the hydrophilic layer include those obtained by subjecting each component of the electrode chip to a hydrophilic treatment, a hydrophilic material layer using a hydrophilic material, and the like. Can be used alone or in combination. The hydrophilic layer may be obtained by subjecting the surface of the hydrophilic material layer to a hydrophilic treatment.
Examples of the hydrophilic treatment target include the substrate described above.
Examples of the hydrophilization treatment include a treatment for forming a moth-eye structure having fine protrusions on the substrate surface, a fluorine gas treatment, and the like.
Examples of the hydrophilic material include a hydrophilic resin material and a metal material.
The method for forming the hydrophilic layer may be any method that can form the hydrophilic layer in a desired shape, and may be the same as the method for forming the dam portion as the reagent storage unit. When the hydrophilic layer is formed using the same metal material as the electrode, it can be formed in the same process as the electrode.

The location where the hydrophilic layer is formed may be in the measurement region, and differs depending on the position where the reagent is immobilized. For example, when the immobilization position includes the surface of the substrate, the formation location may include the surface of the substrate. Moreover, when the said fixing position includes on the side surface of the said insulating layer, the said formation location shall include on the side surface of the said insulating layer.
The outer peripheral shape surrounding the outer periphery of the hydrophilic layer can include a portion overlapping the reagent in plan view, and is the same shape as the outer peripheral shape of the reagent immobilization position. It is preferable that the reagent solution has the same shape as the wet spreading region. This is because the hydrophilic layer can stably immobilize the reagent.
The planar shape of the hydrophilic layer may be the one that overlaps the entire surface of the reagent immobilization position in plan view, and has a frame shape that has an opening at a location that overlaps the immobilization position in plan view. There may be.

(C) Hydrophobic layer The hydrophobic layer is formed so as to surround the immobilization position of the reagent, and has low wettability with the reagent solution.

The degree of hydrophobicity of the hydrophobic layer may be any as long as it is more hydrophobic than the periphery of the hydrophobic layer. The reagent solution is usually an aqueous solution in which the components of the reagent are dispersed or dissolved. For this reason, since the hydrophobic layer is higher in hydrophobicity than the periphery of the hydrophobic layer, the reagent solution can be stably immobilized in the region surrounded by the hydrophobic layer.
The static contact angle of pure water on the surface of the hydrophobic layer is preferably greater than 50 ° by the θ / 2 method, for example, preferably 150 ° or less, and particularly 80 ° to 100 °. It is preferably within the range of °. This is because when the static contact angle is within the above range, the hydrophobic layer can stably fix the reagent solution.

The hydrophobic layer is not particularly limited as long as it has a desired hydrophobicity. For example, each of the components of the electrode chip may be subjected to a water repellent treatment.
Examples of the target for the hydrophobic treatment include the substrate and the like.
Examples of the hydrophobic treatment include silicone oil, silicone-based, hydrocarbon-based, fluorocarbon-based, wax-based, polyethyleneimine-octadecyl isocyanate-based, poly (meth) acrylic acid ester-based, polystyrene-based, polyethylene-based, and polypropylene-based resins. May be applied by appropriately dissolving or dispersing in an organic solvent or water. Moreover, water repellency can also be expressed by giving smoothness by grinding | polishing.

The location where the hydrophobic layer is formed is different as long as the region surrounded by the hydrophobic layer is within the measurement region, and differs depending on the immobilization position of the reagent. For example, when the immobilization position includes the surface of the substrate, the formation location may include the surface of the substrate. Moreover, when the said fixing position includes on the side surface of the said insulating layer, the said formation location shall include on the side surface of the said insulating layer.
The shape of the hydrophobic layer in plan view may be a frame shape surrounding the reagent immobilization position, and the outer peripheral shape of the reagent immobilization position is the same as the inner peripheral shape of the hydrophobic layer. It is preferable that That is, because the shape in plan view is the same shape as the area where the reagent solution spreads out, the reagent can be stably immobilized.

(3) Flow path The electrode chip may have a flow path having one end connected to the housing portion and the other end connected to the inlet.
The channel may be formed by a groove of the substrate, or may be formed by stacking the substrate and a second substrate in which a groove or an opening corresponding to the channel is formed.
The width and height of the flow path are not particularly limited as long as the subject can be guided to the housing portion, and can be, for example, about 0.1 mm to 10 mm.
8 and 9 described above are examples in which the flow path 14 is formed by laminating the substrate 1 and the second substrate 8 in which the opening 14a corresponding to the flow path 14 is formed. FIG. 10 shows an example in which the flow path 14 is formed by the groove 14 b of the substrate 1.
The formation method of the flow path can be the same as the formation method described in the section “(1) Housing”.

(4) Conductive wire The electrode tip usually includes a conductive wire that electrically connects the electrode and the terminal.
The material of the conducting wire can be the same as the material of the electrode. The conductive wire material only needs to have conductivity that does not affect the detected current value, and a general conductive material can be used. Among them, from the viewpoint of conductivity, Au, Pt, Ag It is preferably a metal such as Pd, Ni.
The method for forming the conducting wire can be the same as the method for forming the electrode.
Moreover, the said conducting wire may be formed simultaneously with an electrode, and may be formed separately from an electrode.

(5) Insulating layer In this invention, an insulating layer can be formed so that a conducting wire may be covered. The insulating layer can suppress oxidation of the conductive wire and prevent a short circuit.
As the material of the insulating layer, for example, a thermosetting resin, a photocurable resin, or the like can be used.
As the method for forming the insulating layer, any method can be used as long as it can form the insulating layer in a pattern so as to cover the conductive wires and not cover the electrodes and terminals. For example, a photolithography method, a screen printing method, a gravure printing method, etc. , Flexographic printing methods, ink jet methods and the like.

(6) Lid part The said electrode chip can have a cover part which covers the said board | substrate upper surface. By having the lid, it is possible to prevent contamination of endotoxin and the like in the air, and the electrode chip is excellent in the accuracy of concentration detection. Further, it is possible to prevent drying of the specimen and the like, and the electrode chip can detect the endotoxin concentration with high accuracy.

The shape of the lid may be any plate that can cover the top surface of the substrate, and may be a plate.
6 to 10 described above show examples in which the lid portion 15 has a plate shape.

The covering portion of the lid on the upper surface of the substrate may be the upper surface of the substrate, and may include, for example, the measurement region.
Moreover, when the said electrode chip has the said accommodating part and the said flow path, the said coating | coated location can include the area | region in which the said accommodating part and the said flow path are formed.
In this invention, it is preferable that the said cover part is what the said terminal exposes. This is because the electrode chip can be measured in a state where the upper surface of the substrate is covered with the lid.
In FIGS. 8 to 10 already described, the lid portion 15 covers the accommodating portion 12 and the flow path 14.

The lid portion may have a through hole. A through hole can be used as the inlet.
The size of the through hole may be any size as long as the subject can be introduced. As illustrated in FIG. 7, the size of the through hole 15 a may be the same as the size of the housing portion 12 in plan view.
The shape of the through hole is not particularly limited, but the shape of the through hole in plan view is preferably a circle or an ellipse because it is easy to form the through hole.
6 and 7 described above show an example in which the lid portion 15 has a through-hole 15a and is used as the introduction port 11. FIG.

  The lid portion may be recloseable. By opening the lid and introducing the analyte into the electrode chip and then closing it again, contamination of endotoxin and the like in the air can be prevented, and the electrode chip is excellent in the accuracy of concentration detection. Because it can be.

(7) Second substrate In the present invention, a second substrate may be disposed on the substrate. The second substrate is provided to form the housing part, the flow path, and the like.
The second substrate can have an opening corresponding to a storage portion or the like. The size and shape of the opening can be the same as the size and shape of the housing and the like.
6 to 10 already described show examples having an opening 12a corresponding to the accommodating portion 12, an opening 14a corresponding to the flow path 14, and the like.

The second substrate is not particularly limited as long as it has insulating properties, and examples thereof include a resin substrate.
The shape of the second substrate is not particularly limited, and may be an arbitrary shape such as a square, a rectangle, a circle, or an ellipse.

The second substrate is disposed on the substrate so that at least the terminals are exposed and the opening of the second substrate is positioned on the electrode.
The said 2nd board | substrate can be affixed on a board | substrate via an adhesive agent or an adhesive, for example.

6). Method for Using Electrode Tip An example of the endotoxin concentration detection method using the electrode tip of the present invention will be described.
For example, in the case of an electrode chip as illustrated in FIGS. 1 and 2, the detection method described above includes subjecting an analyte containing endotoxin onto the reagent 4, the electrode 2, the substrate 1, etc. in the measurement region 5. The reagent 4 is dissolved in the analyte to form a pre-reaction mixture that is a mixture of the analyte and the reagent components, and then the pre-reaction mixture is allowed to proceed with a free reaction for a certain period of time. Next, a method for quantifying the endotoxin concentration based on a current value measured by an electrochemical reaction can be used.
Further, in the electrode tip as illustrated in FIGS. 6 and 7, as a method for forming the pre-reaction mixture, a specimen containing endotoxin is introduced from the introduction port 11 of the electrode tip 10 and is introduced into the channel 14 by capillary action. Inflow and move to the storage unit 12. Thereafter, by bringing the analyte into contact with the reagent, the reagent 4 is dissolved in the analyte, and a method of forming a pre-reaction mixture that is a mixture of the analyte and the components of the reagent can be used.

  Specific examples of the subject include pharmaceuticals such as a dialysis solution, an injection, a transplanted tissue piece, and a culture solution of a fertilized egg of artificial insemination, a medical device, and the like. In addition, when the said test substance is solid, what was melt | dissolved or disperse | distributed in suitable solvents, such as a buffer solution, can be used as a test object.

In order to activate the reaction during the formation of the post-reaction mixture, that is, during the free reaction, it is preferable to warm the pre-reaction mixture. The reaction temperature of the above free reaction is preferably in the range of 20 ° C. to 50 ° C., more preferably in the range of 25 ° C. to 45 ° C., and particularly preferably about 37 ° C.
The reaction time is preferably 30 minutes or longer, more preferably 1 hour or longer, and even more preferably 2 hours or longer. Thereby, a sufficient amount of free redox substances can be obtained.
Incidentally, within the capacity of the post-reaction mixture is 0.1mm ³ ~1000mm ^3, in particular in the range of 0.1 mm ³ 100 mm ^3, if more small, such as in the range of 0.1mm ³ ~50mm ³ is The reaction temperature can be about 30 ° C. to 40 ° C., and the reaction time can be about 15 minutes to 1 hour.

In the quantification method, the endotoxin concentration is quantified in the post-reaction mixture based on the current value measured by an electrochemical reaction. That is, the redox substance released from the peptide compound is present in the mixture of the analyte and the reagent components after the release reaction, and the redox substance undergoes an oxidation reaction or a reduction reaction at a specific potential. There is a correlation between the current value resulting from this oxidation reaction or reduction reaction and the concentration of the redox substance, that is, the concentration of endotoxin, and the concentration of endotoxin is quantified using this correlation.
Note that the measurement by the electrochemical reaction with respect to the mixture after the reaction is not limited to the oxidation-reduction reaction of the redox substance itself released from the peptide compound with an electrode, but the reaction with the oxidation-reduction substance. It also includes those in which the second redox substance after reacting with the redox substance is subjected to an oxidation-reduction reaction with an electrode using a possible second redox substance.

  The measurement method by electrochemical reaction is not particularly limited as long as it is a method capable of quantifying the concentration of endotoxin based on the current value measured by electrochemical reaction, and examples thereof include amperometry and voltammetry. Examples of the amperometry method include the chronoamperometry method. Examples of the voltammetry method include a normal pulse voltammetry method, a differential pulse voltammetry method, a cyclic voltammetry method, and a linear sweep voltammetry method. Among these, since the measurement is simple, the amperometry method is preferable as the measurement method.

  In measurement by the amperometry method, for example, an electrode is placed in the mixture after the reaction, and measurement based on the amperometry method is performed. That is, the flowing current is measured with a constant potential applied to the working electrode. The potential is controlled with respect to the reference electrode, and current flows between the working electrode and the counter electrode. When the current value is small, the reference electrode may be provided with the role of the counter electrode without providing the counter electrode. By using a graph in which the voltage application time is plotted on the horizontal axis and the current value is plotted on the vertical axis, the current after a certain time has elapsed since the application of the constant potential to the electrode is measured. The endotoxin concentration can be calculated from the measured current value by preparing in advance a calibration curve showing the correlation between the endotoxin concentration and the current value after a predetermined time.

B. Next, a method for manufacturing an electrode chip according to the present invention will be described.
An electrode chip manufacturing method according to the present invention includes a substrate, an electrode formed on one surface of the substrate, and a terminal formed on one surface of the substrate and connected to the electrode. A layered body preparing step for preparing a body, a coating step for coating a reagent solution containing a peptide compound in which a factor C and a redox substance are bound on one surface of the substrate after the layered body preparing step, and the reagent A drying step of drying the solution to form a reagent, wherein the coating step applies the reagent solution in a measurement region in which the electrode is arranged and a current value is measured by an electrochemical reaction. It is characterized by being.

Such a method for manufacturing an electrode tip according to the present invention will be described with reference to the drawings. FIG. 11 is a process diagram showing an example of a method for producing an electrode chip according to the present invention. As illustrated in FIG. 11, the electrode chip manufacturing method of the present invention is formed on a substrate 1, an electrode 2 formed on one surface of the substrate 1, and one surface of the substrate 1, A laminate 20 having a terminal 3 connected to the electrode 2 is prepared (FIG. 11 (a)), and then a peptide compound in which a factor C and a redox substance are bonded to one surface of the substrate 1; The reagent solution 4a containing is applied to the measurement region 5 where the electrode 2 is arranged and the current value is measured by an electrochemical reaction (FIG. 11B), and then applied to the measurement region 5. In addition, the reagent solution 4a is dried to form a reagent (FIG. 11 (c)), thereby obtaining the electrode chip 10 on which the reagent 4 is immobilized (FIG. 11 (d)).
11A shows the laminate preparation step, FIG. 11B shows the coating step, and FIGS. 11C and 11D show the drying step.
Note that the reference numerals in FIG. 11 indicate the same members as those in FIGS. 6 and 7 already described, and thus the description thereof is omitted here.

  According to the present invention, by having the coating step and the drying step, it is possible to easily manufacture an electrode chip in which a reagent containing a factor C and a peptide compound in which a redox substance is bound is immobilized in the measurement region. .

The manufacturing method of the electrode chip of this invention has the said laminated body preparation process, the said application | coating process, and the said drying process.
Hereinafter, each process of the manufacturing method of the electrode chip of this invention is demonstrated in detail.

1. Laminate Preparation Step The laminate preparation step in the present invention includes a substrate, an electrode formed on one surface of the substrate, a terminal formed on one surface of the substrate and connected to the electrode, It is the process of preparing the laminated body which has this.
The substrate, electrodes and terminals used in this step, and the method for forming the electrodes and terminals can be the same as the contents described in the above section “A. Electrode chip”, and thus the description thereof is omitted here. .

2. Application Step The application step in the present invention is a step of applying a reagent solution containing a C compound and a peptide compound having a redox substance bound thereto on one surface of the substrate after the laminate preparation step.
Moreover, the said application | coating process is a process of apply | coating the said reagent solution in the measurement area | region where the said electrode is arrange | positioned and the measurement of the electric current value by an electrochemical reaction is performed.
The factor C and the peptide compound used in this step can be the same as those described in the above section “A. Electrode chip”, and thus the description thereof is omitted here.

The reagent solution contains the factor C and the peptide compound, and usually contains a solvent that disperses or dissolves the factor C and the peptide compound.
The solvent is not particularly limited as long as it can stably disperse or dissolve both of the factor C and the peptide compound, and examples thereof include distilled water and a buffer solution.
Examples of the buffer solution include the buffer solution described in the above section “A. Electrode chip”.
The reagent solution can contain other components as necessary. Examples of such other components include factor B, coagulase precursors and other components described in the above section “A. Electrode chip”.

The reagent solution may be prepared by any method as long as it can stably disperse or dissolve the factor C and the peptide compound in the solvent. For example, the peptide compound is contained in a container containing the factor C. And a method of adding and mixing the above solvent.
The mixing method may be any method that can sufficiently mix the above components, and examples thereof include a vibration treatment that applies vibration to the container.

  The method for applying the reagent solution is not particularly limited as long as it is an application method that can apply the reagent solution in the measurement region, and the method described in “1. Reagent” of “A. The application | coating method as described in an item can be mentioned.

3. Drying step The drying step in the present invention is a step of drying the reagent solution to form a reagent.
The drying method in this step is not particularly limited as long as it is a drying method capable of forming the reagent by drying the reagent solution. For example, it is described in the section “1. Reagent” in “A. Electrode chip”. The drying method can be mentioned.
The reagent formed in this step can be the same as that described in the section “1. Reagent” of “A. Electrode chip”, and the description thereof is omitted here.

4). Electrode Chip Manufacturing Method The electrode chip manufacturing method of the present invention includes the laminate preparation step, the coating step, and the drying step, but may include other steps as necessary. .
As said other process, when the said electrode chip has an accommodating part, a reagent fixing | fixed part, etc., the accommodating part formation process performed before the said application | coating process, a reagent fixing | fixed part forming process, etc. can be mentioned.
Moreover, when the said electrode chip has a cover part, the said other process shall have a cover part arrangement | positioning process which arrange | positions the said cover part after the said application | coating process.

  The electrode chip manufactured by the electrode chip manufacturing method of the present invention can be the same as that described in the above section “A. Electrode chip”, and thus the description thereof is omitted here.

C. Endotoxin concentration measuring apparatus Next, the endotoxin concentration measuring apparatus of the present invention will be described.
The endotoxin concentration measuring device of the present invention is an endotoxin concentration measuring device having a connection terminal portion used for electrical connection with the terminal of the electrode chip, and the electrode chip is disposed on the substrate and one surface of the substrate. The formed electrode, the terminal formed on one surface of the substrate and connected to the electrode, and the reagent immobilized on the one surface of the substrate, the reagent, It comprises a peptide compound in which factor C and a redox substance are bound, and further, the electrode is disposed and is immobilized in a measurement region where a current value is measured by an electrochemical reaction. is there.

  Such an endotoxin concentration measuring apparatus of the present invention will be described with reference to the drawings. FIG. 12 is a schematic view showing an example of the electrode tip 30 of the present invention. As illustrated in FIG. 12, the endotoxin concentration measuring device 30 of the present invention includes a connection terminal portion 31 used for electrical connection with a terminal of an electrode chip, and the electrode chip includes a substrate, the above-mentioned An electrode formed on one surface of the substrate, the terminal formed on one surface of the substrate and connected to the electrode, and a reagent immobilized on the one surface of the substrate. The reagent includes a factor C and a peptide compound in which a redox substance is bound, and the electrode is disposed and is immobilized in a measurement region in which a current value is measured by an electrochemical reaction. Is.

  FIG. 13 is a process diagram showing an example of a method for using the endotoxin concentration measuring apparatus of the present invention. As illustrated in FIG. 13, the endotoxin concentration measuring device of the present invention is prepared by preparing the endotoxin concentration measuring device 30 of the present invention and the electrode chip 10, and using the electrode chip 10 as the endotoxin concentration measuring device. 30 is pressed with a predetermined force p (FIG. 13A) to bring the terminal 3 of the electrode chip 10 into contact with the connection terminal portion 31 of the endotoxin concentration measuring device 30, and then the electrochemical reaction The endotoxin concentration can be quantified based on the current value measured by (Fig. 13 (b)).

  According to the present invention, the endotoxin concentration measuring apparatus can be used to detect the endotoxin concentration with high accuracy by being used together with the above-described electrode chip.

The endotoxin concentration measuring apparatus of the present invention has the connection terminal portion.
Hereafter, each structure of the endotoxin concentration measuring apparatus of this invention is demonstrated in detail.

1. Connection terminal part The connection terminal part in this invention is used for electrical connection with the terminal of an electrode chip.
Further, the number of connection terminals included in the connection terminal portion varies depending on the number of terminals of the electrode chip, and when the electrode of the electrode chip is a two-electrode type electrode, the number is two. In the case where the electrode of the electrode tip is a three-electrode type electrode, the number can be three.
As a constituent material of the connection terminal portion, for example, the same material as that of the electrode described in the section “A. Electrode chip” can be used.

  The number of the connection terminal portions may be at least 1 or more per one endotoxin concentration measuring device, but may be 2 or more. This is because when the number of the connection terminal portions is 2 or more, the endotoxin concentration can be simultaneously measured using two or more electrode chips.

  The electrode chip can be the same as that described in the section “A. Electrode chip”, and the description is omitted here.

2. Endotoxin concentration measuring apparatus The endotoxin concentration measuring apparatus of the present invention has the connection terminal portion.
Such an endotoxin concentration measuring device is not particularly limited as long as it can quantitate the concentration of endotoxin based on the current value measured by an electrochemical reaction, and a device generally used for electrochemical measurement is not limited. Examples thereof include a potentiostat, a current amplifier, and a device having a function equivalent to these.

  The present invention is not limited to the above embodiment. The above-described embodiment is an exemplification, and the present invention has substantially the same configuration as the technical idea described in the claims of the present invention, and any device that exhibits the same function and effect is the present invention. It is included in the technical scope of the invention.

  Hereinafter, the present invention will be specifically described using examples and comparative examples.

[Preparation]
1. Reagent for measurement The reagent for measurement used in the present Example is as follows.

(1) Standard endotoxin solution The endotoxin standard product is manufactured by Seikagaku Corporation E.I. Coli O113: USP Reference Standard Endotoxin derived from H10 strain was used. The standard endotoxin solution was prepared using endotoxin-free water attached to Endospecy ES24S manufactured by Seikagaku Corporation, and vigorously stirred for 30 minutes immediately before use. The endotoxin concentration was shown as the final concentration in the measurement solution.

(2) Lysate reagent As the lysate reagent, Endspecy ES24S manufactured by Seikagaku Corporation, in which the lysate reagent was sealed in a dedicated test tube for each test in a lyophilized state, was used.

(3) Peptide compound p-aminophenol (pAP) was used as the redox substance, and Boc-Leu-Gly-Arg-pAP (LGR-pAP) was used as the peptide compound.
LGR-pAP was dissolved in water for injection manufactured by Otsuka Pharmaceutical Co., Ltd. and stored at −20 ° C. as a 10 mM stock solution.

(4) Buffer solution The following buffer solutions a to d were prepared as buffer solutions.
(A) Buffer solution a: Buffer solution attached to Endospecy manufactured by Seikagaku Corporation (b) Buffer solution b: Tris-HCl (after dissolving Tris-hydroxymethylaminomethane (MW 121.14 g / mol) in ultrapure water ( 0.1M), pH adjusted to 7.9 with hydrochloric acid.)
(C) Buffer c: PBS (The pH is adjusted to 7.9 after dissolving commercially available Dulbecco's phosphate buffer (-) in 500 ml of ultrapure water.)
(D) Buffer d: HEPES (MW: 121.14 g / mol) and KCl (MW 74.55 g / mol) are dissolved in ultrapure water (0.1 M), and the pH is adjusted to 7 with an aqueous NaOH solution. Adjusted to .9. )

2. Apparatus and Measurement Method The apparatus and measurement method used in the electrochemical measurement method in this example are as follows.

(1) Measuring device Potentiostat CompactStat made by Ivium Technologies

(2) Measurement method Unless otherwise specified, in cyclic voltammetry (CV), the scanning speed was set to 20 mV / s, and the scanning range was changed from 0 V to 0.5 V.

[Example 1]
1. Preparation of electrode chip (1) Formation of conductive wire and terminal Conductive wire and terminal by applying Ag paste in a pattern on a PET substrate (Toray Industries, Lumirror 350H10) by screen printing and baking at 130 ° C. for 30 minutes Formed.

(2) Formation of electrode Next, a carbon paste was applied in a pattern on the PET substrate by screen printing and baked at 120 ° C for 15 minutes to form a working electrode and a counter electrode. Further, an Ag / AgCl paste made by Lhasa Industries was applied in a pattern on the PET substrate by screen printing, and baked at 80 ° C. for 10 minutes to form a reference electrode.

(3) Formation of insulating layer Next, for the purpose of covering the conductive wire, a UV curable resin is applied to the PET base material in a pattern by screen printing, and exposed for 100 seconds with a Deep UV lamp manufactured by USHIO. Cured.

(4) Formation of flow path and accommodating portion A 25 μm thick acrylic adhesive sheet was bonded to both surfaces of a 100 μm thick PET base material (A4100, manufactured by Toray Industries, Inc.) to form a laminated base material (second substrate). Next, the laminated base material is cut with a cutting machine manufactured by GRAPHTEC in order to form a flow path, an accommodating portion and an introduction port through which the analyte liquid passes, and the flow path, the accommodating portion and the introduction of the laminated base material are cut. By removing a region corresponding to the mouth, an opening portion corresponding to the flow path, the accommodating portion, and the introduction port was formed.
Next, the laminated base material (second substrate) in which openings corresponding to the flow path, the accommodating portion, and the introduction port are formed is replaced with the laminator on the PET base material (substrate) in which the above-described conductive wires, terminals, electrodes, and the like are formed. Were laminated together to form a flow path and a housing part having a height of 150 μm.

(5) Immobilization of Reagent Next, 10 mM LGR-pAP (20 μL) and buffer solution a (180 μL) were added to a test tube containing a lysate reagent in a lyophilized state and stirred to obtain a reagent solution. Next, 50 μL of the reagent solution was dropped onto the electrode arranged in the container using a dispenser, and then freeze-dried to remove water. As a result, an electrode chip having the reagent immobilized in the measurement region was formed.
Next, a hydrophilic cover film (lid part) manufactured by 3M Healthcare Co., Ltd. was bonded to the laminated base material so as to cover the flow path and the housing part, thereby obtaining an electrode chip.
The lyophilization was performed for a minimum of 3 hours. The lyophilization conditions were set to −30 ° C. or lower and 30 Pa or lower. Specifically, it was set to −44 ° C. and 12 Pa.

[Comparative Example 1]
An electrode chip was obtained in the same manner as in Example 1 except that the reagent solution was dropped on the electrode and then not lyophilized, that is, the reagent solution was protected in the form of a solution.

[Evaluation]
50 μL of an endotoxin standard solution having an endotoxin concentration of 500 EU / L is introduced from the inlet of the electrode tip obtained in Example 1, and the reaction is performed at 37 ° C. for a maximum of 60 minutes, and 10 minutes, 30 minutes, and 60 minutes after the start of the reaction. At this timing, the endotoxin concentration was detected by the cyclic voltammetry method (CV method).
In Comparative Example 1, a reaction solution was formed in the same manner as in the evaluation method of Example 1 except that 50 μL of an endotoxin standard solution having an endotoxin concentration of 1000 EU / L was added from the electrode chip inlet. The metaly characteristics were measured and the maximum output current value was measured.

In FIG. 14 (Example 1) and FIG. 15 (Comparative Example 1), the reaction time was set to (A) 10 minutes, (B) 30 minutes, and (C) 60 minutes, respectively, and an endotoxin standard solution having a concentration of 500 EU / L. The voltammogram when reacting with lysate reagent is shown. A peak derived from the oxidation reaction of pAP released from LGR-pMA by the LAL cascade reaction was confirmed around 0.3V.
16 and 17 show the voltammograms of Example 1 and Comparative Example 1 with reaction times of 30 minutes and 60 minutes, respectively.
Further, Table 1 and FIG. 18 show a table and a graph showing the peak current value (nA) with respect to the reaction time (min).

  14 to 18 and Table 1, it was confirmed that Example 1 had a faster reaction rate than that of Comparative Example 1. It was also confirmed that the output current sensitivity was high.

[Example 2]
1. Production of electrode chip (1) Production of substrate with electrode After forming a 50 nm palladium (Pd) film on the PET substrate (substrate) by sputtering, etching is performed by photolithography to form a working electrode and a counter electrode. Except for the above, a PET base material (substrate) was formed in the same manner as in Example 1, in which conductive wires, terminals, electrodes, and the like were formed, and in addition, the formation of flow paths and storage portions was formed.

(2) Immobilization of Reagent Next, 10 mM LGR-pAP (20 μL) and buffer solution a (180 μL) were added to a test tube containing the lysate reagent in a lyophilized state and stirred to obtain a reagent solution. Next, 50 μL of the reagent solution was dropped onto the electrode arranged in the container using a dispenser, and then freeze-dried to remove water. As a result, an electrode chip having the reagent immobilized in the measurement region was formed.
Next, a hydrophilic cover film (lid part) manufactured by 3M Healthcare Co., Ltd. was bonded to the laminated base material so as to cover the flow path and the housing part, thereby obtaining an electrode chip.

2. Evaluation 50 μL of an endotoxin standard solution having an endotoxin concentration of 500 EU / L is introduced from the inlet of the electrode tip obtained in Example 2, and the reaction is performed at 37 ° C. for a maximum of 60 minutes. The endotoxin concentration was detected by the cyclic voltammetry method (CV method) at the timing of minutes.
As a result, a current value similar to that in Example 1 was obtained. That is, substantially the same current value as that of Example 1 in FIGS.

[Example 3]
1. Preparation of Electrode Chip Buffer b (270 μL) and 10 mM LGR-pAP (30 μL) were mixed, 200 μL of which was added to a test tube containing a lysate reagent in a lyophilized state, and stirred to obtain a reagent solution.
Next, an electrode tip was obtained in the same manner as in Example 1 except that 50 μL of the reagent solution was dropped on the electrode.

2. Evaluation 50 μL of an endotoxin standard solution having an endotoxin concentration of 1000 EU / L is introduced from the electrode chip inlet obtained in Example 3, and the reaction is performed at 37 ° C. for a maximum of 60 minutes. After the reaction starts, 0 minutes, 15 minutes, and 30 minutes. The linear sweep voltammetry characteristics were measured at the timing of 60 minutes, and the maximum output current value was measured. The voltage range at the time of linear sweep voltammetry measurement was set to 0V → 0.4V. The resolution was adjusted as appropriate. The results are shown in FIG.

[Comparative Example 2]
1. Preparation of reagent An electrode tip was obtained in the same manner as in Example 3 except that 25 μL of the reagent solution was dropped on the electrode and lyophilization was not performed, that is, the reagent solution was protected in the form of a solution. .

2. Evaluation The maximum output current value was measured in the same manner as in Example 3 except that 25 μL of an endotoxin standard solution having an endotoxin concentration of 2000 EU / L was introduced from the inlet of the electrode chip obtained in Comparative Example 2. The results are shown in FIG.

[Example 4]
Except that the buffer solution c was used instead of the buffer solution b, an electrode tip was formed in the same manner as in Example 3, the linear sweep voltammetry characteristics were measured, and the maximum output current value was measured. The results are shown in FIG.

[Comparative Example 3]
An electrode tip was formed in the same manner as in Comparative Example 2 except that the buffer c was used instead of the buffer b, the linear sweep voltammetry characteristics were measured, and the maximum output current value was measured. The results are shown in FIG.

[Example 5]
An electrode tip was formed in the same manner as in Example 3 except that the buffer solution d was used instead of the buffer solution b, the linear sweep voltammetry characteristics were measured, and the maximum output current value was measured. The results are shown in FIG.

[Comparative Example 4]
An electrode tip was formed in the same manner as in Comparative Example 2 except that the buffer d was used instead of the buffer b, the linear sweep voltammetry characteristics were measured, and the maximum output current value was measured. The results are shown in FIG.

[Summary of Examples 3 to 5 and Comparative Examples 2 to 4]
19 to 21, it was confirmed that Examples 3, 4 and 5 had higher output current sensitivity than Comparative Examples 2, 3 and 4. It was confirmed that by lyophilizing the reagent solution containing the buffer solution, the CV characteristic oxidation output current significantly increased as compared with the reagent without lyophilization. Moreover, it has confirmed that the same effect was acquired in the wide buffer solution.

DESCRIPTION OF SYMBOLS 1 ... Board | substrate 2 ... Electrode 3 ... Terminal 4 ... Reagent 5 ... Measurement area | region 6 ... Conductor 7 ... Insulating layer 8 ... 2nd board | substrate 10 ... Electrode chip 11 ... Inlet 12 ... Storage part 13 ... Reagent fixing | fixed part 14 ... Channel 15 … Lid 15a… Through hole 20… Laminate 30… Endotoxin concentration measuring device 31… Connection terminal

Claims (7)

A substrate,
An electrode including at least a working electrode and a counter electrode formed on one surface of the substrate;
A terminal formed on one surface of the substrate and connected to the electrode;
A reagent immobilized on one surface of the substrate;
An accommodation part formed on one surface of the substrate, the electrode and the reagent being arranged, and having a height capable of accommodating a predetermined capacity;
Have
The reagent includes a factor C and a peptide compound to which a redox substance is bound, and further, the electrode is disposed without contacting the electrode in a measurement region in which a current value is measured by an electrochemical reaction. An electrode chip that is fixed.   The electrode chip according to claim 1, wherein the reagent contains a buffer component.   The electrode tip according to claim 2, wherein the reagent has a porous structure. The electrode tip according to claim 1 , wherein the measurement region is defined by the housing portion . The electrode chip according to any one of claims 1 to 4 , wherein the storage part includes a dam part that holds the reagent having a height lower than the height of the storage part .   The electrode chip according to any one of claims 1 to 5, further comprising a lid that covers the upper surface of the substrate. A substrate; an electrode including at least a working electrode and a counter electrode formed on one surface of the substrate ; a terminal formed on one surface of the substrate and connected to the electrode; and one surface of the substrate A laminated body preparing step of preparing a laminated body formed on the electrode and the reagent, and having a container having a height capable of accommodating a predetermined capacity ;
After the laminate preparation step, an application step of applying a reagent solution containing a peptide compound in which a factor C and a redox substance are bonded to one surface of the substrate;
A drying step of drying the reagent solution to form a reagent;
Have
The electrode tip is characterized in that the application step is to apply the reagent solution without contact with the electrode in a measurement region where the electrode is arranged and a current value is measured by an electrochemical reaction. Manufacturing method.