JP2007006880A - Method for measuring enzyme and apparatus for measuring the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for measuring an enzyme, capable of giving a result on the spot, without requiring a waiting time due to an induction period, capable of continuously collecting and measuring a sample, and capable of following a change of a concentration of the enzyme in the sample with the lapse of time, and to provide an apparatus for measuring the same. <P>SOLUTION: This method for measuring the enzyme comprises utilizing autocatalytic reaction in which the enzyme acts as a catalyst and calculating an amount of the enzyme contained in a sample solution based on a time by which the reaction has rapidly proceeded, wherein the sample solution is mixed with a reagent participating in the autocatalytic reaction, so as to form a mixed solution, the mixed solution is poured into a channel at a prescribed flow rate, the autocatalytic reaction is made to arise in the channel, and the amount of the enzyme contained in the sample solution is calculated based on a state of a change of a color developed in the channel in accordance with the autocatalytic reaction. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to an enzyme measurement method and a measurement apparatus. Enzyme measurement is performed in various fields as a technique for enzyme-labeling target organisms and chemical substances and detecting them in an analysis called enzyme immunoassay (EIA), for example. If this invention is used, it will become possible to measure an enzyme rapidly, simply and with high sensitivity. The fields of application include all fields that measure enzymes, and include a wide range of fields including clinical, pharmaceutical, pharmaceutical, food, and environmental analysis. Specific uses include detection of endocrine disrupting substances (environmental hormones), measurement of hormone-like effects, identification of causative substances for atopic dermatitis, measurement of cedar pollen scattering, and measurement of microorganisms such as viruses and bacteria Is mentioned.

  The immunoassay is a technique using an antigen-antibody reaction, which is a specific immune reaction in a living body, and is an excellent analytical technique capable of measuring various targets such as allergic reaction causative substances, viruses and bacteria with high specificity. The measurement principle is to indirectly quantify the antigen to be measured by measuring the amount of labeling substance bound to the antigen.

  As the labeling substance, radioactive substances, fluorescent substances, luminescent substances, enzymes and the like are used. In particular, an analytical method using an enzyme as a label is called an enzyme immunoassay (EIA), which is safer and easier to operate than other methods and has features such as high sensitivity and selectivity, and is widely used. Yes. As a method for measuring a labeling enzyme, a method of measuring absorbance using a coloring reagent and a method of using chemiluminescence are widely used.

However, in these methods, there is a problem in that the measurement accuracy, labor, and cost of the labeling enzyme measurement system are largely limited for the entire measurement system. On the other hand, the prior art described in Patent Document 1 focuses on peroxidase (hereinafter referred to as POD), which is a representative labeling enzyme, and utilizes an autocatalytic reaction in which POD acts as a catalyst. And providing a method for determining the amount of POD from the time until the reaction proceeds abruptly (induction period). In this prior art, the autocatalytic reaction in which the POD acts as a catalyst is a sulfite-hydrogen peroxide reaction or a Co- (5-Br-PAPS) ₂ -peroxo-sulfuric acid reaction.

In addition, the end point of the induction period can be visually recognized as a rapid change in pH or a sudden change in the color state accompanying the progress of decomposition of Co (5-Br-PAPS) ₂ , without requiring a special detection device. Convenient. By this technology, simple and low-cost enzyme measurement is realized.
JP 2001-333797 A

  However, in the method of Patent Document 1, since the reaction proceeds batchwise, it is necessary to measure time with, for example, a stopwatch and wait for discoloration during the induction period. In addition, this prior art is a method for measuring the enzyme concentration of one sample and has practical limitations such as continuous monitoring is not possible.

  Therefore, in the present invention, without waiting for the induction period, the result can be known on the spot, the sample can be continuously collected and measured, and the change in the enzyme concentration in the sample over time can be tracked. It is an object of the present invention to provide an enzyme measuring method and a measuring apparatus that can be used.

  In order to achieve the above object, the enzyme measuring method according to claim 1 of the present invention uses an autocatalytic reaction in which the enzyme acts as a catalyst, and is based on the time (induction period) until the reaction proceeds rapidly. An enzyme measuring method for determining the amount of enzyme contained in a sample solution, wherein a reagent involved in an autocatalytic reaction is mixed with the sample solution, and the mixed solution is allowed to flow through the channel at a constant flow rate. It is characterized in that an autocatalytic reaction is caused and the amount of enzyme contained in the sample liquid is obtained from the discolored state appearing in the flow path accompanying the autocatalytic reaction.

  The method for measuring an enzyme according to claim 2 of the present invention shows the method for measuring the amount of enzyme according to claim 1, and is the length of the discoloration region that appears in the flow path with the autocatalytic reaction, or The amount of the enzyme contained in the sample solution is measured from either one of the lengths of the non-discolored region.

  The enzyme measurement method according to claim 3 of the present invention is an apparatus for carrying out the enzyme measurement method according to claims 1 and 2, comprising means for holding a reagent, means for holding a sample solution, It is characterized by comprising a liquid means, a flow path for flowing the reagent, the sample liquid and a mixture thereof, a means for mixing the reagent and the sample liquid, and a means for detecting an autocatalytic reaction.

  The enzyme measuring method according to claim 4 of the present invention specifies the properties of the flow channel according to claim 3, and the portion of the flow channel through which the mixed solution of the reagent and the sample solution flows is transparent. Features.

  The enzyme measuring method according to claim 5 of the present invention specifies the shape of the flow channel according to claim 4, and the portion of the flow channel through which the mixed liquid of the reagent and the sample solution flows has a helical structure. It is characterized by that.

  The enzyme measurement method according to claim 6 of the present invention is an invention that enables measurement of a plurality of samples, and is an apparatus according to claims 3 to 5, comprising means for holding a plurality of sample solutions, and a plurality of samples. The flow path is provided.

  A method for measuring an enzyme according to claim 7 of the present invention is an invention for realizing a calibration function, and is the apparatus according to claims 3 to 6, wherein the calibration solution does not contain an enzyme (zero calibration solution). And a calibration solution (span calibration solution) containing an enzyme having a known concentration, or both.

  According to the present invention, without waiting for the induction period, the result can be known on the spot, the sample can be continuously collected and measured, and the change in the enzyme concentration in the sample over time can be traced. Provided are an enzyme measurement method and a measurement apparatus which can be used.

  Embodiments of the present invention will be described below, but the scope of the present invention is not limited to this content.

Autocatalytic reaction An autocatalytic reaction is a chemical reaction in which the number of reactants increases or the number of substances that catalyze the reaction increases as the reaction proceeds.
The characteristic of this reaction is that the reaction rate increases exponentially with the progress of the reaction, and the reaction proceeds rapidly when a certain time elapses. A substance that becomes a reaction initiator in this reaction (for example, an enzyme that catalyzes the reaction) is called a trigger. The amount of the trigger can be obtained from the time from the reaction start to the reaction end point (induction period). In the autocatalytic reaction, even if mixing is started and the reaction is started, the induction period proceeds as if there is almost no reaction, and the reaction rapidly proceeds and completes after such an induction period. Therefore, the induction period is a period from the mixing of the reactant and the necessary catalyst in this way until the reaction is completed, and the reaction does not substantially proceed until just before the lapse of the period. This means that the reaction is almost instantaneously completed.

There is a sodium sulfite / hydrogen peroxide reaction system as a system for detecting the progress of reaction by utilizing a change in pH with time in an autocatalytic reaction. In this reaction system, the reaction of formula (1) occurs, and one hydrogen sulfite ion reacts with one hydrogen ion to generate two hydrogen ions.
HSO ³ + H ₂ O ₂ + H ⁺
→ 2H ⁺ + SO ₄ ^2- + H ₂ O (1)

  Since the generated hydrogen ion becomes an element on the reaction side of the formula (1), as the reaction proceeds, the equilibrium is pushed to the generation side, and the reaction rate increases. As a result, hydrogen ions increase exponentially as time elapses, and the pH of the reaction solution rapidly decreases when a certain time (induction period) elapses. Therefore, if a pH indicator such as bromothymol blue (BTB) is added to this solution, the color of the solution instantly changes from blue to yellow as the pH of the solution changes after the induction time has elapsed. . This change in the color state can be sufficiently visually confirmed, and no special detection device or the like is required.

Another example of the autocatalytic reaction is a Co- (5-Br-PAPS) ₂ / peroxo-sulfuric acid reaction system. In this reaction system, the reaction of the formula (2) occurs and Co ²⁺ ions are generated.

（２）
Ｃｏ²⁺イオンは、式（２）の反応に対して触媒として働くため、結果的に、上記の（１）の反応と同様、反応進行とともに反応速度が高まっていく。Ｃｏ−（５−Ｂｒ−ＰＡＰＳ）₂ は紫色、その分解生成物は無色であるため、誘導期間が経過した後、紫色だった反応溶液は無色透明になる。

(2)
Since Co ²⁺ ions act as a catalyst for the reaction of the formula (2), as a result, the reaction rate increases as the reaction proceeds, as in the reaction of the above (1). Since Co- (5-Br-PAPS) ₂ is purple and its decomposition product is colorless, the reaction solution which is purple after the induction period has passed becomes colorless and transparent.

  In both of the two autocatalytic reaction systems described above, horseradish peroxidase (hereinafter abbreviated as HRP), which is a kind of enzyme peroxidase (POD), works as a trigger. HRP is an enzyme often used for labeling antibodies in EIA. The autocatalytic reaction can quantify HRP to a very small amount and is very excellent as a signal detection means of EIA.

3. Form of enzyme measuring method according to the present invention In the enzyme measuring method according to the present invention, an autocatalytic reaction is performed in a flow path. Reagents related to autocatalytic reaction and sample liquid are mixed by flowing into the flow path using a pump or the like, for example, in a flow path (coil) in which a polytetrafluoroethylene tube is wound spirally around a glass rod. Pass through. In such a form, a change (for example, discoloration of a solution) occurs due to an autocatalytic reaction at a position of an arbitrary length of the coil.

For example, the place where the sample solution and the reagent are mixed is set to x = 0, the distance from the place where the change occurs to x, the cross-sectional area at each part of the pipeline is S _(X) , the flow rate is V (constant), When the period is T, these relationships can be expressed by Equation (3).
∫ S _(X) dx = V x T (3)
In equation (3), if the cross-sectional area of the pipe is a constant S,
S x x = V x T
x = V / S × T
And this is
Distance = Flow velocity x Time (4)
Can also be interpreted. That is, not only the flow rate but also the flow rate is constant.
When a system in which BTB is added to the autocatalytic reaction of formula (1) is used, if the HRP concentration in the sample solution is different, the length of the blue band on the glass rod becomes shorter as the concentration increases. This is because as the HRP concentration increases, the speed of the autocatalytic reaction increases and the induction period becomes shorter, resulting in a shorter distance to the place where the change occurs. Based on this fact, the present invention has established a method for detecting an autocatalytic reaction in a flow system.

The autocatalytic reaction proceeds rapidly after a certain induction time. Therefore, as described above, the reaction solution flowing through the flow path at a predetermined flow rate (a constant flow rate in a region where the tube diameter is constant) depends on the induction period (the proportion is proportional in the region where the tube diameter is constant). ) And change. For this reason, at a position of an arbitrary length, the solution is divided into a solution before the reaction suddenly progresses and a solution after the progress.
When the tube diameter is constant, the distance and the induction period are in a proportional relationship, and based on this, the HRP concentration can be specified.
Even if the pipe diameter changes, if the flow rate is constant, the total flow rate during the induction period is measured, and the HRP concentration can be specified because the total flow rate during the induction period and the induction period are in a proportional relationship. it can.
The present inventors conducted BTB / sulfite / hydrogen peroxide autocatalytic reaction, which is an autocatalytic reaction system utilizing pH change, through a flow path. As a result, a blue band and a yellow band appeared with an arbitrary length in the stirring coil as a boundary. In this autocatalytic reaction system, the pH of the reaction solution drops rapidly after a certain induction time. Therefore, the blue band solution corresponds to a high pH solution (before reaction progress), and the yellow band solution corresponds to a low pH solution (after reaction progress). The variation in the position of the boundary between the blue band and the yellow band was about ± 1 mm after 6 hours of continuous operation. For this reason, in the present invention, it is not necessary to monitor the solution until the moment when the reaction proceeds abruptly, unlike the batch autocatalytic reaction in which the color change time is measured visually and with a stopwatch.

Next, regarding the enzyme measuring method and measuring apparatus according to the present invention, the relationship between the examples and the claims will be shown, and further examples will be described.
Example 1. Reagents BTB, sodium sulfite, and HRP were manufactured by Wako Pure Chemical Industries. Hydrogen peroxide manufactured by Kanto Chemical was used. Unless otherwise noted, commercially available special grade reagents were used after diluting with distilled water.

2. A plunger pump can be used as the device liquid feeding means. In this example, two double plunger pumps TCI-NOX1000ω made by Tokyo Chemical Industry were used. As a pump other than the plunger type, a peristaltic pump is suitable when importance is attached to the flow rate ratio, and a piezo pump is suitable for miniaturization.

  A polytetrafluoroethylene tube (outer diameter 1/16 inch, inner diameter 1 mm) manufactured by GL Science was used for the flow path. This tube is translucent, and the color of the liquid in the flow path can be detected visually. Further, a glass tube having a diameter of 7 mm and a length of 300 mm was used for winding the polytetrafluoroethylene tube in a spiral shape. This is because by making the tube spiral, it becomes easy to recognize the discoloration region during visual observation.

3. Preparation of each solution As the sulfite / BTB aqueous solution, 0.64 g of sodium sulfite and 15 mg of BTB were dissolved in distilled water, diluted to 50 mL, and this solution was further diluted 5 times when used in the flow path. Hydrogen peroxide water was used by diluting 4 mL of 30% hydrogen peroxide water to 100 mL with distilled water.
The HRP sample solution was diluted with distilled water, and the HRP concentration adjusted to 100 μgL ⁻¹ was used as a mother liquor, which was further diluted with distilled water.

4). FIG. 1 shows an outline of a flow system apparatus that is an embodiment of the present invention. Using the plunger pump 1, the BTB / sulfite aqueous solution 2 was passed through the flow path A and the hydrogen peroxide solution 3 was flowed through the flow path B at a flow rate of 0.5 mL · min ⁻¹ . Similarly, the HRP sample solution 4 was passed through the channel C at a flow rate of 1.0 mL · min ⁻¹ . 1 is an embodiment for carrying out the present invention, and the apparatus for carrying out the present invention is not limited to that shown in FIG.

  In the apparatus of FIG. 1, the solution mixed in the flow path flows through a reaction coil 5 wound around a glass rod. Since the pH of the solution is weakly alkaline at first, it exhibits a blue color of BTB (region 6 in the figure). From a certain length, the pH rapidly decreases due to autocatalytic reaction and turns yellow. (Region 7 in the figure). For this reason, it can visually discriminate | determine a mode that the reaction coil on a glass rod was divided into the blue area | region before discoloration, and the yellow area | region after a food change. In addition to visual observation, the discolored state can be observed by a CCD camera or a CMOS camera. In addition, a linear image sensor can be used. The apparatuses indicated by reference numerals 8 and 9 are mixers for mixing the reagent and the sample solution, and may be used as necessary. After the measurement, the waste liquid 10 is discharged from the apparatus.

  In this example, using the apparatus of FIG. 1, the length from the left end of the reaction coil to the boundary between blue and yellow, that is, the length of the blue region 6 is measured to measure the HRP concentration in the sample solution. went.

5. In this example, when the HRP concentration in the sample solution was changed to 0 ppm, 1 ppm, 2 ppm, 1 ppm, and 0 ppm, the length of the blue region 6 with the passage of time. Was measured. The results are shown in FIG. As the HRP concentration increased, the length of the blue region 6 became shorter. When the concentration decreased and returned to the initial value of 0 ppm, the length of the blue region was restored.

  HRP catalyzes a reaction in which hydrogen peroxide is decomposed to generate hydrogen ions. As understood from the result of FIG. 2, the present invention can not only measure the enzyme concentration of one sample but also track the concentration change of the sample in real time.

6). Calibration curve The length of the blue region 6 varies depending on the induction period. The present inventors recognize that the induction time of the sulfite / hydrogen peroxide autocatalytic reaction is proportional to the negative common logarithm of the HRP concentration. The coincidence of the correlation with the HRP concentration can be derived from the relationship of the equation (4) in which the distance is proportional to time when the flow rate is constant. The relationship between the length of the blue region 6 and the logarithmic value of the HRP concentration showed good linearity between 1 and 50 ppm (FIG. 3). Thus, the length of the blue region 6 was proportional to the negative common logarithm of the HRP concentration.
In the example according to FIG. 3, the relationship between the length of the blue region 6 and the logarithmic value of the HRP concentration is shown. Thus, when the tube diameter is constant, the distance (the length of the blue region 6) and the induction period are in a proportional relationship, and based on this, the HRP concentration can be specified. That is, a calibration curve can be obtained.
However, as described above, even if the pipe diameter changes, if the flow rate is constant, the total flow rate during the induction period is measured, and the total flow rate during the induction period is proportional to the induction period. The HRP concentration can be specified. That is, a calibration curve can be obtained based on the flow rate.
For example, if the reaction time is slow, the path is made thicker and the flow rate is reduced at the beginning of the reaction, and the radius is set to be close to the arrival time when the reaction appears (the end time of the induction period). It is also possible to configure the tube (tube) so as to reduce the thickness and increase the flow rate. It is also possible to gradually narrow the tube and gradually increase the pulling flow rate at the same flow rate. Even in this form, the total flow rate during the induction period can be measured. In this way, the flow rate is not necessarily constant, the flow rate is constant, the total flow rate during the induction period is measured, and the HRP concentration is specified from the proportional relationship between the total flow rate during the induction period and the induction period. This is also included in the technical scope of the present invention.

7). Inhibition of Blank Reaction In the autocatalytic reaction represented by the formula (1) used in this example, when reagents 2 and 3 are mixed, the reaction proceeds even if HRP is not present. In order to reduce the influence of this so-called blank reaction, it is preferable that the sample solution 4 is brought close to the mixing timing of the reagent and the sample solution. The most preferred method is to mix the reagent and sample solution simultaneously.

Next, in addition to the embodiments described with reference to FIGS. 1 to 3, modifications for implementing the present invention will be described.
A. Measurement of multiple samples When measuring multiple samples, configure multiple containers holding sample solutions and their associated channels, add a channel switching mechanism such as a solenoid valve, and add the desired sample solution. What is necessary is just to comprise an apparatus so that liquid feeding can be carried out.

B. Calibration Function Assuming that the apparatus of the present invention is put into practical use, it is important to realize the calibration function. The configuration of the apparatus having a calibration function is basically the same as the configuration of the apparatus used in the embodiments according to FIGS. If one of the containers holding the sample solution is provided with a calibration solution (zero calibration solution) that does not contain an enzyme, zero calibration can be performed using that solution. Further, if a calibration solution (span calibration solution) containing an enzyme with a known concentration is provided, span calibration can be performed. A calibration function can be realized by providing either or both of these.

C. Eliminating the effects of temperature fluctuations The reaction rate of autocatalytic reactions is affected by temperature. By adjusting the apparatus to a constant temperature, it is possible to avoid the influence of the ambient temperature.
In addition, if the influence of temperature change on the reaction rate for a given autocatalytic reaction is known in advance, a temperature sensor can be provided in the apparatus, and the temperature influence can be corrected by calculation based on the temperature. This method is preferable for simplifying the apparatus because the temperature control mechanism of the apparatus can be omitted.

D. Micronization In the enzyme measuring method according to the present invention, reagents are continuously consumed during the measurement. In order to reduce reagent consumption, micro measurement systems are effective. If a microchip flow system is realized, it is expected to be an environmentally harmonious measurement method that reduces the load on the environment.

  The system using the batch-type autocatalytic reaction of Patent Document 1 is a system in which the measurement result is known after the induction period is over. In contrast, in the enzyme measurement method according to the present invention, since the measurement result always appears during the measurement, the result can be known on the spot without waiting for the induction period.

  Moreover, in the enzyme measuring method according to the present invention, the sample can be continuously collected and measured. As a result, it is possible to track changes in the enzyme concentration in the sample over time. The technique of Patent Document 1 is expected to have a great effect in practice compared to simply measuring the concentration of a single sample.

  Note that the autocatalytic reaction can be sufficiently detected visually as a change in the color state of the reaction solution, and does not require a special detector. This feature is extremely effective in realizing a simple and low-cost measurement system. This effect that can be expected in the invention described in Patent Document 1 can also be expected in the present invention.

  The enzyme measurement method and apparatus according to the present invention can be applied not only to an autocatalytic reaction system but also to other chemical measurement methods using a time measurement system, such as a vibration reaction system and a clock reaction system.

BRIEF DESCRIPTION OF THE DRAWINGS It is a conceptual diagram which shows the outline about one embodiment about the measuring method and apparatus of the enzyme based on this invention. It is a graph which shows the change of the length of a blue region when an HRP density | concentration is changed. It is a graph which shows the relationship between HRP density | concentration and the length of a blue region.

Explanation of symbols

1 Plunger pump 2 BTB / sulfite aqueous solution 3 Hydrogen peroxide solution 4 HRP sample solution 5 Reaction coil 6 Blue region 8, 9 Mixer 10 Waste liquid

Claims (7)

An enzyme measurement method that uses an autocatalytic reaction in which an enzyme acts as a catalyst and determines the amount of enzyme contained in a sample solution based on the time until the reaction proceeds rapidly,
Reagents involved in autocatalytic reaction are mixed with the sample solution to make a mixture,
Flowing the mixture at a constant flow rate through the flow path;
Causing an autocatalytic reaction in the flow path;
A method for measuring an enzyme, characterized in that the amount of enzyme contained in the sample solution is obtained from a discolored state appearing in the flow path in association with an autocatalytic reaction.   2. The amount of enzyme contained in the sample solution is measured from either the length of the color changing region appearing in the flow path in association with the autocatalytic reaction or the length of the non-color changing region. The method for measuring an enzyme according to 1. An apparatus for carrying out the enzyme measurement method according to claim 1 or 2,
Means for holding the reagent;
Means for holding the sample solution;
Liquid feeding means;
An enzyme measuring apparatus comprising: a flow path for flowing the reagent, the sample liquid, and a mixture thereof; means for mixing the reagent and the sample liquid; and means for detecting an autocatalytic reaction.   4. The enzyme measuring apparatus according to claim 3, wherein a portion of the flow path through which the mixed solution of the reagent and the sample solution flows is transparent.   The apparatus for measuring an enzyme according to claim 4, wherein a portion through which the mixed solution flows has a helical structure. Means for holding a plurality of sample solutions;
A plurality of flow paths;
The enzyme measuring apparatus according to claim 3, further comprising a switching mechanism for the flow path. A calibration solution that does not contain enzymes, and
7. The enzyme measuring apparatus according to claim 3, comprising either one or both of a calibration solution containing an enzyme having a known concentration.

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