JP2018072331A - Endotoxin detection chip and endotoxin density quantitative method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an endotoxin detection chip capable of stably detecting 1EU/L of endotoxin.SOLUTION: An endotoxin detection chip at least includes: a support base material 2; an electrode part 6 formed on the support base material and having two action electrodes; and a terminal part 8 formed on the support base material and connected to the electrode part. The two action electrodes 3 are alternate comb-shaped electrodes, and an amplification factor at inducing redox cycling is 5 times or more.SELECTED DRAWING: Figure 1

Description

  The present invention relates to an endotoxin detection chip capable of stably detecting, for example, 1 EU / L 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 such as dialysis solution, injections, transplanted tissue pieces, cultured solutions of fertilized eggs of artificial insemination and medical devices are contaminated with microbial contaminants such as endotoxin, even a trace amount can cause serious results. The amount of contamination of microbial contaminants such as endotoxin must be strictly controlled. However, microbial contaminants such as endotoxins are ubiquitous in the environment, and furthermore have 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 and a turbidimetric method, a colorimetric method, and a fluorescence method are generally known. In either method, a complicated procedure and a long time are required to perform highly accurate detection.

  In particular, for dialysate for artificial dialysis, the dialysate water quality standard for endotoxin activity in dialysate is specified to be less than 1 EU / L, and a rapid and highly accurate endotoxin detection method is required. The reason is that, as a new technology, hemodiafiltration (HDF therapy) has been developed. By adding dialysate into the blood and filtering the increased amount with the dialysate, low molecular weight proteins that could not be removed by conventional dialysis technology It is possible to remove unnecessary substances such as, but in online HDF, a large amount of normal dialysis fluid is put into the body as a replacement fluid, so the management of dialysis water (RO water) and dialysis fluid at each dialysis facility is very This is because it became important. Therefore, it is very important to measure 1 EU / L endotoxin simply, quickly and with high accuracy.

  As a method that can be used to detect 1 EU / L endotoxin currently practiced in the medical field, a colorimetric method using an endotoxin standard solution and a lysate reagent can be exemplified, but even if an endotoxin test kit is used. Since the operation is complicated, it is necessary to master the accurate measurement. In addition, since the measurement time requires at least 30 minutes and cannot be shortened any further, a simpler and quicker endotoxin detection method is required.

  Furthermore, in the above colorimetric method, since the reagent (lysate reagent) prepared from the blood cell extract component of horseshoe crab is expensive, it is difficult to carry out the number of measurements necessary for prevention management of endotoxin contamination.

  In addition, since the colorimetric method measures absorbance at 545 nm, there is a problem in that an object with low transparency and an object containing multiple components cannot be measured.

  Therefore, the present inventors can reduce the amount of expensive lysate reagent used, compared to a colorimetric method for a sample containing only a trace amount of endotoxin or a sample that cannot be measured by a colorimetric method. An endotoxin detection method for detecting endotoxin by an electrochemical reaction that enables quantification of endotoxin at low cost and simply and an endotoxin detection chip used therefor have been found (Patent Document 1).

Japanese Patent No. 5561613

However, when the electrochemical detection method using the electrode tip shown in Patent Document 1 is used, there is a problem that 1 EU / L endotoxin may not be detected stably.
The present invention has been made in view of the above problems. For example, it is a main object of the present invention to provide an endotoxin detection chip capable of stably detecting 1 EU / L endotoxin.

As a result of repeated studies to solve the above problems, the present inventors can detect a small amount of current even when the endotoxin concentration is 0 EU / L when the current measuring device is connected to an electrode chip. In addition, when the endotoxin concentration is a concentration such as 1 EU / L as currently required in the medical field, the current value output from endotoxin is the endotoxin concentration of 0 EU / L. It was newly found that the current value may be lower than the detected current value in some cases.
Specifically, when an electrode chip having only one working electrode is used, when a sample with an endotoxin concentration of 0 EU / L is measured, a current value of 25 nA is detected, whereas an endotoxin concentration of 1 EU / L is detected. It was newly discovered that the current value output from the power source is about 10 nA, which may be less than half the noise.

  In order to achieve the above object, the present invention provides a support base material, an electrode portion formed on the support base material and having two working electrodes, and formed on the support base material and connected to the electrode portion. An endotoxin detection chip having at least a terminal portion, wherein the two working electrodes are alternating comb electrodes, and the amplification factor upon induction of redox cycling is 5 times or more. Providing chips.

  According to the present invention, since the amplification factor at the time of induction of redox cycling is 5 times or more, for example, it is possible to stably detect a current value output derived from 1 EU / L endotoxin.

In the present invention, the area of the two working electrodes is preferably 2 mm ² or more. This is because the endotoxin detection chip of the present invention can easily detect endotoxin and can be easily reduced in size because the area is in the above range.

  In the present invention, it is preferable that a reagent containing a factor C and a peptide compound to which a redox substance is bound is immobilized in a measurement region in which the comb tooth portion of the comb electrode used as the working electrode is disposed. . By immobilizing the reagent in the measurement region, simply introducing the analyte into the endotoxin detection chip, the mixture after the reaction to be measured for the current value in the electrochemical reaction, that is, for example, It becomes possible to obtain a mixture of the analyte, the activated factor C, the unactivated factor C, the peptide compound, the redox substance, and the peptide compound to which the unreacted redox substance is bound. Therefore, the endotoxin detection chip can easily detect, for example, 1 EU / L endotoxin.

  In this invention, it is preferable that the said reagent contains the composition for multistage reactions containing the said C factor, B factor, and a coagulation enzyme precursor. This is because the redox substance can be efficiently released from the peptide compound.

  In the present invention, the reagent is preferably an LAL reagent. This is because endotoxin can be detected with high accuracy.

In the present invention, a reaction current value when the concentration of endotoxin using the subject sample is 0EU / L and C _A, a reaction current value when the concentration of endotoxin using the subject sample is 1 EU / L C _In the case of _B , it is preferable that the value of C _B / C _A in a state where the subject sample is held in the measurement region for 35 minutes is 1.5 or more. This is because endotoxin can be detected with high accuracy.

  In the present invention, the material for the two working electrodes is preferably Pd, and the reagent is preferably an LAL reagent. This is because endotoxin can be detected with high accuracy.

  In the present invention, it is preferable to have an accommodating portion that can accommodate a subject, and the accommodating portion has the electrode portion disposed therein. By forming the housing portion, the endotoxin detection chip can stably hold the analyte in the measurement region, and can stably maintain a current value due to an electrochemical reaction with respect to the analyte. This is because measurement is possible.

  In the present invention, there is provided a method for quantifying endotoxin concentration, characterized by using the aforementioned endotoxin detection chip.

  According to the present invention, the endotoxin concentration can be accurately quantified by using the above-described endotoxin detection chip.

  In the present invention, the endotoxin detection chip is fixed with a reagent containing a C compound and a peptide compound in which a redox substance is bound in a measurement region where the comb teeth of the comb electrode used as the working electrode are arranged. The material of the two working electrodes is Pd, the reagent is an LAL reagent, and the current value immediately before pulse application in the oxidation potential side current obtained by measurement by the amperometry method, It is preferable to quantify the endotoxin concentration by using the difference from the current value after application as the reaction current value. This is because the endotoxin concentration can be accurately quantified.

  The present invention has an effect that, for example, an endotoxin detection chip capable of stably detecting 1 EU / L endotoxin can be provided.

It is the schematic plan view which shows an example of the chip | tip for an endotoxin detection of this invention, and the schematic sectional drawing in the AA line. It is the schematic plan view which shows the other example of the chip | tip for an endotoxin detection of this invention, and the schematic sectional drawing in CC line. It is the schematic plan view which shows the other example of the chip | tip for an endotoxin detection of this invention, and the schematic sectional drawing in the DD line. It is explanatory drawing explaining the amplification factor in this invention. It is explanatory drawing explaining the comb-shaped electrode in this invention. It is a schematic diagram which shows an example of the multistage reaction in this invention. It is a schematic diagram which shows the other example of the multistage reaction in this invention. 2 is a cyclic voltammogram of evaluation 1 in Example 1. FIG. 3 is an amperogram of evaluation 2 in Example 1. 4 is an amperogram of evaluation 3 in Example 1. 4 is an amperogram of evaluations 5 and 6 in Example 2. FIG. 4 is a graph showing the relationship between time (minutes) and reaction current (nA) in Example 2. 10 is an amperogram of evaluation 8 in Example 3. 10 is an amperogram of evaluation 9 in Example 3.

  Hereinafter, the endotoxin detection chip and endotoxin concentration quantification method of the present invention will be described in detail.

A. Endotoxin Detection Chip The endotoxin detection chip of the present invention comprises a support base material, an electrode portion formed on the support base material and having two working electrodes, and formed on the support base material. An endotoxin detection chip having at least connected terminal portions, wherein the two working electrodes are alternating comb-shaped electrodes, and the amplification factor upon induction of a redox cycle is 5 times or more It is.

The endotoxin detection chip of the present invention will be described below with reference to the drawings.
FIG. 1 is a schematic plan view showing an example of the endotoxin detection chip of the present invention and a schematic cross-sectional view taken along the line AA. As illustrated in FIGS. 1A and 1B, an endotoxin detection chip 1 is formed on a support base 2 and the support base 2, and has an electrode portion having two working electrodes 3 and a counter electrode 4. 6 and a terminal portion 8 formed on the support base material 2 and connected to the electrode portion 6, the two working electrodes 3 are alternating comb electrodes, and at the time of redox cycle induction The amplification factor is 5 times or more.

According to the present invention, since the amplification factor at the time of inducing the redox cycle is 5 times or more, for example, it is possible to stably detect the current value output from 1 EU / L endotoxin.
More specifically, the current value output as noise, that is, the current value output when measuring a sample with an endotoxin concentration of 0 EU / L, and the current value output from an endotoxin of 1 EU / L And can be distinguished stably.
For example, when an endotoxin detection chip having only one working electrode is used, that is, when the amplification factor is 1 time, as described above, it is output when a sample having an endotoxin concentration of 0 EU / L is measured. While the current value (noise) is 25 nA, the current value output from endotoxin having an endotoxin concentration of 1 EU / L is about 10 nA, which may be less than half of the noise.
Here, the current value due to noise is proportional to the area of the working electrode, and the current value output from endotoxin is proportional to the area of the working electrode and the amplification factor.
For this reason, when an endotoxin detection chip in which the amplification factor of the two working electrodes is 5 times is used, a sample with an endotoxin concentration of 0 EU / L (reference sample) and a sample with an endotoxin concentration of 1 EU / L (measurement sample 1) Is measured, the reference sample can detect a current value of approximately 25 nA as noise, and the measurement sample 1 can detect a current value of approximately 50 nA as an output current derived from endotoxin.
Furthermore, due to the variation between the endotoxin detection chips, for example, even if the variation in the current value of about 20% is taken into account, the current value of about 25 nA ± 5 nA is detected as noise in the reference sample, and the measurement sample 1 is derived from endotoxin. It can be assumed that a current value of approximately 50 nA ± 10 nA can be detected as the output current.
As a result, the endotoxin detection chip in which the amplification factor of the two working electrodes is 5 times has an upper limit of the current value detected as noise and a current value detected from a sample having an endotoxin concentration of 1 EU / L. The lower limit is not stably overlapped.
Therefore, when the amplification factor is 5 times or more, the endotoxin detection chip of the present invention has a current value output as noise and a current output derived from, for example, 1 EU / L endotoxin. It is possible to stably distinguish the value from the current value, and it is possible to prevent the current value derived from endotoxin from being buried in the noise.
As described above, the present invention does not simply increase the amplification factor to amplify the current value detected from endotoxin, but in the vicinity of 1 EU / L, which has recently been required in the medical field. In the detection of endotoxin, the purpose is to eliminate the influence of noise that has been revealed for the first time.

FIG. 2 is a schematic plan view showing another example of the endotoxin detection chip of the present invention and a schematic cross-sectional view taken along the line CC. As illustrated in FIGS. 2 (a) and 2 (b), the endotoxin detection chip 1 of the present invention exposes at least the insulating layer 9 formed so as to cover the conductor portion 7, and at least the terminal portion 8, And an accommodating portion 15 formed by the second base material 10 disposed so that the opening is positioned on the electrode portion 6.
In this example, the endotoxin detection chip 1 of the present invention shows an example in which the measurement region M is included in the storage unit 15, and the storage unit and the measurement region are the same in plan view.
FIG. 2A omits the description of the second base material 10 for ease of explanation.

  In this example, the endotoxin detection chip of the present invention has the above-described housing portion, so that the subject can be stably held in the measurement region, and can be stably subjected to electrochemical reaction with respect to the subject. The current value can be measured.

FIG. 3 is a schematic plan view showing another example of the endotoxin detection chip of the present invention and a schematic cross-sectional view taken along the line DD. As illustrated in FIGS. 3A to 3C, the endotoxin detection chip 1 of the present invention includes an insulating layer 9 formed so as to cover the conductor portion 7 and the connecting portion 3 b, and at least a terminal portion 8. And an accommodating portion 15 formed by the second base material 10 which is exposed and disposed so that the opening is positioned on the electrode portion 6.
Further, in FIG. 3, the endotoxin detection chip 1 of the present invention shows an example in which the measurement region M is included in the storage unit 15, and the storage unit 15 and the measurement region M are the same in plan view. In the measurement region M in which the comb tooth portion 3a of the comb electrode included as the working electrode 3 is disposed, a reagent 11 containing a factor C and a peptide compound in which a redox substance is bound is immobilized. is there.
In this example, the endotoxin detection chip 1 of the present invention has a lid 12 and an inlet 13 formed in the lid 12.

  In this example, the endotoxin detection chip of the present invention is coated with a reagent containing a factor C and a peptide compound to which a redox substance is bound in the measurement region where the comb teeth are arranged. By simply introducing the sample into the endotoxin detection chip, the measurement of the current value can be started immediately after the formation of the mixture after the reaction. Further, the endotoxin detection chip of the present invention can prevent contamination of endotoxin and the like in the air by having the lid portion, and further from the analyte, the pre-reaction mixture, the post-reaction mixture, and the like. The volatilization of the solvent can be prevented.

  Hereinafter, each configuration in the endotoxin detection chip of the present invention will be described.

1. Amplification factor The two working electrodes in the present invention have an amplification factor that allows the current value output from the endotoxin concentration to be larger than the current value output as noise. Redox cycling The amplification factor during induction is 5 times or more.
Here, the amplification factor during induction of redox cycling is the ratio of the limiting current value (current value at measurement) when redox cycling is induced to the limiting current value (reference current value) when redox cycling is not induced (current value at measurement) / Reference current value).

More specifically, as a method for measuring the reference current value and the current value at the time of measurement, a pAP aqueous solution in which p-aminophenol (pAP) is dissolved in a 0.1 M KCl aqueous solution to make the pAP concentration 0.5 mM is prepared. Then, this pAP aqueous solution is dropped so as to cover all the exposed portions of the two working electrodes in plan view, and only one working electrode has a potential scanning speed of 100 mV / s and a potential (0V → 0.6V → The current value at 0.6V obtained from the single-mode cyclic voltammogram measured by scanning -0.2V → 0V) is used as the reference current value, and the reduction potential is fixed to 0V and applied to the other working electrode. , Obtained from a dual mode cyclic voltammogram measured by scanning the potential (0V → 0.6V → −0.2V → 0V) at one working electrode at a potential scanning speed of 100 mV / s. It is possible to use a method to measure the time of current value a current value of 0.6V in one of the working electrode is an electrode of scanning potential.
4 (a) and 4 (b) show examples of a single mode cyclic voltammogram and a dual mode cyclic voltammogram when pAP is used as the redox material, respectively. May be indicated by A2 / A1 in FIG.
In the result shown in FIG. 4, the amplification factor is calculated as 16.69 μA / 1.241 μA≈13.4.

The amplification factor in the present invention is not limited as long as the current value output from the endotoxin concentration can be larger than the current value output as noise, and more specifically, 1 EU / The L endotoxin can be stably detected.
Such an amplification factor can be, for example, 5 times or more, and in the present invention, it is preferably in the range of 5 to 20 times, particularly in the range of 5 to 15 times. It is preferable that
This is because, when the amplification factor is within the above-described range, for example, the endotoxin detection chip of the present invention can stably detect 1 EU / L endotoxin.
Moreover, as a method for increasing the amplification factor, for example, there is a method of narrowing the space between the comb tooth portions of two adjacent comb electrodes as two working electrodes.
For this reason, when the amplification factor is within the above-described range, the endotoxin detection chip of the present invention can easily form a comb-shaped electrode as a working electrode, for example, and can be easily reduced in size. Because it becomes possible.

In the present invention, a reaction current value when the concentration of endotoxin using the subject sample is 0EU / L and C _A, reaction current value when the concentration of endotoxin using the subject sample is 1 EU / L the when the C _B, it is preferred that the larger the value of C _{B /} C _a in the state of holding 35 minutes analyte sample in the measurement region. This is because endotoxin can be detected accurately in a short time. The value of C _B / C _A is, for example, 1.2 or more, 1.5 or more, or 2 or more. The reaction current values C _A and C _B are usually current values obtained from the same conditions, and the method for obtaining the reaction current values is not particularly limited. For example, as described later, a Pd electrode is used as a working electrode, a LAL reagent is used as a reagent, and an oxidation potential side current obtained by measurement by an amperometry method is measured immediately after the pulse application. The difference from the current value is preferably used as the reaction current value.

2. Electrode part The electrode part in this invention is formed on a support base material, and has two working electrodes.
Moreover, in this invention, as shown in FIG. 1, the electrode part may have a counter electrode and a reference electrode.
Hereinafter, each structure of the electrode part in this invention is demonstrated in detail.

(1) Working electrode The working electrode in the present invention includes two working electrodes. The working electrode is generally an electrode for performing electrochemical measurement by causing an electrochemical reaction of a target object.
In the present invention, the two working electrodes are alternating comb electrodes.
Here, the alternating comb-shaped electrodes are two comb-shaped electrodes arranged so that the comb-tooth portions are adjacent in the width direction, that is, the comb-tooth portions are meshed with each other. Since the two working electrodes are such alternating comb-shaped electrodes, it becomes easy to arrange a wide area portion of the two working electrodes in the same plane within a limited area within a distance close to each other. .
Then, by arranging the comb teeth portions of the two comb electrodes so as to mesh with each other, the working electrode is oxidized or reduced by the oxidation or reduction reaction generated at one working electrode. Since the distance traveled to the other working electrode is shortened, the number of cycles in redox cycling in which the redox substance moved to the other working electrode is reduced or oxidized can be increased, and loss can be reduced. .
Therefore, the working electrode can easily adjust the amplification factor.

  Moreover, since the said comb-shaped electrode has a wide contact area with a test object, the capture rate of the oxidation-reduction substance on the electrode surface can be improved. Therefore, in the endotoxin detection chip of the present invention, the working electrode can efficiently amplify the detected current value.

The area of the working electrode in plan view may be any as long as it can obtain a current value that can be detected by the measuring device. The area is, for example, an area where the current value of noise becomes 10 nA or more, more specifically, 180 μL of the buffer solution attached to the LAL reagent (End Spacey ES-24M), 200 μL of the attached distilled water, 10 mM LGR-pAP Was added dropwise so as to cover all of the two working electrodes, and the potential (0V → 0.6V → −0.2V → 0V) was applied to only one working electrode at a potential scanning speed of 100 mV / s. The area at which the current value at 0.6 V obtained from a single-mode cyclic voltammogram measured by scanning is 10 nA or more can be obtained.
The area, more specifically, 2 mm ² or more and it is possible to, inter ^alia, preferably in the range of 2mm ² ~50mm 2, in ^particular, be in the range of 4 mm 2 to 10 mm ² preferable. Since the area is within the above range, the endotoxin detection chip of the present invention stably stabilizes the current value output as noise and the current value output from, for example, 1 EU / L endotoxin. This is because it is possible to distinguish between the two, and the size can be easily reduced.
The area in the plan view of the working electrode refers to the area of the exposed portion of the working electrode that can come into contact with the post-reaction mixture or the like in the plan view.
Therefore, for example, as shown in FIG. 1A described above, when all of the working electrodes are exposed, the area is equal to two comb-shaped electrodes indicated by the shaded portion A shown in FIG. It can be set as the total planar view area. Further, as shown in FIG. 2A, when only a part of the working electrode is exposed, such as when the working electrode is arranged in the housing portion, the area is shaded as shown in FIG. The total area of the exposed regions of the comb-shaped electrode indicated by the part A can be set as a planar view area. FIG. 5B shows an example in which only the comb-teeth portion is exposed in the comb-shaped electrode, that is, an example in which the area is the sum of the areas of the comb-teeth in a plan view.

In the present invention, the space between the comb tooth portions of the two comb electrodes is not limited as long as the two comb electrodes have a desired amplification factor. For example, the amplification factor is five times or more. From the point of view, it can be in the range of 1 μm to 15 μm, preferably in the range of 3 μm to 10 μm, particularly preferably in the range of 3 μm to 9 μm. This is because, when the space is within such a range, the comb electrode can easily have a desired amplification factor.
In addition, the space between the comb-tooth parts of the two comb-shaped electrodes refers to S in FIG.

Examples of other shapes of the comb-shaped electrode include the width, length, and number of the comb-tooth portions, the width between the adjacent comb-tooth portions, and the width of the connecting portion. Any shape can be used as long as it can accurately form a comb-shaped electrode having a desired amplification factor.
Specifically, the width and length of the comb teeth, the width between adjacent comb teeth, and the width of the connecting portion are indicated by a, b, c and d in FIG. Is. In addition, FIG. 1A already described shows an example in which the number of comb teeth per comb electrode is four.
The width of the comb tooth portion (a in FIG. 1 (a)) is not particularly limited as long as it can be used as a chip for detecting endotoxin with desired accuracy, but it is in the range of 1 μm to 10 μm. It is preferable that it is in the range, and it is preferable that it exists in the range of 3 micrometers-9 micrometers especially.

  The length of the comb tooth part (b in FIG. 1 (a)) can be in the range of 50 μm to 10,000 μm, and in the present invention, in the range of 80 μm to 10,000 μm, In particular, it is preferably within a range of 100 μm to 10,000 μm.

  The number of the comb teeth can be in the range of 50 to 300 per comb electrode.

  The width between adjacent comb teeth in one comb-shaped electrode (c in FIG. 1A) can be in the range of 3 μm to 30 μm.

  The width of the connecting portion (d in FIG. 1A) can be in the range of 50 μm to 10,000 μm.

  The thickness of the working electrode (e in FIG. 1B) is preferably in the range of 10 nm to 150 nm, more preferably in the range of 30 nm to 100 nm, and particularly preferably in the range of 30 nm to 50 nm.

  In addition, the material and manufacturing method of the working electrode can be the same as those of an electrode portion of a general biosensor described later.

(2) Counter electrode The counter electrode in the present invention is an electrode for compensating for excess and deficiency of electrons in the solution caused by reaction at the working electrode and maintaining electrical neutrality. That is, the counter electrode is used to prevent the reaction on the working electrode from being delayed because the electrical neutrality is maintained.
The form of the counter electrode can be the same as that generally used for the electrode part of the biosensor.
The counter electrode material and the manufacturing method can be the same as those of an electrode section of a general biosensor described later.

(3) Reference Electrode The reference electrode is generally an electrode that serves as a reference when determining the potential of the working electrode. In the present invention, the reference electrode serves as a reference for determining the potentials of the two working electrodes.
The planar view shape of the reference electrode can be the same as that generally used for the electrode part of the biosensor.
In addition, the material and manufacturing method of the reference electrode can be the same as those of an electrode portion of a general biosensor described later.

(4) Material of electrode part The material of the electrode part is not particularly limited as long as it has desired conductivity. For example, a noble metal material having non-corrosive properties (hereinafter simply referred to as a noble metal material). And other metal materials other than the noble metal materials can be used.
In addition, the material is not limited to a metal material, and conductive carbon, conductive particles dispersed in a cured binder resin, or the like can also be used.

  Examples of such noble metal materials include gold (Au), platinum (Pt), palladium (Pd), rhodium (Rh), iridium (Ir), ruthenium (Ru), and osmium (Os). Since these noble metal materials have a low ionization tendency and are electrochemically stable, they are not easily corroded by moisture, hydrogen peroxide, etc., and have high conductivity. It is suitable as. In the present invention, the material of the two working electrodes is preferably Pd.

  The other metal material may be anything other than the noble metal material. For example, copper (Cu), iron (Fe), cobalt (Co), aluminum (Al), chromium (Cr), nickel (Ni) , Metals such as titanium (Ti), cerium (Ce), tantalum (Ta), tin (Sn), alloys containing the above metals such as stainless steel (SUS), metal compounds such as indium tin oxide (ITO), etc. Can be used. Among these, nickel is preferable from the viewpoint of corrosion resistance.

  In the present invention, for example, gold, platinum and conductive carbon are preferably used for the working electrode and the counter electrode, and palladium is preferably used for the reference electrode. This is because these materials are excellent in corrosion resistance and conductivity.

(5) Manufacturing method The method for forming the electrode part is not particularly limited as long as the electrode part can be formed in a desired pattern on a support base material. Examples include a method of patterning the conductive film by photolithography after preparing a support substrate and forming a conductive film on the entire surface, a mask vapor deposition method, a screen printing method, a gravure printing method, a flexographic printing method, an inkjet method, and the like. .

3. Terminal part The terminal part in this invention is connected with the said electrode part, and is for connecting with the measuring apparatus which quantifies an endotoxin density | concentration based on the electric current value measured by an electrochemical reaction.
The material of the terminal part can be the same as the material of the electrode part. The material of the terminal portion only needs to ensure conductivity that does not affect the detection current value and the applied voltage, and a general conductive material can be used. Among them, from the viewpoint of conductivity, Au, A noble metal such as Ag, Pt or Pd and a metal such as Cu or Ni are preferred.

Further, the terminal portion preferably has a low contact resistance and has a strength that does not peel off when brought into contact with the connector. For example, a desired conductive material is formed by forming a thin film of a conductive material and laminating conductive carbon. And a terminal portion with low contact resistance can be formed.
The method for forming the terminal portion can be the same as the method for forming the electrode portion.
The terminal portion may be formed simultaneously with the electrode portion or may be formed separately from the electrode portion.

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.
Moreover, it is preferable that the current value output as noise is less than 40 nA in the measuring apparatus in the present invention, and it is particularly preferable that the current value is within the range of 5 nA to 30 nA.
This is because the endotoxin detection chip of the present invention can exhibit the effect that, for example, 1 EU / L endotoxin can be stably detected by using the measurement apparatus.
In addition, the noise described above covers, for example, 180 μL of the buffer solution attached to the LAL reagent (Endspace ES-24M), 200 μL of the attached distilled water, and 20 μL of 10 mM LGR-pAP covering all the two working electrodes. Obtained from a single-mode cyclic voltammogram measured by scanning the potential (0V → 0.6V → −0.2V → 0V) at a potential scanning speed of 100 mV / s on only one working electrode. Current value at 0.6V.
Moreover, the current value output as the noise can be a current value detected when the area of the working electrode is 2 mm ² , for example.

4). Support base material The support base material used for this invention supports an electrode part and a terminal part, and the surface in which the said electrode part and a terminal part are formed has insulation.
As the support substrate, for example, a resin substrate, paper, a ceramic substrate, a glass substrate, a semiconductor substrate having at least a surface insulation, a metal substrate, or the like can be used. The support base material may have rigidity or may have elasticity. Among them, it is preferable to have electrical insulation and elasticity, and for example, a film such as polyethylene terephthalate (PET) resin, vinyl chloride resin, polystyrene (PS) resin, polypropylene (PP) resin can be suitably used.
In addition, the support base material may have flexibility and does not need to have it. Further, it does not matter whether the supporting substrate is transparent.

  Further, for the purpose of improving wettability, adhesion, etc. between the supporting base material and the conductive material used for the electrode part or the like, the supporting base surface may be treated or modified. Examples of such treatment and modification of the surface of the supporting substrate include corona treatment, UV treatment, antifogging treatment, and modification by coding a material having a sulfonic acid group or a sulfuric acid group.

The shape, size, thickness, and the like of the support substrate can be appropriately set depending on the shape of the connecting portion of the measuring instrument that uses the endotoxin detection chip of the present invention.
In addition, when the endotoxin detection chip of the present invention is manufactured with multiple faces, the support substrate may be long or single-wafer. When the supporting substrate is long, the electrode part, the wiring part, and the terminal part can be continuously formed by a roll-to-roll method.

5. Other Configurations The endotoxin detection chip of the present invention has at least a support base, the electrode part formed on the support base, and the terminal part. It may have.
As said other structure, the accommodating part which can accommodate a test object fixed amount, the 2nd base material provided in order to form an accommodating part, the conducting wire part which electrically connects the said electrode part and the said terminal part, In addition, an insulating layer that covers the conductor portion and the connecting portion can be used.

(1) Storage Unit The endotoxin detection chip of the present invention preferably has a storage unit that can store a subject, and the electrode unit is disposed inside the storage unit. By forming the housing portion, the endotoxin detection chip can stably hold the analyte in the measurement region, and can stably maintain a current value due to an electrochemical reaction with respect to the analyte. This is because measurement is possible.

As such a housing part, it may be formed by a groove of the support base material, and an insulating material such as a second base material in which a groove or an opening corresponding to the support base material and the housing part is formed. You may form by laminating | stacking a member. The insulating member may include an insulating layer described later. That is, the housing portion can be a region surrounded by the second base material, the insulating layer, and the like.
The constituent material of the second base material may be a material different from the support base material or the same material.
2 to 3 that have already been described, the housing portion 15 is formed by laminating the support base material 2, the second base material 10 in which an opening corresponding to the housing portion 15 is formed, and the insulating layer 9. The example formed by this is shown.

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.
The capacity of the housing portion ^is preferably in a range of 1 mm 3 to 200 mm ^3, among them ^{1 mm} 3 in the range of 100 mm ^3, and particularly preferably in the range of ^{1mm 3 ~50mm} 3. In addition, as the size of the housing portion, for example, when the shape of the housing 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. This is because the electrochemical endotoxin concentration detection method using differential pulse voltammetry or the like can measure by introducing a sample of several μL to several tens of μL.
2 and 3 which have already been described show examples in which the shape of the accommodating portion 15 is rectangular.

The location for forming the accommodating portion is not particularly limited as long as the subject can be accommodated and the electrode portion can be disposed therein. For example, 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, and the formation location of the storage portion in the plan view is the measurement region. A broader range can be included. The measurement area will be described later.
In the present invention, it is particularly preferable that the location where the housing portion is formed is the same as the measurement region. This is because the mixture after the reaction of the analyte can be stably brought into contact with the electrode portion. 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 part.
2 and 3 described above show an example in which the housing portion 15 includes the electrode portion 6 (working electrode 3, counter electrode 4 and reference electrode 5) in plan view. An example in which the location is the same as the measurement region M in plan view is shown.

  The constituent material constituting the housing part is not particularly limited as long as it can stably form the housing part and does not inhibit the liberation reaction, inorganic materials such as silicon and glass, acrylic resin, Examples thereof include silicone resins such as polydimethylsiloxane (PDMS) and resin materials such as epoxy resins. In addition, the constituent material may be a single material or a combination of two or more materials.

The method for forming the housing part is not particularly limited as long as it can stably form the housing part having a desired shape, and can be appropriately selected depending on the constituent material of the housing part.
For example, when the constituent material is silicon, glass, or the like, the forming method includes a concave portion used as the housing portion or the like using anisotropic dry etching, wet etching using hydrofluoric acid, or the like. The method of forming can be mentioned.
Further, when the constituent material is a silicone resin or the like, as the forming method, a photoresist formed in an inverted shape corresponding to the accommodating portion or the like on a silicon wafer using a normal photolithography method is used as a mold. After forming and pouring a mixture of an unpolymerized silicone resin and a polymerization initiator into this mold and heating and curing it, the cured silicone resin is peeled off from the mold, and thus the above-mentioned accommodating portion and the like The method of forming the recessed part used as can be mentioned.
When the constituent material is a resin material, the resin material is pressed by pressing a mold in which concave and convex shapes corresponding to the housing portion are formed, and the resin material is cured by irradiation with ultraviolet rays or heating. Next, a method of forming a concave portion used as the housing portion or the like by peeling the cured product of the resin material from the mold can be exemplified.
The forming method may be a method of forming a recess in the constituent material using a laser or the like.
In addition, the forming method includes preparing two substrates on which recesses corresponding to the housing portions are formed, and bonding them so that the recesses of the two substrates face each other, and an opening as the recess. A method of bonding a single formed substrate and a flat substrate may be used.

(2) 2nd base material In this invention, the 2nd base material may be arrange | positioned on the said support base material. A 2nd base material is provided in order to form the said accommodating part.
The second base material can have an opening corresponding to the accommodating portion. The size and shape of the opening can be the same as the size and shape of the housing.

As said 2nd base material, if it has insulation, it will not specifically limit, For example, a resin base material etc. are mentioned.
The shape of the second base material is not particularly limited, and may be any shape such as a square, a rectangle, a circle, an ellipse, or the like.

The second base is disposed on the support base so that at least the terminals are exposed and the opening of the second base is located on the electrode.
The said 2nd base material can be affixed on a support base material through an adhesive agent or an adhesive, for example.

(3) Lid The endotoxin detection chip of the present invention may include a lid that covers the upper surface of the housing. Mixing of endotoxin and the like in the air can be prevented, and further, volatilization of the solvent from the analyte, the pre-reaction mixture, the post-reaction mixture, and the like can be prevented. Therefore, the endotoxin detection chip of the present invention can detect the endotoxin concentration with high accuracy.
Note that FIG. 3 described above shows an example in which the endotoxin detection chip 1 has the lid portion 12, and the lid portion 12 is supported by the second base material 10 with respect to the support base material 2. The example formed is shown.

The shape of the lid portion may be any shape as long as it can cover the upper surface of the endotoxin detection chip, and may be a plate shape.
Note that FIG. 3 already described shows an example in which the lid 12 has a plate shape.
Further, when the endotoxin detection chip has a structure in which two base materials in which concave portions corresponding to the housing portions and the like are formed are bonded so that the concave portions face each other, one base material Can be used as the lid.

The lid portion may have a through hole. The through hole can be used as an inlet for the subject. Since the introduction port will be described later, description thereof is omitted here.
Note that FIG. 3 already described shows an example in which the lid portion 12 has the introduction port 13.

  The lid may be closable. By closing the lid and introducing the subject into the container and then closing it again, contamination of endotoxin in the air can be prevented, and the endotoxin detection chip can be used to detect the endotoxin concentration. It is because it can be made excellent.

(4) Inlet The endotoxin detection chip of the present invention may have an inlet for introducing the subject into the lid.
The size of the introduction port may be any size as long as the subject can be introduced, and may be the same as the size of the container in plan view.
The shape of the introduction port is not particularly limited, but the shape of the introduction port in plan view is preferably circular or elliptical because it is easy to form the introduction port.
The introduction port may be covered with a reclosable film or the like. Since it is the same as the reason described in the section “(3) Lid”, the description is omitted here.
A reclosable adhesive layer is preferably formed at the end of the film, and can be the same as a normal reclosable film.

(5) Conductive wire portion The endotoxin detection chip of the present invention usually has a conductive wire portion that electrically connects the electrode portion and the terminal portion.
The material of the conducting wire part can be the same as the material of the electrode part. As the material of the conductive wire portion, it is only necessary to ensure 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, Ag, Pt It is preferable that there are noble metals such as Pd and metals such as Cu and Ni.
The method for forming the conductor portion can be the same as the method for forming the electrode portion.
Moreover, the said conducting wire part may be formed simultaneously with an electrode part, and may be formed separately from an electrode part.

(6) Insulating layer The endotoxin detection chip of the present invention may have an insulating layer so as to cover at least the conductor portion. This is because the endotoxin detection chip of the present invention can suppress oxidation of the conductor portion and the like by the insulating layer.
As a material for the insulating layer, for example, a thermosetting resin, a photocurable resin, or the like can be used.
The insulating layer may be formed by any method as long as it can form the insulating layer in a pattern so as to cover at least the conductor portion and not the electrode portion and the terminal portion. For example, a photolithography method or a screen printing method may be used. , Gravure printing method, flexographic printing method, inkjet method and the like.
In addition, the said insulating layer may be used also as the above-mentioned 2nd base material, for example.

(7) Reagent As illustrated in FIG. 3, the endotoxin detection chip of the present invention has a factor C and a redox substance bound in the measurement region where the comb teeth of the comb electrode used as a working electrode are arranged. It is preferable that a reagent containing a peptide compound is immobilized.
Furthermore, as illustrated in FIG. 3, the endotoxin detection chip of the present invention may have a housing portion so as to include the measurement region, and the reagent may be immobilized in the measurement region.
Here, the measurement region is a region where the electrode part is arranged and a current value is measured by an electrochemical reaction. In addition, the immobilization in the region is not limited to the one formed so as to cover the entire surface of the measurement region in a plan view, but is usually limited to one in the measurement region in a plan view. It is formed in the area of the part.
Since the reagent is immobilized in the measurement region in which the comb tooth portion is arranged, after the reaction, which is a target for measuring the current value in the electrochemical reaction, only by introducing the analyte into the endotoxin detection chip. Thus, it is possible to obtain a mixture of the analyte, the factor C and the peptide compound after the completion of the free reaction.
For this reason, after forming a post-reaction mixture to be measured using a container prepared separately from the endotoxin detection chip, the post-reaction mixture is taken out of the container and introduced into the endotoxin detection chip. The process to perform can be made unnecessary. As a result, when the mixture after the reaction is taken out from the container and introduced into the endotoxin detection chip, contamination of endotoxin in the air can be prevented, and deterioration in the accuracy of endotoxin concentration detection can be suppressed. Can do.
Therefore, the endotoxin detection chip can stably detect, for example, 1 EU / L endotoxin.
Note that that the reagent is immobilized means that the mixture of the components of the reagent is disposed in a dry state.

  In addition, since a reagent containing a factor C and a peptide compound in which a redox substance is bound is immobilized in the measurement region in which the comb teeth are arranged, only the analyte is introduced into the endotoxin detection chip. Thus, the measurement of the current value can be started immediately after the formation of the mixture after the reaction. For this reason, even when the reaction rate of the free reaction is fast, or when the redox substance released from the peptide compound contained in the mixture after the reaction is deactivated in a short time, the endotoxin detection chip is, for example, 1 EU / L endotoxin can be detected stably.

FIG. 6 and FIG. 7 are schematic diagrams showing an example of the case where the liberation reaction is a multistage reaction, in which the redox substance is paramethoxyaniline (hereinafter sometimes simply referred to as pMA) and paraamino. An example of phenol (hereinafter sometimes referred to as a unit pAP) is shown. As illustrated in FIG. 6 and FIG. 7, 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 multistage 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.

  Endotoxin concentration detection using an electrochemical reaction makes it possible to detect the reaction on the electrode surface, and the endotoxin concentration can be measured even with a small amount of sample. Therefore, the above-mentioned endotoxin detection chip not only enables stable detection of, for example, 1 EU / L endotoxin, even in a very small amount of analyte, but also enables rapid detection. 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. Endotoxin can be detected stably.

  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. .
Hereinafter, each configuration of the reagent, the immobilization position, and the release reaction will be described in detail.

(A) Reagent The reagent used for this invention contains the peptide compound which C factor and the redox substance couple | bonded. The reagent may contain a buffer component. This is because the endotoxin detection chip of the present invention can detect the endotoxin concentration of the subject in a short time because the reaction rate of the above-described free reaction can be improved.
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 reaction rate can be improved as compared with the result of the release reaction, the factor C and the peptide that contact the analyte when the analyte contacts the reagent. It can be inferred that the concentration of the compound and the buffer component can be increased.
The reagent is immobilized in the measurement region where the comb teeth are arranged.

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 others, the above-mentioned factor C is used together with factor B and a clotting enzyme precursor, that is, the reagent contains a multistage reaction composition containing factor C, factor B and a clotting enzyme precursor. It is preferable. 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. Such a lysate reagent can be used as “LAL equivalent”.
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. Note that LAL and LAL equivalents may be referred to as LAL reagents.

The content of the factor C or the multistage reaction composition in the reagent is not particularly limited as long as the concentration of endotoxin can be accurately detected, and the analyte in the endotoxin detection chip of the present invention Is appropriately set according to the introduction amount of the container, the presence / absence of the accommodating portion, the volume of the accommodating portion and the like.
As content in the said reagent of the said multistage reaction composition, it can be made into the same quantity 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 bonded is only required to have a redox substance and an oligopeptide compound to which the redox substance is bonded. The redox substance is bonded to one end of the oligopeptide compound. A peptide compound having a protecting group bonded to the other 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 compound, and Y represents the above redox substance.
Examples of the protecting group X include a protecting group of a peptide compound, such as a t-butoxycarbonyl group (Boc), a benzoyl group, and an acetate group.

  The oligopeptide compound is not particularly limited as long as it can release the redox substance by the action of factor C or the multistage reaction composition. Among them, the oligopeptide compound preferably has 2 to 10 amino acids, particularly 2 to 5, more preferably 3 to 4.

  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, as an oligopeptide for a multistage reaction composition, a general formula: R ² -A ¹ -A ² -A
Mention may be made of the tetrapeptide A ¹ -A ² -A ³ -A ⁴ in ^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. A peptide compound of an oligopeptide compound in which 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 is contained in the peptide compound. It becomes easier to recognize the sequence. For this reason, since the compound represented by the general formula (3) bonded to the oligopeptide compound as a redox substance is pMA, the peptide compound has increased reactivity with the activated factor C and the like. It is.

  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, for example, 1 EU / L endotoxin can be stably detected. .

The content of the peptide compound in the reagent is not particularly limited as long as, for example, 1 EU / L endotoxin can be stably detected. Introduction of the analyte in the endotoxin detection chip of the present invention It is appropriately set according to the amount, the presence / absence of the accommodating portion, the volume of the accommodating portion, and the like.
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.

In addition, 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 the endotoxin concentration can be accurately detected. The amount of the analyte introduced in the endotoxin detection chip of the present invention, the presence / absence of the accommodating portion, the volume of the accommodating portion, etc. are appropriately set.
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 endotoxin detection chip of the present invention can be reduced in price.

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 particularly pH 7.0 to 8.0. Preferably, it is a component of 5 buffer solutions. This is because the release amount of the redox substance can be increased.
Examples of the buffer component include Tris-acetate buffer, Tris-HCl (Tris-HCl) buffer, phosphate buffer, HEPES (4- (2-hydroxyethyl) -1-piperazine etheric acid) buffer. , Components such as PIPES (Piperazine-1, 4-bis (2-ethanesulfonic acid) buffer).

The content ratio of the buffer component in the reagent is not particularly limited as long as it can generate a buffer solution in a desired pH range, and the amount of analyte introduced in the endotoxin detection chip of the present invention It is set as appropriate according to the presence / absence of the accommodating portion and the volume of the accommodating portion.
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 peptide compound to which factor C and a redox substance are bound, and further contains factor B, a coagulation enzyme precursor, and a buffer solution component, and other components as necessary. May be included.
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 detection chip of the present invention can detect the endotoxin concentration more accurately by causing the second redox substance after reacting with the redox substance to undergo an oxidation-reduction reaction with an electrode. .
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 fixed in the measurement region where the comb tooth portion is arranged has a porous structure, the reagent quickly dissolves in the subject. 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 of determining whether the reagent has a porous structure or a non-porous structure, when the immobilized reagent is cloudy, it has a porous structure and has transparency. In such a case, there are a method of visually judging that the structure is non-porous, and a method of confirming by observation with a scanning electron microscope (SEM).

(B) Reagent immobilization method The reagent immobilization method is not particularly limited as long as it is a method capable of immobilizing the reagent in the measurement region in which the comb teeth are arranged. Alternatively, after preparing a reagent solution containing the multistage reaction composition, the peptide compound, the buffer component, the other components, and the like, the reagent in the measurement region where the comb teeth are arranged is immobilized. A method of applying the reagent solution to the position and drying it can be used.
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, it is particularly preferable that the drying method is 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 activated. This is because it is possible to suppress the formation of a structure that does not. 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 for drying and immobilizing the reagent solution at a desired position and range can be reduced.

(C) Immobilization position of reagent The reagent is immobilized in the measurement region.
The reagent immobilization position may be a position within the measurement region where the reagent can be brought into contact with the sample to be introduced. Specifically, as shown by the reagent 11 in FIG. 3, it may be formed so as to cover all the comb teeth 3b of the two working electrodes 3, or a part of the comb teeth 3b may be covered. It may be formed as follows. When the analyte is introduced from the introduction port, the reagent may be immobilized immediately below the introduction port.
In addition, the said measurement area | region is an area | region where the said electrode part is arrange | positioned and an electric current value is measured by an electrochemical reaction as mentioned above. Specifically, it refers to a region surrounding the electrode part, and can be a region indicated by M in FIG. The measurement region M preferably includes all the parts (for example, the comb-teeth part 3b of the comb-shaped electrode) involved in the measurement of the electrode part 6 (the working electrode 3, the counter electrode 4, the reference electrode 5 and the like). Specifically, it is preferably a minimum rectangle including an end portion of the portion related to the measurement (for example, the tip portion of the comb tooth portion 3b, the opposite surface facing the working electrode of the counter electrode and the reference electrode). This is because the endotoxin detection chip can be easily reduced in size, and the endotoxin concentration can be accurately detected.

According to the present invention, the reagent containing the buffer solution component together with the peptide compound to which the factor C and the redox substance are bound is immobilized in the measurement region in which the comb tooth portion is arranged. It is possible to obtain the post-reaction mixture, which is a current value measurement target by an electrochemical reaction, simply by introducing the analyte into the housing without adding.
Therefore, for example, the endotoxin detection chip can stably detect 1 EU / L endotoxin.

6). Use The endotoxin detection chip of the present invention can be used for, for example, detection of 1 EU / L endotoxin, but is not limited to 1 EU / L, and is used for detection of endotoxin near 1 EU / L. Can do.
As the vicinity of 1 EU / L, for example, it can be in the range of 0.5 EU / L to 5 EU / L, and in particular, it is preferably in the range of 0.5 EU / L to 3 EU / L. It is preferable to be within the range of 0.5 EU / L to 2 EU / L. This is because the endotoxin within the above-mentioned range as the vicinity of 1 EU / L can effectively exhibit the effect of the present invention that the endotoxin can be stably detected due to the above-described amplification factor.

Examples of analytes measured by the endotoxin detection chip of the present invention include biological samples such as blood, saliva, sweat, urine, environmental test objects, foods, cosmetics, waste liquids, resins, and the like. it can. Examples of the subject include pharmaceuticals such as dialysate and injections, medical tools, and the like.
The analyte is not limited to a liquid sample and can include a gas sample, but is usually a liquid sample.
When the analyte is a solid, a liquid sample dissolved or dispersed in an appropriate solvent such as a buffer solution can be used as the analyte to be measured.

B. Endotoxin concentration quantification method The endotoxin concentration quantification method of the present invention is characterized by using the aforementioned endotoxin detection chip.

  According to the present invention, the endotoxin concentration can be accurately quantified by using the above-described endotoxin detection chip. Since the endotoxin detection chip in the present invention is the same as the contents described in the above-mentioned “A. Endotoxin detection chip”, description thereof is omitted here.

  As described above, since there is a correlation between the current value measured by the electrochemical reaction (for example, the reaction current value measured by the oxidation reaction of pMA or pAP) and the endotoxin concentration, this correlation is shown. By preparing a calibration curve in advance, the endotoxin concentration can be determined from the reaction current value. The endotoxin concentration is preferably quantified after the sample is introduced and held for a predetermined time. The holding time is, for example, 60 minutes or less, 50 minutes or less, or 40 minutes or less. On the other hand, the holding time is, for example, 30 minutes or longer, and may be 35 minutes or longer. The holding temperature is not particularly limited, but is preferably around 37 ° C.

  In particular, in the endotoxin detection chip, a reagent containing a peptide compound in which a factor C and a redox substance are bound is immobilized in a measurement region where a comb tooth portion of a comb-shaped electrode used as the working electrode is disposed. Preferably, the material for the two working electrodes is Pd, and the reagent is an LAL reagent. In addition, the endotoxin concentration can be quantified by using, as the reaction current value, the difference between the current value immediately before the pulse application and the current value after the pulse application in the oxidation potential side current obtained by measurement by the amperometry method. preferable. When the Pd electrode and the LAL reagent are combined, the current value immediately before the pulse application becomes a negative value, and the value also changes depending on the endotoxin concentration. By using the current value as a reference, the endotoxin concentration can be accurately determined. The current value after applying the pulse is the current value when the temporal variation of the current value is so small that it can be ignored in quantifying the endotoxin concentration. For example, it may be 10 seconds after pulse application, 20 seconds later, or 30 seconds later.

  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.

[Example 1]
After forming a 50 nm-thick gold sputtered film on a PET substrate (Toray Co., Ltd., Lumirror 350H10) by sputtering, etching is performed by photolithography, and two working electrodes as shown in FIG. As a pattern, a gold layer having an alternating comb electrode, an electrode portion including one counter electrode and one standard electrode, a terminal portion, and a wiring portion was formed to obtain an endotoxin detection chip. Further, an Ag / AgCl layer was formed on the gold layer of the standard electrode by a screen printing method. Thus, an endotoxin detection chip was obtained.
The two comb-shaped electrodes constituting the alternating comb-shaped electrodes have two comb-shapes, with the width of the comb teeth (a in FIG. 1) and the width between the comb teeth (c in FIG. 1) being 9 μm and 27 μm, respectively. The space between the comb teeth of the electrode (S in FIG. 1) was 9 μm.
The area of the two working electrodes was 4.5 mm ² .

[Evaluation 1]
(Preparation of sample solution 1)
A pAP aqueous solution (sample solution 1) was prepared by dissolving p-aminophenol (pAP) in a 0.1 M KCl aqueous solution to a pAP concentration of 0.5 mM.

(Measurement of cyclic voltammogram)
30 μL of sample solution 1 was dropped so as to cover all the exposed portions of the two working electrodes in plan view, and the potential (0V → 0.6V → −) was applied to only one working electrode at a potential scanning speed of 100 mV / s. Current value at 0.6V (reference current value A1) obtained from a single-mode cyclic voltammogram measured by scanning 0.2V → 0V) and a reduction potential fixed to 0V on the other working electrode One working electrode obtained from a dual-mode cyclic voltammogram measured by scanning the potential (0V → 0.6V → −0.2V → 0V) at one working electrode at a potential scanning speed of 100 mV / s. Current value at 0.6V (current value A2 during measurement).
As a result, the reference current value A1 and the measurement current value A2 obtained from the single-mode and dual-mode cyclic voltammograms were 1.44 μA and 7.36 μA, respectively, and the amplification factor (A2 / A1) was 5.1. It was twice.
In addition, the result of the measured cyclic voltammogram of a single mode and a dual mode is shown to Fig.8 (a) and (b). In FIG. 8B, CH1 represents the oxidation current at one working electrode on the side scanned with the potential (0V → 0.6V → −0.2V → 0V), and CH2 represents the potential on the side where the potential is fixed. It shows the reduction current at the other working electrode.
Further, A1 in FIG. 8A and A2 in FIG. 8B are a reference current value and a measurement current value, respectively.
Further, in FIG. 8, the horizontal axis represents voltage (V), and the vertical axis represents reaction current (μA).
Hereinafter, in this example, Potentiostat CompactStat manufactured by Ivium Technologies was used as a measuring apparatus.

[Evaluation 2]
(Preparation of sample solution 2)
As a lysate reagent, 180 μL of buffer solution, 10 mM LGR-pAP is contained in one test tube of Endspecy ES24-M manufactured by Seikagaku Corporation, in which the lysate reagent is sealed in a dedicated test tube in a lyophilized state for each test. Was added and stirred.
Next, 200 μL of distilled water (0 EU / L) was added and stirred to obtain a sample solution (sample solution 2).
In addition, LGR-pAP which is a peptide compound uses Boc-Leu-Gly-Arg-pAP, which is dissolved in water for injection manufactured by Otsuka Pharmaceutical Co., Ltd. and stored at −20 ° C. as a 10 mM stock solution. did.
LGR-pAP was synthesized using the method described in JP-A-2015-187607.
Moreover, the buffer solution attached to Seikagaku Corporation Endspecy ES24-M was used for the buffer solution.

(Measurement of chronoamperometry characteristics)
After dropping the sample solution 2 so as to cover all the electrode parts of the endotoxin detection chip obtained in Example 1, the chronoamperometry characteristics are measured using the amperometry method, and the output current value at the time of measurement is measured. did. FIG. 9 shows the results measured at the timing after the sample solution 2 was dropped and held at 37 ° C. for 60 minutes.
In FIG. 9, Y1 indicates a reduction potential side current, Y2 indicates an oxidation potential side current, C1 fixes the reduction potential at -0.2V, and the oxidation potential is an initial potential of -0.2V. Shows the output current value at the time of measurement, which is the current value derived from the redox reaction of pAP when the potential is stepped (pulsed) to 0.4 V and then applied for 10 seconds after the voltage is applied for 10 seconds. In FIG. 9, the horizontal axis represents time (seconds), and the vertical axis represents reaction current (A).
In the amperometry method, the reduction potential is fixed at -0.2 V, and the initial potential of -0.2 V is applied for 10 seconds, and then the potential is stepped (pulsed) to 0.4 V to be 10 The current derived from the redox reaction of pAP when applied for 2 seconds was recorded.
Further, from FIG. 9, the measured output current value 10 seconds after the pulse application was 50 nA.

[Evaluation 3]
Endotoxin was mixed with distilled water (0 EU / L) to prepare a 1 EU / L endotoxin solution.
Next, a sample solution (sample solution 3) was obtained in the same manner as in evaluation 2, except that 200 μL of 1 EU / L endotoxin solution was used instead of 200 μL of distilled water (0EU / L).
The chronoamperometry characteristics were measured in the same manner as in Evaluation 2 except that the sample solution 3 thus obtained was used. The result is shown in FIG.
As a result, as shown in FIG. 10, the output current value during measurement 10 seconds after the pulse application was 250 nA.
Note that endotoxin added to distilled water (0 EU / L) was manufactured by Seikagaku Corporation. Coli O113: USP Reference Standard Endotoxin derived from H10 strain was used.

[Summary]
From evaluations 1 to 3, when the amplification factor at the time of redox cycling induction is 5 times or more, for example, the measurement output current value obtained from the measurement result of the chronoamperometry characteristics is 0 EU / L and 1 EU / L. Then, it was confirmed that there was no stable duplication.

[Example 2]
After forming a 50 nm thick Pd sputtered film on a PET substrate (Toray Industries, Lumirror 350H10) by sputtering, etching is performed by photolithography, and two working electrodes as shown in FIG. A pattern-shaped Pd layer having alternating comb-shaped electrodes, an electrode portion including one counter electrode and one standard electrode, a terminal portion, and a wiring portion was formed to obtain an endotoxin detection chip. Pd can be applied as a standard electrode, and it is not necessary to form an Ag / AgCl layer like an Au electrode.
Further, the two comb-shaped electrodes constituting the alternating comb-shaped electrodes have two comb-shapes, with the width of the comb teeth (a in FIG. 1) and the width between the comb teeth (c in FIG. 1) being 9 μm and 27 μm, respectively. The space between the comb teeth of the electrode (S in FIG. 1) was 9 μm.
The area of the two working electrodes was 4.5 mm ² .

[Evaluation 4]
Sample solution 1 was obtained in the same manner as Example 1. Using the obtained sample solution 1, cyclic voltammogram measurement was performed in the same manner as in Example 1. As a result, the reference current value A1 and the measurement current value A2 obtained from the single-mode and dual-mode cyclic voltammograms were 1.4 μA and 7.5 μA, respectively, and the amplification factor (A2 / A1) was 5.3. It was twice.

[Evaluation 5]
The same as in Example 1 except that a reagent using a recombinant protein produced by a gene recombination technique was used as each of the three types of lysate reagents: factor C, factor B, and coagulase precursor. Thus, a sample solution 2 was obtained. The obtained sample solution 2 was used to measure chronoamperometry characteristics. After dripping the sample solution 2 so as to cover all the electrode parts of the endotoxin detection chip obtained in Example 2, the chronoamperometry characteristics were measured by using the amperometry method, and the output current value at the time of measurement (oxidation) The potential side current value) was measured. After the sample solution 2 was dropped, the measurement was carried out at the timing after holding at 37 ° C. for 30 minutes, 35 minutes, and 40 minutes. The pulse application conditions are the same as in Example 1 except that the potential is stepped to 0.2 V at the time of pulse application.

[Evaluation 6]
Endotoxin was mixed with distilled water (0 EU / L) to prepare a 1 EU / L endotoxin solution.
Next, a sample solution (sample solution 3, gene recombination type) was obtained in the same manner as in Evaluation 5, except that 200 μL of 1 EU / L endotoxin solution was used instead of 200 μL of distilled water (0EU / L).
The obtained sample solution 3 was used to measure chronoamperometry characteristics. The measurement conditions are the same as in Evaluation 5.

[Summary]
An example of the measurement result of the chronoamperometry characteristics is shown in FIG. FIG. 11A and FIG. 11B are the results of evaluations 5 and 6 when the holding time is 35 minutes, respectively. As shown in FIGS. 11 (a) and 11 (b), when a ground current of 0 nA is used as a reference, the reaction current value 10 seconds after the pulse application is almost the same as in FIGS. 11 (a) and 11 (b). It was the same and the difference was small. This is because the endotoxin concentration is very low. On the other hand, when the Pd electrode and the LAL reagent are used, the current value immediately before the pulse application (current value at the position indicated by X in the figure) is not 0 nA but a negative value, and the value is also the endotoxin concentration. It changed by.

Therefore, when the current value immediately before the pulse application is used as a reference instead of using the ground current (0 nA) as a reference, the difference in the reaction current value 10 seconds after the pulse application becomes large as shown in FIG. It was. Specifically, C _A was 35 nA, C _B was 63 nA, and C _B / C _A was 1.8. The method for defining the reaction current value is an application method in which the current value immediately before the pulse application changes depending on the endotoxin concentration. Further, FIG. 11 shows the results when the holding time is 35 minutes, but the reaction current values were similarly defined when the holding time was 30 minutes and when the holding time was 40 minutes. The result is shown in FIG. As shown in FIG. 12, as the holding time increased, the value of C _B / C _A also increased, and at the time of holding time of 35 minutes, the value of C _B / C _A became 1.5 times or more.

[Example 3]
In the same manner as in Example 1, an endotoxin detection chip was obtained.

[Evaluation 7]
Sample solution 1 was obtained in the same manner as Example 1. Using sample solution 1, cyclic voltammogram measurement was performed in the same manner as in Example 1. The result was the same as in Example 1.

[Evaluation 8]
Sample solution 1 was obtained in the same manner as Example 2. The obtained sample solution 2 was used to measure chronoamperometry characteristics. After dripping the sample solution 2 so as to cover all the electrode parts of the endotoxin detection chip obtained in Example 3, the chronoamperometry characteristics were measured using the amperometry method, and the output current value at the time of measurement (reduction) The potential side current value) was measured. After the sample solution 2 was dropped, the measurement was performed at a timing after holding at 37 ° C. for 60 minutes. The pulse application conditions are the same as in Example 2.

[Evaluation 9]
A sample solution 3 was obtained in the same manner as in Example 2. The obtained sample solution 3 was used to measure chronoamperometry characteristics. The measurement conditions are the same as in evaluation 8.

[Summary]
The measurement results of the chronoamperometry characteristics are shown in FIG. 13 and FIG. As shown in FIGS. 13 and 14, in the case of 0 EU / L, the measurement output current value (C _A ) after 10 seconds from the pulse application is 21.4 nA, and in the case of 1 EU / L, 10 seconds from the pulse application. The later measurement output current value (C _B ) was 59.9 nA, and C _B / C _A was 2.8 times. Note that the value of C _B / C _A in a state where the subject sample is held in the measurement region for 60 minutes is preferably 2 times or more, and more preferably 3 times or more.

DESCRIPTION OF SYMBOLS 1 ... Endotoxin detection chip | tip 2 ... Support base material 3 ... Working electrode 3a ... Comb-tooth part 3b ... Connection part 4 ... Counter electrode 5 ... Reference electrode 6 ... Electrode part 7 ... Conductor part 8 ... Terminal part 9 ... Insulating layer 10 ... First 2 Substrate 11 ... Reagent 12 ... Lid 13 ... Inlet 15 ... Container

Claims (10)

A support substrate;
An electrode part molded on the support substrate and having two working electrodes;
A terminal part formed on the support substrate and connected to the electrode part;
An endotoxin detection chip having at least
The endotoxin detection chip, wherein the two working electrodes are alternating comb electrodes, and the amplification factor upon induction of redox cycling is 5 times or more. The endotoxin detection chip according to claim 1, wherein the area of the two working electrodes is 2 mm ² or more.   The reagent containing the peptide compound which the C factor and the redox substance couple | bonded is fix | immobilized in the measurement area | region where the comb-tooth part of the comb-shaped electrode used as the said working electrode is arrange | positioned. Alternatively, the endotoxin detection chip according to claim 2.   The chip for endotoxin detection according to claim 3, wherein the reagent comprises a multistage reaction composition containing the factor C, factor B and a coagulation enzyme precursor.   The chip for endotoxin detection according to claim 3 or 4, wherein the reagent is an LAL reagent. The reaction current value when the concentration of endotoxin using the subject sample is 0EU / L and C _A, a reaction current value when the concentration of endotoxin using the subject sample is 1 EU / L in case of the C _B The value of C _B / C _A in a state in which the subject sample is held in the measurement region for 35 minutes is 1.5 or more, 6. The chip for endotoxin detection according to Item. The material of the two working electrodes is Pd;
The chip for endotoxin detection according to any one of claims 3 to 6, wherein the reagent is an LAL reagent. It has a storage unit that can store the subject,
The chip for endotoxin detection according to any one of claims 1 to 7, wherein the electrode part is disposed inside the housing part.   An endotoxin concentration quantification method using the endotoxin detection chip according to claim 1. In the endotoxin detection chip, a reagent containing a peptide compound in which factor C and a redox substance are bound is immobilized in a measurement region in which a comb tooth portion of a comb-shaped electrode used as the working electrode is disposed. The material of the two working electrodes is Pd, the reagent is a LAL reagent,
It is characterized by quantifying endotoxin concentration by using the difference between the current value immediately before pulse application and the current value after pulse application in the oxidation potential side current obtained by the amperometry method as the reaction current value. The method for quantifying endotoxin concentration according to claim 9.