WO2019111551A1 - Temperature measuring device, reactor, and device and method for evaluating degree of activity of enzyme - Google Patents

Abstract

An evaluating device 100 for evaluating a degree of activity of an enzyme is provided with a thin film 102, a container 110, and a thermographic camera 120. An enzyme 2 to be examined is fixed to a top surface of the thin film 102. The container 110 is filled with a substrate solution 4 which reacts with the enzyme 2. The thermographic camera 120 captures an image of the thin film 102 from the rear surface thereof, with the enzyme 2 in contact with the substrate solution 4.

Description

Temperature measurement apparatus, reactor, evaluation apparatus for enzyme activity and evaluation method thereof

The present invention relates to a technique for evaluating enzyme activity.

Biosensors are used to detect specific components in liquid samples. A calorimetric method has been proposed as one of the biosensor methods. A calorimetric biosensor (hereinafter referred to as a calorimetric biosensor or simply a calorimetric sensor) includes a pair of electrodes forming a thermocouple. An enzyme that reacts with the target substance in the solution is applied to one of the reaction electrodes, and heat is generated by the reaction between the enzyme and the target substance. The other reference electrode is thermally isolated from the reaction electrode so that the temperature does not change. When a temperature rise corresponding to the concentration of the target material occurs in the reaction electrode, a potential difference corresponding to the temperature rise is observed.

The ability of an enzyme is expressed in terms of the amount of base that can be degraded in a certain time, which is called the activity. The unit is generally U (μmol / min). It is known that the activity of the enzyme is lost with time, and it is important to grasp the temporal transition of the activity in the application to which the enzyme is applied.

In the measurement of the degree of activity of the enzyme, various drugs are mixed into the enzyme to be measured and the absorbance is mainly measured, and a kit therefor is commercially available.

JP 2012-183034 A Japanese Patent Publication No. 2016-520591

However, conventional methods require expensive kits. In addition, handling required chemical knowledge and skills in handling drugs. For these reasons, it has been difficult to say that the measurement of the activity of the enzyme can be easily introduced by anyone.

The present invention has been made in such a situation, and one of the exemplary objects of one of its embodiments is to provide an apparatus and method capable of easily measuring the activity of an enzyme.

One embodiment of the present invention relates to a temperature measurement device. The temperature measuring device images the thin film from the back side in a state in which the thin film has a region to which the enzyme to be tested is to be fixed on the surface, a container filled with a substrate solution that reacts with the enzyme, and the enzyme contacts the substrate solution. And a thermography camera.

Another aspect of the invention relates to a reactor. The reactor is a container filled with a substrate solution that reacts with the enzyme to be tested, and a test chip that is removably attached to the opening provided in the container, and a thin film having a region on the surface of which the enzyme is to be fixed And an inspection chip having The reactor is configured to be able to image the thin film from the back side with the enzyme in contact with the substrate solution.

It is to be noted that any combination of the above-mentioned constituent elements, or one in which the constituent elements or expressions of the present invention are mutually substituted among methods, apparatuses, etc. is also effective as an aspect of the present invention.

According to an embodiment of the present invention, the temperature can be easily measured in the activity evaluation of the enzyme.

It is a perspective view which shows typically the evaluation apparatus which concerns on embodiment. It is a figure explaining operation | movement of the evaluation apparatus of FIG. FIGS. 3A to 3C are a cross-sectional view, a plan view, and a perspective view of a part of an evaluation device according to one embodiment. 4A and 4B are a cross-sectional view and a perspective view of an inspection chip according to a modification.

(Overview)
An apparatus for evaluating enzyme activity is disclosed herein as an embodiment of the present invention. The evaluation device captures an image of the thin film from the back side in a state in which the thin film has a region to which the enzyme to be tested is to be fixed on its surface, a container filled with a substrate solution that reacts with the enzyme, and the enzyme contacts the substrate solution. And a thermography camera.

When the enzyme reacts with the substrate solution, an exotherm occurs and the temperature of the thin film on which the substrate is fixed rises. The temperature rise at this time becomes smaller as the enzyme deteriorates and the activity decreases. Therefore, by observing the temperature of this thin film from the back side by thermography, the temperature rise can be measured without contact, and the degree of degradation of the activity of the enzyme can be estimated from the measured temperature.

The container may be provided with an opening. The thin film may be provided on a test chip that is removable from the opening of the container.

The inspection chip may include a base that can be fitted to the opening of the container, and a support structure that supports the thin film substantially at the center of the opening provided in the base.
As a result, the heat capacity of the test chip can be reduced, and the heat of the thin film can be made less likely to escape to the substrate.

The support structure may have a beam structure. The width of the beam structure is preferably shorter than one side of the thin film.

The substrate, the support structure, and the thin film may be integrally formed of the same material. This can reduce the manufacturing cost.

The same material may be silicon. Thus, a test chip can be manufactured using a silicon semiconductor process.

The thin film may be a semiconductor or a metal.

Embodiment
Hereinafter, the present invention will be described based on preferred embodiments with reference to the drawings. The same or equivalent components, members, and processes shown in the drawings are denoted by the same reference numerals, and duplicating descriptions will be omitted as appropriate. In addition, the embodiments do not limit the invention and are merely examples, and all the features and combinations thereof described in the embodiments are not necessarily essential to the invention.

The dimensions (thickness, length, width, etc.) of each member described in the drawings may be scaled as appropriate for ease of understanding. Furthermore, the dimensions of a plurality of members do not necessarily represent the magnitude relationship between them, and even if one member A is drawn thicker than another member B in the drawing, the member A is a member B It may be thinner than that.

FIG. 1 is a perspective view schematically showing an evaluation device 100 according to the embodiment. The evaluation device 100 includes a temperature measurement device 101 and a processing device 130. The temperature measuring device 101 comprises a thin film 102, a container 110 and a thermographic camera 120. The thin film 102 has on its surface a reaction area in which the enzyme 2 to be tested is fixed. The method for fixing the enzyme is not particularly limited. For example, the enzyme may be mixed with a water-soluble photosensitive resin (for example, Biosurfine manufactured by Toyo Gosei Co., Ltd.), applied to the thin film 102, and irradiated with ultraviolet light to be cured. Alternatively, an enzyme adhesive layer may be formed on the surface of the thin film 102, and the enzyme may be adsorbed to the adhesive layer.

The container 110 is filled with a substrate solution 4 that reacts with the enzyme 2. The thermography camera 120 images the thin film 102 from the back surface in a state where the enzyme 2 is in contact with the substrate solution 4 and measures the temperature change of the thin film 102.

The output of the thermography camera 120 is input to a processing device 130 such as a computer, a tablet terminal, a microcomputer, an FPGA (Field Programmable Gate Array) or an ASIC (Application Specific Integrated Circuit). The processing device 130 acquires the temperature increase range from the output of the thermography camera 120, generates an index indicating the degree of deterioration according to the increase range, or whether or not the enzyme 2 is still usable Determine

It is desirable that the back surface of the thin film 102 be directly exposed to the space 6 with the thermography camera 120. Therefore, the bottom of the container 110 may be provided with an opening at least at a portion overlapping with the thin film 102.

The top and bottom (upper and lower) of FIG. 1 may be reversed.

The above is the basic configuration of the evaluation device 100. Subsequently, the measurement principle will be described. FIG. 2 is a diagram for explaining the operation of the evaluation device 100 of FIG. A time waveform of the temperature of the thin film 102 is shown in FIG. t ₀ is the reaction start time of the enzyme 2 and the substrate solution 4. Also before time t ₀ is in thermal equilibrium, the reference temperature T ₀ the temperature of the enzyme 2 (and the thin film 102) at that time. When the enzyme 2 and the substrate solution 4 react, the temperature of the enzyme 2 rises, the heat is conducted to the thin film 102, and the temperature of the thin film 102 rises. The temperature rise ΔT at this time depends on the activity of the enzyme, ie, the degree of degradation. Therefore, by measuring the temperature rise ΔT of the thin film 102 by the thermography camera 120, the activity of the enzyme can be measured.

The thermography camera 120 measures the temperature of the thin film 102 from its back surface, rather than measuring the surface temperature of the enzyme. Since the back surface of the thin film 102 is extremely flat, stable and accurate temperature measurement can be performed as compared with the case of measuring the surface of an enzyme having irregularities or a curved surface.

The present invention extends to various apparatuses and methods that can be understood from FIG. 1 or derived from the above description, and is not limited to a specific configuration. Hereinafter, in order not to narrow the scope of the present invention but to help the understanding of the nature of the invention and the circuit operation and to clarify them, more specific configuration examples and modifications will be described.

FIGS. 3A to 3C are a cross-sectional view, a plan view, and a perspective view of a part of the evaluation device 100 according to one embodiment. FIG. 3A shows a cross-sectional view of the reactor 200 in which the enzyme 2 and the substrate solution 4 are reacted. The reactor 200 of FIG. 3A corresponds to the portion of the evaluation device 100 of FIG. 1 other than the thermographic camera 120 and the processing device 130.

The reactor 200 includes an inspection chip 210 and a container 220. The container 220 corresponds to the container 110 of FIG. 1, and the container 220 is provided with an opening. The interior of the container 220 is filled with the substrate solution 4. Although the substrate solution 4 may be injected from the opening, separately, the container 220 is provided with a flow path 222 for supplying the substrate solution 4 to the inside.

The inspection chip 210 is, for example, a rectangle of several millimeters square, and is detachable from the opening of the container 220. The inspection chip 210 is provided with the thin film 102 described above. In this embodiment, the rectangle of the thin film 102 is about 1 mm square, and the whole is a reaction area to which the enzyme is applied.

The top view of the test | inspection chip 210 is shown by FIG.3 (b), and the perspective view of the test | inspection chip 210 is shown by FIG.3 (c). The inspection chip 210 includes a substrate 212 and a support structure 214 in addition to the thin film 102. The base 212 is engageable with an opening provided in the container 110. An opening 213 is provided substantially at the center of the base 212. The support structure 214 supports the thin film 102 substantially at the center of the opening 213 of the substrate 212. The support structure 214 also has a membrane structure in the same manner as the thin film 102. In addition, the support structure 214 has a beam structure. The width of the beam structure is narrower than the length of one side of the thin film 102. The thin beam 102 can be thermally isolated from the substrate 212 by reducing the width of the beam.

As shown in FIG. 3B, the thin film 102 has a rectangular shape. The rectangle of membrane 102 is inclined 45 degrees with respect to aperture 213 which is also rectangular, and the beams of support structure 214 are formed along the diagonal of aperture 213. By inclining the rectangle of the thin film 102 by 45 degrees, the length of the beam of the support structure can be increased compared to the case where the inclination is not 45 degrees. Further, by forming the beams of the support structure along the diagonal lines, the length of the beams of the support structure can be made longer than in the case where they are formed in parallel with one side of the opening 213. By lengthening the length of the beam, the heat of the enzyme 2 is less likely to be conducted from the thin film 102 to the substrate 212. Thereby, the temperature of only the thin film 102 can be raised, and the detection sensitivity can be enhanced.

The substrate 212, the support structure 214, and the thin film 102 may be integrally formed of the same material. For example, the material can use silicon. By employing silicon, it is possible to manufacture the inspection chip 210 using a semiconductor manufacturing process.

The thickness of the base 212 may be determined so as to obtain rigidity to such an extent that it can be fitted to the opening of the container 110, and is, for example, several hundred μm.

The thickness of the support structure 214 and the thin film 102 is preferably thinner than that of the substrate 212, and is 100 μm or less, preferably 20 μm or less, and may be about 3 to 5 μm. The thinner the film 102, the smaller the heat capacity of the film 102 and thus the sensitivity of the enzyme 2 to the temperature can be increased.

The surface of the inspection chip 210 may be sealed except for the thin film 102.

Although the present invention has been described based on the embodiments, the embodiments only show the principles and applications of the present invention, and the embodiments deviate from the concept of the present invention defined in the claims. However, many variations and modifications of the arrangement are permitted.

<Modification>
In the embodiment of FIG. 3, a beam structure is used as the support structure 214, but it is not limited thereto. FIGS. 4A and 4B are a cross-sectional view and a perspective view of an inspection chip 210 according to a modification. In the variation of FIG. 4 (a), the thin film 102 is supported by a sheet-like support structure 214a. An opening 215 is provided at the center of the support structure 214 a, and the thin film 102 is provided in the opening 215.

The material of the thin film 102 is not particularly limited, and a semiconductor or metal having high thermal conductivity can be used. Specifically, in addition to silicon, metals such as gold, aluminum and copper, and alloys can be used.

Reference Signs List 2 enzyme 4 substrate solution 100 evaluation apparatus 101 temperature measurement apparatus 102 thin film 110 container 120 thermography camera 130 processing apparatus 200 reactor 210 inspection chip 212 base 213 opening 214 support structure 220 container 222 flow path

The present invention can be used for evaluation of enzyme activity.

Claims (11)

A thin film having a region on the surface of which an enzyme to be tested is to be fixed;
A container filled with a substrate solution that reacts with the enzyme;
A thermographic camera for imaging the thin film from the back side in a state where the enzyme is in contact with the substrate solution;
A temperature measuring device comprising: The container is provided with an opening,
The temperature measuring device according to claim 1, wherein the thin film is provided on a test chip that is detachable with respect to the opening of the container. The inspection chip is
A base matable with the opening of the container;
A support structure for supporting the thin film substantially at the center of an opening provided in the substrate;
The temperature measurement device according to claim 2, comprising: The temperature measurement device according to claim 3, wherein the support structure has a beam structure. The temperature measuring device according to claim 4, wherein the width of the beam structure is narrower than the width of the thin film. The temperature measurement device according to any one of claims 3 to 5, wherein the substrate, the support structure, and the thin film are integrally formed of the same material. The temperature measuring device according to claim 6, wherein the same material is silicon. The temperature measuring device according to claim 1, wherein the thin film is a semiconductor or a metal. An apparatus for evaluating enzyme activity comprising the temperature measurement apparatus according to any one of claims 1 to 8. A container filled with a substrate solution that reacts with the enzyme to be tested;
An inspection chip removably mounted on an opening provided in the container, the inspection chip having a thin film having a region on the surface of which the enzyme is to be fixed;
Equipped with
A reactor characterized in that the thin film can be imaged from the back side while the enzyme is in contact with the substrate solution. Fixing the enzyme to be tested on the surface of the thin film;
Contacting the enzyme with a substrate solution that reacts with the enzyme;
Imaging the thin film from the back side with a thermography camera;
A method of evaluating enzyme activity characterized by comprising:

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