JP2016151482A - Electrochemical concentration measurement method of endotoxin - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide means capable of easily measuring concentration of endotoxin with higher sensitivity (100 pg/mL order) compared to conventional electrochemical measurement.SOLUTION: The electrochemical concentration measurement method is a method to measure the concentration of endotoxin contained in a sample, the method including the steps of: (1) bringing a sample into contact with a carrier on which a first endotoxin recognition molecule is fixed to capture and concentrate the endotoxin in the sample on the carrier; (2) bringing an endotoxin recognition probe, which contains a heavy metal compound and a second endotoxin recognition molecule, into contact with the carrier to capture the endotoxin recognition probe on to the endotoxin on the carrier; (3) allowing the endotoxin recognition probe to react with an acid solution to elute heavy metal ion from the heavy metal compound of the probe; and (4) measuring the amount of the eluted heavy metal ion in an electrochemical manner using a working electrode for measuring the heavy metal ion. The concentration of the endotoxin in the sample is measured by using the amount of the heavy metal ion as the index.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a method for measuring the concentration of endotoxin possessed by gram-negative bacteria, and a kit and a device for carrying out this method.

  Endotoxin is a general term for toxic substances that constitute the outer membrane of Gram-negative bacteria such as Escherichia coli and Salmonella, and its main body is Lipopolysaccharide (LPS).

  Endotoxins can be released by the destruction (killing or lysis) of bacteria that inhabit the production process of pharmaceuticals, and can be mixed into pharmaceuticals. For example, parenteral pharmaceuticals such as infusion solutions and injectable preparations are produced mainly by fermentation technology or genetic recombination technology, and there is concern about contamination from host cell wall components in the production process. When endotoxin is mixed in blood even in a very small amount (for example, on the order of ng / mL), it causes fever, shock death, intravascular blood pseudo-solidification, or sepsis. For this reason, the endotoxin content in these preparations is legally regulated, and it is essential to measure the endotoxin content simply and with high sensitivity in order to ensure the safety of these pharmaceuticals.

  As a method for measuring and quantifying endotoxin concentration, the Limulus test has become the mainstream. This Limulus test utilizes the phenomenon that the blood cell extract of horseshoe crab coagulates with endotoxin. The clotting reaction of Limulus reagent occurs in a cascade reaction similar to that of mammalian blood pseudosolid systems. Therefore, a small amount of endotoxin signal is amplified to cause quasi-solidity, which is a very sensitive measurement system (see, for example, Patent Document 1). Recently, a method has been developed in which a genetically modified luciferase and a luminescent synthetic substrate are used to measure the endotoxin concentration in a sample from the amount of luminescence (see, for example, Patent Document 2).

  However, the conventional limulus test and the measurement method based on the limulus test as described above are expensive for both the measuring apparatus and the reagent, and it is difficult to perform on-site measurement. In addition, as is clear from the fact that the measurement principle is the change over time in the coagulation process that undergoes multiple stages of reaction, the endotoxin concentration, especially at lower concentrations, requires more coagulation time (measurement time), and moreover it is measured by the measurer. There were problems such as variations in values.

  By the way, in a method for quantifying a target component in a sample, there is an electrochemical measurement method as a simple and highly sensitive measurement method. This method is characterized in that a target component in a solution or a specific component whose concentration changes in response to a change in the concentration of the target component is converted and output as a current signal on the electrode.

  From the background as described above, the present invention provides a simple and highly sensitive method for measuring endotoxin by an electrochemical method, which is a simple and optimal method for the purpose of high sensitivity, as a new method for measuring endotoxin replacing the Limulus test. Is to provide. However, since the main components constituting the endotoxin to be measured are polysaccharides and lipids, and they themselves do not show any electrochemical activity, the present inventors have made boronic acid and polymyxin B that recognize endotoxin. Developed a compound with reversible redox ability, which causes the formation of a complex with endotoxin, and as a result, the amount of endotoxin causing the change is quantitatively, easily, and highly selectively determined by an electrochemical method. It was found that it can be measured (Patent Document 3, Non-Patent Documents 1 and 2). The measurement methods described in Patent Document 3 and Non-Patent Documents 1 and 2 enable electrochemical measurement of endotoxin, and when boronic acid and its derivatives are used, the detection limit concentration is 100 to 500 ng / mL, The detection limit concentration was 50 ng / mL when using polymyxin B, which was an excellent measurement method. Recently, with the aim of further improving the detection sensitivity of endotoxin concentration measurement, the following new detection methods have been proposed (Patent Document 4, Non-Patent Document 3). That is, (1) a step of adding endotoxin on the electrode on which the first endotoxin recognition molecule is immobilized and capturing the endotoxin on the electrode; (2) reversible redox substance or reversible on the electrode capturing the endotoxin. Adding a second endotoxin recognition molecule modified with one of the enzymes capable of generating a redox substance, and capturing the second endotoxin recognition molecule; (3) a change in current when a voltage is applied to the electrode; An endotoxin electrochemical measurement method comprising the step of measuring the concentration of endotoxin from the measured value based on the amount of reversible redox material concentrated on the electrode or the amount of reversible redox material produced by the concentrated enzyme. is there. However, the methods of Patent Document 4 and Non-Patent Document 3 are electrochemical measurement methods capable of measuring up to about 1.67 to 2 ng / mL endotoxin. However, as a method for measuring endotoxin causing sepsis and the like on the order of ng / mL, Further improvement in detection sensitivity has been desired.

Japanese Unexamined Patent Publication No. 06-324047 International Publication 2009/063840 Pamphlet JP 2007-093378 A JP 2014-52224

Biosensors and Bioelectronics 22 (2007) 1527-1531 Biosensors and Bioelectronics 26 (2011) 2080-2084 Electroanalysis 26 (2014) 618-624

  From the background as described above, the present invention provides a method capable of measuring the endotoxin concentration more easily and with higher sensitivity than conventional electrochemical measurements, and an endotoxin concentration measurement kit and device for carrying out this method. It is an issue to provide.

  Based on the development of the electrochemical endotoxin measurement method described above (Patent Documents 3 and 4), the present inventors have conducted extensive research. As a result, the first endotoxin recognition molecule that recognizes endotoxin with high selectivity. After capturing and concentrating the endotoxin in the sample on the surface of a carrier such as microparticles modified with, the endotoxin recognition probe modified with a heavy metal compound capable of obtaining an electrochemical signal is captured and concentrated, and finally captured. The present inventors have completed the present invention by finding that the concentration of endotoxin can be determined from the amount of electrochemical signal by measuring heavy metal ions eluted from the probe obtained on an electrode that can be deposited and desorbed. In the present invention, a combination of a two-stage concentration process of concentrating endotoxin on the surface of a fine particle carrier and concentrating heavy metal ions on an electrode, and a working electrode that can finally measure heavy metal ions stably to a low concentration ( For example, the carbon electrode: Japanese Patent No. 4531093) makes it possible to amplify the electrochemical response even with a low concentration of endotoxin and to measure with high sensitivity.

That is, the invention of the present application is a method for measuring the concentration of endotoxin contained in a sample as a first invention for solving the above-mentioned problems, comprising the following steps:
(1) A step of contacting the sample with a carrier on which the first endotoxin recognition molecule is immobilized, and capturing and concentrating the endotoxin in the sample on the carrier,
(2) contacting an endotoxin recognition probe comprising a heavy metal compound and a second endotoxin recognition molecule with a carrier, and capturing the endotoxin recognition probe on the endotoxin on the carrier;
(3) A step of reacting an endotoxin-recognizing probe with an acidic solution to elute heavy metal ions from the heavy metal compound of the probe, and (4) electrochemically measuring the amount of heavy metal ions eluted using a working electrode that measures heavy metal ions. Measuring process,
And measuring the endotoxin concentration in the sample using the amount of heavy metal ions as an indicator.

  This endotoxin concentration measurement method can measure endotoxin at a concentration of 200 pg / mL or more.

  One of the embodiments in this method for measuring the concentration of endotoxin is that the endotoxin recognition molecule is selected from poly ε lysine, poly α lysine, polymyxin B, cetyl pyrinium, protein, polypeptide, oligopeptide, oligonucleotide, polyethyleneimine and polyallylamine. The first endotoxin recognition molecule and the second endotoxin recognition molecule are the same or different species.

  In another embodiment, the heavy metal compound of the endotoxin recognition probe is a heavy metal complex compound, and the heavy metal in the heavy metal complex compound is zinc, silver, copper, cadmium, nickel, cobalt, lead, selenium or indium.

In another embodiment, the working electrode has 40% or more sp ³ carbon bonds, and has a wide potential window on the reduction side sufficient to measure heavy metal ions by electrochemical reaction based on reduction deposition and oxidation elution reaction. The carbon thin film is a boron-doped diamond thin film, a conductive diamond-like carbon thin film, or a sputtered carbon thin film.

  In the above endotoxin concentration measuring method, endotoxin is at least one selected from the group consisting of endotoxin, lipopolysaccharide, pyrogen, and pyrogen.

  A second invention is a kit for carrying out the above endotoxin concentration measuring method, comprising a carrier on which the first endotoxin recognition molecule is immobilized, an endotoxin recognition probe comprising a heavy metal compound and a second endotoxin recognition molecule, and an acid An endotoxin concentration measurement kit comprising a solution.

  A third invention is a device for carrying out the above-mentioned endotoxin concentration measuring method, an electrochemical cell comprising a carrier on which the first endotoxin recognition molecule is immobilized and a working electrode, means for applying voltage to the electrode, and current It is an endotoxin concentration measuring device characterized by including a measurement means.

  According to the endotoxin concentration measurement method, concentration measurement kit, and concentration measurement device of the present invention, electrochemical measurement is utilized, regardless of the skill level of the user, easily and with high sensitivity (ng / mL order or less). Can measure the concentration of endotoxin.

It is the conceptual diagram which showed the principle of the density | concentration measuring method of endotoxin in this invention. In Example 1, the rectangular wave voltammogram at the time of measuring zinc ion of various density | concentrations using a sputter carbon thin film electrode as a working electrode is shown. Moreover, FIG.2 (b) is a figure which shows the relationship between the peak electric current value obtained from the voltammogram of Fig.2 (a), and zinc ion concentration. In Example 2, when a sputtered carbon thin film electrode was used as a working electrode, a sample solution containing endotoxin was allowed to act on ET cleans, and after the endotoxin-recognizing probe was captured, the solution eluting zinc ions was measured. 5 is a graph showing the relationship between the endotoxin concentration and response current value.

1 Measurement System Using Endotoxin Recognition Particles and Endotoxin Recognition Probe 2 Carrier 3 First Endotoxin Recognition Molecule 4 Endotoxin 5 Endotoxin Recognition Probe 6 Electrode 7 Heavy Metal Ion

  The present invention is a method for measuring the concentration of endotoxin contained in a sample by an electrochemical measurement method. The principle of endotoxin concentration measurement of the present invention will be described below. FIG. 1 is a schematic diagram illustrating the principle of the endotoxin concentration measurement method in the present invention.

  As illustrated in FIG. 1, in the present invention, the carrier (2) on which the first endotoxin recognition molecule (3) is chemically cross-linked and immobilized is used as a capture material for the endotoxin (4). The carrier of the present invention is not limited as long as the first endotoxin-recognizing molecule can be immobilized, and may be spherical or irregularly shaped particles or fine particles, and may be in the form of a fiber, a film, a plate, or a column. good. The carrier may be porous or nonporous. Considering the preparation for availability, fine particles for immunoassay, spherical or monolithic chromatographic carriers, filters, fibers and the like can be preferably used. Add endotoxin sample to carrier (2) and capture. Next, an endotoxin recognition probe (5) having a heavy metal compound site is allowed to act on the endotoxin (4). At this time, the endotoxin recognition molecular probe (5) is captured according to the amount of endotoxin (4) captured on the carrier (2). Subsequently, by adding an acid solution to the carrier (2) on which the probe (5) is captured, heavy metal is eluted from the probe (5) as heavy metal ions (7). An eluate containing heavy metal ions (7) is added to a predetermined buffer solution, and a working electrode (6) capable of electrochemical measurement of the heavy metal ions (7) is used to determine the concentration of heavy metal ions in the eluate. Quantify. Here, the electrochemical measurement of heavy metal ions is an anodic stripping voltammetry (ASV) method. The ASV method is an analytical method that detects the oxidation current value obtained by reducing and depositing metal ions once on the electrode and oxidizing the deposited metal. This is an analysis method suitable for on-site analysis.

  Therefore, by creating a calibration curve between the known endotoxin amount and the heavy metal ion amount constituting the heavy metal compound in the endotoxin recognition probe captured accordingly, and measuring the heavy metal ion, the amount of endotoxin in the sample can be calculated. It becomes possible to know.

  In the present invention, the amount of heavy metal ions is measured by measuring the current flowing through the electrode, which is basically caused by the transfer of electrons, and the amount of endotoxin is ascertained. Through the process of deposition (concentration) of heavy metal ions. Therefore, even if the endotoxin recognition probe trapped on endotoxin, that is, the amount of heavy metal component is small, it contains the effect of concentrating heavy metal ions on the electrode surface, which makes it possible to measure the amount of endotoxin with high sensitivity. Have

  These series of reactions were made possible not only by concentrating the endotoxin-recognizing molecule on the carrier surface, but also by depositing and concentrating the heavy metal ions constituting the endotoxin-recognizing probe on the working electrode. This results in an improvement in the detection limit of the measured concentration, which was impossible to achieve with the endotoxin concentration measurement method in US Pat.

  Although there is no restriction | limiting in particular as a material of the electrode utilized in this invention, A carbon-type material is used conveniently from a viewpoint of the electrode raw material which has a wide electric potential window which can measure ASV of final heavy metal ions.

  The first endotoxin recognition molecule immobilized on the carrier surface is mainly a biomolecule or polycation synthesis molecule composed of protein, polypeptide, polyamino acid, and nucleic acid. Specific binding to endotoxin, such as α-lysine, polymyxin B, cetylpyridinium, LPS binding protein, endotoxin neutralizing protein, anti-LPS antibody, polypeptide, oligopeptide, oligonucleotide (aptamer), polyethyleneimine, polyallylamine A recognition molecule with the ability can be used. The specific adsorptivity of polyε-lysine to LPS is described in, for example, JP-A-2002-263486 and Analytical Biochemistry 385 (2009) 368-370.

  In the endotoxin recognition probe composed of the second endotoxin recognition molecule and the heavy metal compound used in the present invention, the second endotoxin recognition molecule constituting the probe includes at least the substance group having endotoxin recognition ability described above. A molecule composed of one kind can be used as appropriate. The heavy metal compound constituting the endotoxin recognition probe is not particularly limited as long as the eluted heavy metal ions are finally deposited on the electrode, the desorption reaction proceeds, and is a heavy metal species that can be electrochemically measured. Stable and inexpensive ones are advantageously used. Specifically, metal complexes in which zinc, nickel, copper, etc., which are easy to synthesize and form heavy metal compounds are coordinated (for example, complexes composed of ligands such as picolyl groups and heavy metals) and nanoparticles can be formed. Peptide residues consisting of histidine residues that can form coordinate bonds with quantum dots composed of silver, cadmium, lead, zinc, selenium, indium, etc., heavy metals such as nickel, zinc, and copper (His-tag) Etc. can be used as appropriate. Furthermore, as a heavy metal used in the present invention, zinc, silver, copper, cobalt, selenium and the like can be more preferably used from the viewpoint of relatively low toxicity and safe use. As an example, the structural formula of an endotoxin recognition probe composed of a dipicolylamine-zinc complex that is a heavy metal compound and cetylpyridinium bromide that is an endotoxin recognition molecule is shown.

  Next, an embodiment of the present invention will be described in detail with an example. Here, a sputtered carbon thin film will be described as an example. First, an endotoxin sample was dropped onto endotoxin capture particles (ET Cleans, manufactured by JNC Corporation) in which poly ε-lysine, the first endotoxin recognition molecule, was chemically cross-linked and fixed on cellulose fine particles (diameter 40-130 μm). ET cleans captured on the surface. Next, an endotoxin recognition probe composed of cetylpyridinium bromide, which is a second endotoxin recognition molecule, and a dipicolylamine-zinc complex, which is a heavy metal compound, is allowed to act on the endotoxin. At this time, the endotoxin recognition probe is captured according to the amount of endotoxin. An acid solution is added here, zinc in the adsorbed probe is eluted as zinc ions, and the eluate is recovered. The endotoxin is obtained by adding the sample solution containing zinc ions thus treated to an appropriate buffer solution and performing electrochemical measurement in combination with a working electrode, an appropriate counter electrode, and a reference electrode. Depending on the amount, the oxidation current value of the trapped zinc ions is obtained.

  There is no particular limitation on the concentration of the endotoxin recognition probe that acts on the endotoxin captured on the surface of the particulate carrier, but a concentration of about 1-100 μmol / L is advantageous as a concentration that can give a clear current response. In addition, regarding the type of buffer solution used and its pH, it is necessary to use an electrolyte that is easy to deposit on the electrode and an electrolyte that forms a complex with the molecules that make up the buffer solution after elution of heavy metal ions in the probe to be used. preferable. For example, when an endotoxin recognition probe having the above zinc complex is used, a weakly alkaline ammonia buffer having a pH of 8.0 and a concentration of 10-100 mmol / L is advantageously used.

Specifically, when an endotoxin recognition probe having a zinc complex is used as an endotoxin recognition probe, when ASV measurement is performed at pH 8.0, the following reaction occurs on the electrode.
(A) Zn ²⁺ + 2e → Zn (deposition reaction on the electrode)
Next, when a positive voltage is applied to the electrode, the following oxidation reaction occurs (metal elimination reaction).
(B) Zn → Zn ²⁺ + 2e
At this time, the amount of e in the above reaction formula (b), that is, the current in the electrode depends on the zinc ion present in the sample, and the applied voltage and applied voltage time to the working electrode required for the deposition reaction of (a). To do.

  That is, an oxidation current is applied when zinc concentrated and deposited on the electrode surface causes a desorption reaction. The oxidation current at this time is substantially proportional to the amount of zinc deposited on the electrode surface. Therefore, as the amount of zinc deposited on the electrode surface increases as the endotoxin concentration in the sample increases, the resulting current also increases. In other words, the amount (concentration) of endotoxin present can be determined from the degree of increase in current.

  In the above embodiment, an example of a combination of a zinc complex and cetylpyridinium bromide has been described. Needless to say, a zinc complex-LPS recognition peptide, or another heavy metal compound-LPS recognition peptide such as a heavy metal compound capable of ASV measurement and LPS It may be a combination of other probes composed of recognition molecules.

  The endotoxin to be measured in the present invention is not particularly limited. For example, endotoxin (a toxin that is a component of the cell wall and is not secreted actively), lipopolysaccharide, pyrogen, and pyrogen are included. If it is, it can measure more efficiently.

  The endotoxin measurement kit and device of the present invention include an electrochemical cell, an endotoxin recognition probe, and a heavy metal for extracting at least a solid support such as a fine particle or a substrate on which an endotoxin recognition molecule is immobilized and a working electrode for measurement. It is characterized by having an acid reagent and a buffer solution for measuring heavy metals. Furthermore, it is good also as a measurement kit or device combining the member for constructing the measurement system of this invention, such as a washing | cleaning liquid, a pH adjuster, an electric current measuring apparatus, the pump of flow cell construction, or a reagent.

  The measurement method, measurement kit, and measurement device of the present invention as described above are used for the manufacture, storage, analysis, use, etc. of pharmaceuticals such as culture solution, biological sample, injection solution, infusion solution, dialysate, and purified water that is a raw material thereof. It is used in the related fields. For example, parenteral drugs such as injectable preparations are mainly produced by fermentation technology or genetic recombination technology, and there is concern about contamination from host cell wall components. It is necessary to remove the endotoxin that causes it. In the treatment of septic patients, blood endotoxin is removed by artificial dialysis by extracorporeal circulation. In order to determine whether or not these endotoxins have been removed as intended, a method for simply measuring a minute amount of endotoxins is required. Furthermore, endotoxin confirmation tests are indispensable also in the manufacture of medical devices such as syringes and dialysis membranes. Under such circumstances, the measurement method and measurement kit of the present invention are extremely effective in measuring these endotoxins, and in the measurement kit of the present invention, regardless of the skill level of the user, Endotoxin concentration can be measured. The specific form in that case is not specifically limited to the said measurement kit, For example, you may use as a detector for flow injection analysis. In this case, it is more preferable to use a form that is incorporated in the final step after treatment such as artificial dialysis.

  Moreover, unless otherwise defined, terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Terms used herein should be interpreted as having a meaning consistent with the meaning in this specification and the related technical field, unless otherwise defined, idealized, or overly formal. It should not be interpreted in a general sense.

  Hereinafter, although this invention is demonstrated in detail using an Example and a comparative example, this invention is not limited to these Examples. As an electrochemical measurement method, an example using rectangular wave voltammetry will be described below, but other methods such as cyclic voltammetry, differential pulse voltammetry, normal pulse voltammetry, and alternating current voltammetry can be used as appropriate.

Example 1 Evaluation of Zinc Ion Measurement Performance with Sputtered Carbon Thin Film Electrode A sputtered carbon thin film electrode formed on a silicon wafer was used as a working electrode. An insulating tape with a hole having a diameter of 2 mm was attached to the produced sputtered carbon thin film to obtain a sputtered carbon thin film electrode having a known electrode area. This thin film electrode was inserted into a 100 mmol / L ammonia buffer (pH 8.0), and a potentiostat (CHI Instruments, ALS1240B) was subjected to a salt bridge (silver-silver chloride: Ag / AgCl) and a counter electrode (platinum: Pt).

Next, adjust the concentration of zinc ions (Zn ²⁺ ) in the ammonia buffer to be 0 to 1000 ng / mL, and set the reduction deposition potential to -1.5 V and the deposition time to 240 seconds. The zinc ions in the solution were deposited on the electrode surface. Subsequently, the frequency was set to 40 Hz, the potential increment was 2 mV, and the amplitude was 25 mV, and rectangular wave voltammetry measurement was performed for each concentration. The measurement results are shown in FIG. In FIG. 2 (a), the horizontal axis represents the applied voltage, and the vertical axis represents the oxidation current accompanying zinc elution. From the obtained measurement results, an increase in oxidation current with zinc concentration was clearly observed. Actually, as shown in Fig. 2 (b), the relationship between zinc concentration and oxidation current value shows linearity in the concentration range of 5 to 1000 ng / mL, and quantitative zinc ions up to a low concentration range of 5 ng / mL. It was found that it was possible to detect.
Example 2: Endotoxin concentration measurement with ET Cleans and zinc complex probe
Weigh 10 mg of ET Cleans in a microtube, drop 200 μL of Japanese Pharmacopoeia standard endotoxin standard with different concentration here, shake at room temperature for 120 minutes to adsorb endotoxin, and then clean the surface of ET Cleans with Tris buffer physiology. Washed with saline (TBS). Subsequently, 200 μL of 100 μmol / L endotoxin recognition probe (comprised of dipicolylamine-zinc complex and cetylpyridinium bromide) TBS solution was added dropwise, shaken at room temperature for 60 minutes, and the probe was ET Cleans surface via the adsorbed endotoxin. After adsorbing on the surface, the surface of ET Cleans was washed with TBS. By this operation, the probe can be captured on the ET clean s surface. Further, 200 μL of a 0.1 mol / L sulfuric acid aqueous solution was dropped here and shaken for 10 minutes to elute zinc in the probe as zinc ions, and the eluate was collected. A solution obtained by diluting the eluate 100 times with 50 mM ammonia buffer (pH 8.0) was used as a measurement solution.

  The above-described measurement solution was subjected to the operation described in Example 1 using a sputtered carbon thin film electrode as a working electrode, and rectangular wave voltammetry measurement was performed on the measurement solution obtained by causing each concentration of endotoxin to act. The measurement results are shown in FIG. In FIG. 3, the horizontal axis represents the endotoxin concentration, and the vertical axis represents the response current value. As apparent from FIG. 3, an increase in current depending on the concentration of endotoxin added to ET Cleans was observed. In this method, it was estimated that about 200 pg / mL endotoxin could be quantified.

Claims (10)

A method for measuring the concentration of endotoxin contained in a sample, comprising the following steps:
(1) A step of contacting the sample with a carrier on which the first endotoxin recognition molecule is immobilized, and capturing and concentrating the endotoxin in the sample on the carrier,
(2) contacting an endotoxin recognition probe comprising a heavy metal compound and a second endotoxin recognition molecule with a carrier, and capturing the endotoxin recognition probe on the endotoxin on the carrier;
(3) A step of reacting an endotoxin-recognizing probe with an acidic solution to elute heavy metal ions from the heavy metal compound of the probe, and (4) electrochemically measuring the amount of heavy metal ions eluted using a working electrode that measures heavy metal ions. Measuring process,
And measuring the endotoxin concentration in the sample using the amount of heavy metal ions as an indicator.   The method for measuring an endotoxin concentration according to claim 1, wherein an endotoxin concentration of 200 pg / mL or more can be measured.   The endotoxin recognition molecule is at least one selected from the group consisting of poly-ε-lysine, poly-α-lysine, polymyxin B, cetylpyridinium, protein, polypeptide, oligopeptide, oligonucleotide, polyethyleneimine, and polyallylamine; The endotoxin concentration measuring method according to claim 1 or 2, wherein the one endotoxin recognition molecule and the second endotoxin recognition molecule are the same or different.   The method for measuring an endotoxin concentration according to claim 1 or 2, wherein the heavy metal compound of the endotoxin recognition probe is a heavy metal complex compound.   The endotoxin concentration measuring method according to claim 4, wherein the heavy metal in the heavy metal complex compound is zinc, silver, copper, cadmium, nickel, cobalt, lead, selenium or indium. The working electrode is a carbon thin film having sp ³ carbon bonds of 40% or more and having a wide potential window on the reduction side sufficient to measure heavy metal ions by electrochemical reaction based on reduction deposition and oxidation elution reaction. 1 or 2 endotoxin concentration measurement method.   The endotoxin concentration measuring method according to claim 6, wherein the carbon thin film is a boron-doped diamond thin film, a conductive diamond-like carbon thin film, or a sputtered carbon thin film.   The endotoxin concentration measuring method according to any one of claims 1 to 7, wherein the endotoxin is at least one selected from the group consisting of endotoxins, lipopolysaccharides, pyrogens, and pyrogens.   A kit for carrying out the endotoxin concentration measuring method according to any one of claims 1 to 8, comprising a carrier on which the first endotoxin recognition molecule is immobilized, an endotoxin recognition probe comprising a heavy metal compound and a second endotoxin recognition molecule, and An endotoxin concentration measurement kit comprising an acidic solution.   A device for carrying out the endotoxin concentration measuring method according to any one of claims 1 to 8, comprising a carrier on which a first endotoxin recognition molecule is immobilized and a working electrode, and means for applying a voltage to the electrode. And an endotoxin concentration measuring device characterized by comprising current measuring means.