JPWO2021106124A1 - Preparation equipment, detection system, and detection method - Google Patents

Abstract

The preparation instrument (2) is used in a detection system (1) for detecting a non-proteinogenic substance contained in a sample obtained from a living body, and prepares a sample (20) for detection. The preparation instrument (2) includes a container (21) having a container (22) capable of accommodating a sample and a squeezing body (23) capable of squeezing a sample contained in the container (22), and an accommodating body. It is contained in (22) and includes an ion exchanger for removing impurities from the sample, and a protein flocculant contained in the container (22) for removing protein from the sample. With the sample contained in the container (22), the extract extracted when the sample is squeezed by the squeezed body (23) is reacted with the ion exchanger and the protein flocculant and used as the sample (20). As a result, the contained substances contained in the sample are detected.

Description

The present invention relates to a preparation instrument, a detection system, and a detection method used in a detection system for detecting a substance contained in a living body.

Various devices and methods for detecting various non-proteinogenic substances contained in a living body (hereinafter referred to as contained substances) have been proposed. For example, Patent Document 1 discloses a method for quantifying histamine. In this quantification method, first, a histamine-containing sample solution is prepared from a sample such as seafood or meat by a trichloroacetic acid extraction method, a methanol extraction method, or a neutral phosphate buffer heat extraction method. Next, a predetermined amount of histamine oxidase composed of an arbitrary enzyme is added to a predetermined amount of the histamine-containing sample solution, and the mixture is heated and mixed. As a result, H ₂ O ₂ is generated in the reaction solution. Then, by measurement using a three-electrode method, a current value according to the amount of H ₂ O ₂ is measured electrochemically. Finally, histamine is quantified based on the measurement results.

Japanese Unexamined Patent Publication No. 2001-99803

The above method requires steps such as preparation of a histamine-containing sample solution, mixing of histamine oxidase, and measurement. On the other hand, there is a demand for accurate detection of contained substances by a simpler method.

An object of the present invention is to provide a preparation instrument, a detection system, and a detection method used in a detection system for simply and accurately detecting a substance contained in a living body.

The preparation instrument according to the first aspect of the present invention is used in a detection system for detecting a non-proteinaceous substance contained in a sample obtained from a living body, and is a preparation instrument for preparing a sample for detection. A container provided with a container capable of accommodating the sample and a squeezed body capable of squeezing the sample contained in the container to extract an extract, and a container contained in the container and contaminated from the extract. The sample is provided with an ion exchanger for removing a substance and a protein flocculant for removing a protein from the extract, which is contained in the container, and the sample is contained in the container. By reacting the extract extracted when the sample is squeezed by the squeezed body with the ion exchanger and the protein flocculant and using the sample as the sample, the contained substance contained in the sample is detected. It is characterized by that.

According to the first aspect, the sample contained in the container is squeezed by the squeezed body, and then the extract reacts with the ion exchanger and the protein flocculant. In this case, the contaminants contained in the extract are removed by the ion exchanger, and the proteins contained in the extract are removed by the protein flocculant. Therefore, by using the obtained sample, the contained substance contained in the sample can be detected easily and accurately.

In the first aspect, the detection system may detect the contained substance contained in the sample by electrochemical measurement using the sample. In this case, since the sample from which impurities and proteins have been removed can be prepared by the preparation instrument, the contained substances contained in the sample can be detected accurately by electrochemical measurement.

In the first aspect, the contained substance may be an amine. In this case, the preparation instrument can prepare a sample for detecting the amine.

In the first aspect, the amine may be histamine. In this case, the preparation instrument can prepare a sample for detecting histamine.

In the first aspect, the ion exchanger may be an anion exchanger. In this case, the preparation instrument can appropriately remove the anion contaminants contained in the sample by the anion exchanger.

In the first aspect, the protein flocculant may be either polyethylene glycol or dextran. In this case, the preparation instrument can appropriately remove the protein contained in the sample by either polyethylene glycol or dextran.

In a first aspect, a pH regulator that is housed in the container and adjusts the pH of the sample may be further provided. In this case, the preparation instrument can be adjusted to a pH suitable for quantification, so that the accuracy of the detected contained substance can be improved.

The detection system according to the second aspect of the present invention is characterized by comprising a preparation instrument according to the first aspect and a detector for detecting the contained substance using the sample prepared by the preparation instrument. ..

According to the second aspect, the detection system can easily and accurately detect the contained substance by the detector using the sample prepared by the preparation instrument.

In the second aspect, the detector may perform an electrochemical measurement using the sample and detect the concentration of the contained substance based on the amount of electricity applied to the sample. In this case, the detection system can easily detect the contained substance by utilizing the correlation between the concentration of the contained substance contained in the sample and the current value when the voltage is applied.

The detection method according to the third aspect of the present invention is a detection method for detecting a non-proteinaceous substance contained in a sample obtained from a living body, and is housed in a container capable of accommodating the sample and the container. A container equipped with a squeezable body capable of squeezing the sample, an ion exchanger contained in the container for removing impurities, and a protein flocculant contained in the container for removing proteins. The sample is contained in the container, and the sample is squeezed by the squeezed body to extract an extract, and the sample is extracted when the sample is squeezed by the squeezing step. It is characterized by comprising a detection step of detecting a substance contained in the sample by reacting the extract with the ion exchanger and the protein flocculant and using the sample as a sample.

According to the third aspect, the sample contained in the container is squeezed by the squeezed body, and then the extract reacts with the ion exchanger and the protein flocculant. In this case, the contaminants contained in the extract are removed by the ion exchanger, and the proteins contained in the extract are removed by the protein flocculant. Therefore, by using the obtained sample, the contained substance contained in the sample can be measured easily and accurately.

In the third aspect, the detection step may detect the contained substance contained in the sample by electrochemical measurement using the sample. In this case, since the sample from which impurities and proteins have been removed can be prepared, the contained substances contained in the sample can be accurately detected by electrochemical measurement.

In the third aspect, the contained substance may be an amine. In this case, a sample for detecting amine can be prepared.

In the third aspect, the amine may be histamine. In this case, a sample for detecting histamine can be prepared by electrochemical measurement.

In the third aspect, the ion exchanger may be an anion exchanger. In this case, the anion contaminants contained in the extract can be appropriately removed by the anion exchanger.

In the third aspect, the protein flocculant may be either polyethylene glycol or dextran. In this case, the protein contained in the extract can be appropriately removed by either polyethylene glycol or dextran.

In a third aspect, a pH regulator that is housed in the container and adjusts the pH of the sample may be further provided. In this case, since the pH can be adjusted to an appropriate level for quantification, the accuracy of the detected contained substance can be improved.

In the third aspect, the detection step may detect the concentration of the contained substance based on the amount of electricity applied to the sample. In this case, the contained substance can be easily detected by utilizing the correlation between the concentration of the contained substance contained in the sample and the current value when the voltage is applied.

It is a figure which shows the outline of the detection system 1. It is a figure which shows the enzyme reaction. It is a flowchart which shows the detection method. It is a graph which shows the relationship between the concentration of histamine and the current value. It is a figure which shows the outline of the detection system 9 including the preparation instrument 5 in the modification.

Embodiments embodying the present invention will be described in order with reference to the drawings. The drawings to be referred to are used to explain the technical features that can be adopted by the present invention, and the configuration and the like of the devices described are not intended to be limited thereto, but are merely explanatory examples.

<Overview of detection system 1>
The outline of the detection system 1 will be described with reference to FIG. The detection system 1 includes a preparation instrument 2 and a detector 3. In the detection system 1, a sample is prepared from a sample obtained from a living body such as fish meat by the preparation instrument 2. The detector 3 detects non-proteinogenic substances contained in the sample by using the sample prepared by the preparation instrument 2. In this embodiment, the non-proteinogenic substance detected by the detection system 1 is an amine, more specifically histamine.

<Preparation equipment 2>
As shown in FIG. 1, the preparation instrument 2 includes a container 21, an ion exchanger, a protein flocculant, and a pH adjuster. The container 21 has a container 22 and a squeezed body 23. The accommodating body 22 can accommodate a sample inside the tubular outer cylinder 22A. The squeezed body 23 has a pusher 23A and a gasket 23B. The squeezed body 23 can squeeze the sample contained in the container 22 to extract the extract. The ion exchanger, the protein flocculant, and the pH adjuster are pre-contained in the container 22.

The ion exchanger has granules. The ion exchanger is an anion exchange resin capable of removing contaminants of anions in the extract. Anion exchange resin is an ion-exchange resin in which an amino group has been introduced as a functional group, Cl ion, SO ₄ ions, the contaminants anions such as amino acids can be removed. The mass of the ion exchanger contained in the container 22 is, for example, 400 mg.

The protein flocculant has a powdery form. The protein flocculant is a substance capable of removing the protein in the extract, and either polyethylene glycol or dextran is used. The mass of the protein flocculant contained in the container 22 is, for example, 150 mg.

The pH regulator is in powder form. The pH regulator can adjust the pH of the extract to neutral to weakly alkaline. Phosphoric acid is used as an example of a pH adjuster. The mass of the pH regulator contained in the container 22 is, for example, 4 mg.

The sample 20 is obtained by reacting the extract contained in the container 22 with the squeezing body 23 with the ion exchanger, the protein aggregate agent, and the pH adjusting agent. The sample 20 is discharged to the outside through the tube tip 22B of the container 22.

<Detector 3>
As shown in FIG. 1, the detector 3 includes a sensor chip 3A and a main body 3B. The sensor chip 3A has a measuring unit 31 and an electrode 32. A thin film of enzyme (hereinafter referred to as "enzyme film 32A") is formed on the surface of the electrode 32. The sample 20 prepared by the preparation instrument 2 is dropped onto the enzyme membrane 32A. The measuring unit 31 can measure the current value of the current flowing through the sample 20 when a voltage is applied to the electrode 32. The measured current value is output to the main body 3B. The main body 3B detects the concentration of histamine contained in the sample in the sample 20 based on the current value output from the sensor chip 3A. The detected histamine concentration is displayed on the display unit 33 of the main body unit 3B. The detector 3 may be composed of only the sensor chip 3A. In this case, the measuring unit 31 may measure the current value of the current flowing through the sample 20 when a voltage is applied to the electrode 32, and detect the concentration of histamine contained in the sample in the sample 20. The sensor chip 3A may display the detected concentration on a display unit (not shown).

<Decomposition reaction of histamine by enzyme>
As shown in FIG. 2, histamine is oxidized, deaminated and decomposed by reacting with an enzyme. By this reaction, imidazole acetaldehyde is produced from histamine (arrow Y11), and the enzyme is displaced from the oxidized form to the reduced form by the reduction reaction (arrow Y12). In addition, oxygen is decomposed by the oxidation reaction of the reduced enzyme (arrow Y13), and hydrogen peroxide is generated from the decomposed oxygen and water (arrow Y14). That is, in response to the decomposition reaction of one molecule of histamine by the enzyme, one molecule each of histamine and oxygen disappears, and one molecule each of imidazole acetaldehyde and hydrogen peroxide is produced.

<Histamine detection method>
As shown in FIG. 1, a sample obtained from a living body such as fish meat is housed in the container 22 of the container 21 of the preparation device 2 (arrow Y21). As shown in FIG. 3, the sample is squeezed by the squeezing body 23 (S11). Extraction is extracted from the sample by squeezing. The extract reacts with the anion exchange resin contained in the container 22, and the anion contaminants are removed (S13). Further, the extract reacts with the protein flocculant contained in the container 22, and the protein is removed (S15). Further, the extract reacts with the pH adjusting agent contained in the container 22 to adjust the pH (S17). As a result, sample 20 (see FIG. 1) is prepared from the extract. As shown in FIG. 1, the sample 20 is discharged to the outside through the tube tip 22B of the accommodating body 22 and dropped onto the enzyme film 32A on the surface of the electrode 32 in the sensor chip 3A of the detector 3 (arrow Y22, S19 (see FIG. 3)).

At this time, as shown in FIG. 2, the histamine contained in the sample 20 reacts with the enzyme of the enzyme membrane 10 and is decomposed to produce imidazole acetaldehyde (arrow Y11). In addition, oxygen in the sample 20 is consumed by the oxidation reaction by the enzyme that decomposes histamine, and hydrogen peroxide is generated (arrow Y14).

As shown in FIG. 3, in the sensor chip 3A of the detector 3, a voltage is applied to the electrode 32 by the measuring unit 31 (S21). At this time, imidazole acetaldehyde is decomposed (arrow Y15, see FIG. 2), and a current flows through the electrode 32. The measuring unit 31 measures the current value of the flowing current. The measured current value is output from the measuring unit 31 of the detector 3 (arrow Y23, see FIG. 1), and is acquired by the main body unit 3B (S21).

The current value changes depending on the amount of imidazole acetaldehyde remaining in the sample 20. Further, the amount of imidazole acetaldehyde remaining in the sample 20 becomes so large that histamine is decomposed in the sample 20. That is, there is a correlation between the amount of histamine contained in the sample 20 and the current value of the current flowing through the electrode 32 when a voltage is applied. FIG. 4 is a graph showing the correspondence between the current value and the histamine concentration. The x-axis defines the concentration of histamine in the sample. The y-axis defines the current value of the current flowing when a voltage is applied to the electrode 32. As shown in FIG. 4, the histamine concentration (variable x) and the current value (variable y) have a substantially linear relationship. A function showing a straight line that linearly approximates the relationship between the histamine concentration and the current value satisfies the relationship in Eq. (1). However, the unit of the variable x is ppm, and the unit of the variable y is nA.
y = 0.7838x + 65.898 (1)

In the main body 3B of the detector 3, the current value acquired in the step of S21 is substituted into the variable y of the equation (1). The value of the variable x calculated at this time is detected as the concentration of histamine (S23). The detected histamine concentration is displayed on the display unit 33 of the main body unit 3B (S25).

When a voltage is applied to the electrode 32 by the measuring unit 31 in the sensor chip 3A of the detector 3 (S21), hydrogen peroxide may be decomposed. The measuring unit 31 may acquire the current value of the current that flows when hydrogen peroxide is decomposed (S21). The main body 3B of the detector 3 may detect the concentration of histamine based on the current value acquired from the sensor chip 3A (S23).

<Action and effect of this embodiment>
As described above, the sample contained in the container 22 of the preparation device 2 is squeezed by the squeezing body 23 (S11). The extract obtained by squeezing the sample reacts with the ion exchanger and the protein flocculant in the container 22. Contaminants contained in the extract are removed by an ion exchanger (S13), and proteins contained in the extract are removed by a protein flocculant (S15). Therefore, by using the extract obtained as described above as the sample 20, histamine contained in the sample can be detected with high accuracy. Also, the removal of contaminants and proteins and the adjustment of pH are carried out in the container 22 at the same time as the squeezing by the squeezing body 23. Therefore, in the detection system 1, by using the preparation instrument 2, the histamine contained in the sample can be detected easily and accurately by the detector 3.

The detector 3 uses the sample 20 prepared by the preparation instrument 2 and detects histamine by electrochemical measurement (S23). In this case, anionic contaminants and proteins that may reduce accuracy during electrochemical measurement can be easily removed by the preparation instrument 2. Therefore, the detection system 1 can accurately detect the histamine contained in the sample by performing the electrochemical measurement using the sample 20 prepared by the preparation instrument 2.

In the detection system 1, the preparation instrument 2 is used as an instrument used for detecting the concentration of histamine. Therefore, the preparation instrument 2 can easily prepare the sample 20 for detecting histamine by the above method.

An anion exchange resin is preliminarily housed in the container 22 of the preparation instrument 2 as an ion exchanger. In this case, the preparation instrument 2 can appropriately remove anion impurities contained in the extract by using an anion exchange resin. The container 22 of the preparation device 2 contains either polyethylene glycol or dextran as a protein flocculant. In this case, the preparation instrument 2 can appropriately remove the protein contained in the extract by either polyethylene glycol or dextran.

The container 22 of the preparation instrument 2 contains a pH adjuster that adjusts the pH of the sample. The pH of the extract obtained by squeezing the sample is adjusted by a pH adjuster, and then the concentration of histamine is detected by the detector 3. In this case, the preparation instrument 2 can adjust the pH of the sample 20 to a value at which an enzymatic reaction is likely to occur, so that the accuracy of the detected histamine concentration can be improved.

The concentration of histamine contained in the sample correlates with the current value obtained by the electrochemical measurement by the detector 3. Therefore, the detector 3 performs an electrochemical measurement using the sample 20 and detects the concentration of histamine based on the current value of the current flowing through the sample 20. In this case, the detection system 1 can easily and accurately detect the concentration of histamine.

<Modification example>
The present invention is not limited to the above embodiment, and various modifications can be made. FIG. 5 shows the detection system 9 in the modified example. The detection system 9 differs from the detection system 1 (see FIG. 1) in that the preparation instrument 5 is used instead of the preparation instrument 2 (see FIG. 1). The detector 3 used in the detection system 9 is the same as the detector 3 in the detection system 1. In FIG. 5, the detector 3 is omitted. As shown in FIG. 5, the preparation instrument 5 has a first preparation instrument 5A and a second preparation instrument 5B.

The first preparation instrument 5A includes a container 50 and an ion exchanger. The container 50 has an accommodating body 51 and a squeezed body 52. The container 50, the container 51, and the squeezed body 52 correspond to the container 21, the container 22, and the squeezed body 23 in the preparation instrument 2 (see FIG. 1), respectively. Unlike the preparation instrument 2, the squeezing body 52 squeezes the sample in the container 51 by rotating the handle 52A. The ion exchanger is pre-accommodated in the accommodating body 51. The ion exchanger is the same as the ion exchanger used in the detection system 1.

The second preparation instrument 5B includes a container 55, a protein flocculant, and a pH adjuster. The container 55 has an accommodating body 56. Unlike the first preparation instrument 5A, the container 55 of the second preparation instrument 5B does not have a squeezed body. The protein flocculant and the pH adjuster are pre-contained in the container 51. The protein flocculant and the pH adjuster are the same as the protein flocculant and the pH adjuster used in the detection system 1.

The method for detecting histamine in the detection system 9 is as follows. A sample obtained from a living body such as fish meat is contained in the container 51 of the container 50 of the first preparation device 5A (arrow Y31). The sample is squeezed by the squeezer 52 (S11, see FIG. 3). Extraction is extracted from the sample by squeezing. The extract reacts with the anion exchange resin contained in the container 51 to remove anion contaminants (S13, see FIG. 3). After the reaction with the anion exchange resin, the extract is released from the container 51 to the outside and is housed in the container 56 of the second preparation device 5B (arrow Y32).

The extract contained in the container 56 of the second preparation device 5B reacts with the protein flocculant contained in the container 56 to remove the protein (S15, see FIG. 3). Further, the extract reacts with the pH adjusting agent contained in the container 56 to adjust the pH (see S17, FIG. 3). As described above, the sample 20 is prepared from the extract. The sample 20 is discharged to the outside from the housing 56 and dropped onto the enzyme membrane 32A on the surface of the electrode 32 in the sensor chip 3A of the detector 3 shown in FIG. 1 (see S19 and FIG. 3). The method for detecting the concentration of histamine by the detector 3 is the same as that for the detection system 1 (S21 to S25, see FIG. 3).

In the above, the ion exchanger and the protein flocculant may be contained in the container 51 of the first preparation device 5A, and the pH adjuster may be housed in the container 56 of the second preparation device 5B. Further, the protein flocculant may be contained in the container 51 of the first preparation device 5A, and the ion exchanger and the pH adjuster may be housed in the container 56 of the second preparation device 5B.

<Other variants>
Hereinafter, a modification of the detection system 1 will be described, but it goes without saying that it can be appropriately applied to the detection system 9. The method for detecting histamine by the detector 3 is not limited to the method by electrochemical measurement. For example, the detector 3 may detect histamine by various well-known methods using the sample 20 prepared by the preparation instrument 2. For example, the detector 3 may detect histamine by a fluorescence intensity method (AOAC method), an HPLC method, an enzyme immunoassay method, or the like.

The detection target detected by the detection system 1 is not limited to histamine. For example, the detection system 1 may detect amines containing histamine. Further, the detection system 1 may detect a non-proteinogenic substance contained in a sample obtained from a living body.

The ion exchanger housed in the container 22 of the preparation instrument 2 is not limited to the anion exchange resin, and a cation exchanger may be used under the condition that the contained substance is not adsorbed. Further, both the anion exchange resin and the cation exchange resin may be contained in the accommodating body 22. The ion exchanger is not limited to the resin. The protein flocculant contained in the container 22 of the preparation device 2 is not limited to polyethylene glycol and dextran, and may be other materials. The pH adjuster contained in the container 22 of the preparation instrument 2 is not limited to the phosphoric acid powder, and other substances capable of making the sample 20 neutral to weakly alkaline may be used. The pH regulator does not have to be contained in the container 22.

The method for quantifying histamine in the step of S23 is not limited to the method using the formula (1). For example, a table showing the correspondence between the current value and the concentration of histamine may be prepared in advance. In this case, it may be specified by acquiring the concentration of histamine corresponding to the current value acquired in the step of S21 in the table.

<Others>
The treatment of S11 is an example of the "squeezing step" of the present invention. The process of S23 is an example of the "detection step" of the present invention.

1, 9: Detection system 2, 5: Preparation instrument 3: Detector 5A: First preparation instrument 5B: Second preparation instrument 21, 50, 55: Container 22, 51, 56: Containment 23, 52: Squeezed body

Claims (17)

It is used in a detection system that detects non-proteinogenic substances contained in samples obtained from living organisms, and is a preparation device for preparing samples for detection.
A container provided with a container capable of accommodating the sample and a squeezed body capable of squeezing the sample contained in the container to extract an extract.
An ion exchanger that is housed in the container and for removing impurities from the extract,
A protein flocculant that is contained in the container and for removing the protein from the extract,
Equipped with
With the sample contained in the container, the extract extracted when the sample is squeezed by the squeezed body is reacted with the ion exchanger and the protein flocculant and used as the sample. A preparation instrument characterized in that the contained substance contained in the sample is detected. The detection system is
The preparation instrument according to claim 1, wherein the contained substance contained in the sample is detected by an electrochemical measurement using the sample. The preparation instrument according to claim 1 or 2, wherein the contained substance is an amine. The preparation instrument according to claim 3, wherein the amine is histamine. The preparation instrument according to any one of claims 1 to 4, wherein the ion exchanger is an anion exchanger. The preparation instrument according to any one of claims 1 to 5, wherein the protein flocculant is any one of polyethylene glycol and dextran. The preparation device according to any one of claims 1 to 6, further comprising a pH adjuster which is contained in the container and adjusts the pH of the sample. The preparation device according to any one of claims 1 to 7.
A detection system comprising a detector for detecting the contained substance using the sample prepared by the preparation instrument. The detector is
The detection system according to claim 8, wherein an electrochemical measurement is performed using the sample, and the concentration of the contained substance is detected based on the amount of electricity applied to the sample. It is a detection method for detecting non-proteinogenic substances contained in samples obtained from living organisms.
A container provided with an accommodating body capable of accommodating the sample and a squeezing body capable of squeezing the sample contained in the accommodating body, an ion exchanger housed in the accommodating body for removing impurities, and the above-mentioned With the sample housed in the container, the sample is squeezed by the squeezing body to extract the extract using a preparation device that is housed in the container and equipped with a protein flocculant for removing the protein. Squeezing process and
The present invention comprises a detection step of detecting a substance contained in the sample by reacting the extract extracted when squeezed by the squeezing step with the ion exchanger and the protein flocculant and using the sample as a sample. A detection method characterized by that. The detection step is
The detection method according to claim 10, wherein the contained substance contained in the sample is detected by electrochemical measurement using the sample. The detection method according to claim 10 or 11, wherein the contained substance is an amine. The detection method according to claim 12, wherein the amine is histamine. The detection method according to any one of claims 10 to 13, wherein the ion exchanger is an anion exchanger. The detection method according to any one of claims 10 to 14, wherein the protein flocculant is any one of polyethylene glycol and dextran. The detection method according to any one of claims 10 to 15, further comprising a pH adjuster which is contained in the container and adjusts the pH of the sample. The detection step is
The detection method according to claim 11, wherein the concentration of the contained substance is detected based on the amount of electricity applied to the sample.

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