JP5964555B2 - Microchip, and measurement system and measurement method using the same - Google Patents

Description

  The present invention relates to a microchip, and a measurement system and a measurement method using the microchip. The microchip is used for biochemical tests such as DNA, proteins, cells, immunity and blood, environmental analysis, synthesis of chemical substances, and the like.

  In recent years, the importance of detecting or quantifying biological substances such as DNA (Deoxyribo Nucleic Acid), enzymes, antigens, antibodies, proteins, viruses and cells, and chemical substances has increased in the fields of medicine, health, food, and drug discovery. Various biochips and microchemical chips (hereinafter collectively referred to as microchips) that can easily measure them have been proposed.

  The microchip can perform a series of experiments or analysis operations performed in the laboratory within a chip of several cm to 10 cm square and a thickness of several mm to several cm. There are many advantages such as low cost, high reaction rate, high throughput testing or analysis, and the ability to obtain test results immediately at the sample collection site.

  The microchip has a fluid circuit therein, and the fluid circuit mixes or reacts with a specimen (for example, blood or the like) to be examined or analyzed, or tests the specimen. A reagent holding unit for holding the reagent; a measuring unit for measuring the sample and the test reagent; a mixing unit for mixing the sample and the test reagent; a detection unit for performing inspection or analysis on the mixed solution, etc. And each of these parts and a fine flow path that appropriately connects these parts. The microchip is typically used by being mounted on a device capable of applying a centrifugal force thereto. By applying a centrifugal force in the appropriate direction to the microchip, the sample (or a specific component in the sample) and / or the test reagent are weighed, the sample (or a specific component in the sample) is mixed, and the test reagent is obtained. Processing such as introduction of the mixed liquid into the detection unit can be performed. Transfer and measurement of various liquids (specimens, specific components in the specimens, test reagents, or a mixture or reaction product of two or more of these) made in the microchip to other parts Hereinafter, the processing such as mixing may be referred to as “fluid processing”.

  Various microchips provided with such a fluid circuit have been disclosed (for example, refer to Japanese Patent Application Laid-Open No. 2006-239538). However, in such a measurement method using a conventional microchip, only one inspection item can be measured by one detection unit (optical cell). For this reason, in order to measure a plurality of inspection items, a plurality of optical cells (cuvettes) and an optical system are required.

JP 2006-239538 A

  An object of the present invention is to provide a microchip capable of measuring a plurality of types of objects to be measured (inspecting a plurality of items) with a single detection unit, and a measurement system and a measurement method using the microchip. To do.

The present invention is a microchip used for measuring a plurality of types of objects to be measured,
Comprising at least a reagent holding part and a detection part,
The reagent holding unit includes a plurality of types of test reagents corresponding to the plurality of types of objects to be measured,
The microchip is characterized in that a plurality of time courses of changes in detection values in the detection unit caused by a reaction between each of the test reagents and the corresponding object to be measured are all different. The number of the detection units is preferably one.

  Both of the plurality of time courses are a positive time course in which the detected value tends to increase with the passage of time, and a negative time course in which the detected value tends to decrease with the passage of time. It is preferable to contain.

  It is preferable that all of the plurality of time courses are time courses suitable for a rate method in which the object to be measured is detected based on a change speed of the detection value at a time when a predetermined time has elapsed.

  The plurality of time courses are a time course suitable for a rate method for detecting the object to be measured based on a change speed of the detection value when a predetermined time has elapsed, and a predetermined time with respect to an initial value of the detection value. It is preferable that both of the time courses suitable for the endpoint method for detecting the object to be measured based on the amount of change in the detected value at the time when elapses.

  The plurality of types of test reagents preferably include at least one latex reagent and at least one gold colloid reagent.

  It is preferable that the plurality of types of objects to be measured are influenza A virus and influenza B virus, and the plurality of types of test reagents are latex reagents and gold colloid reagents.

The detected value is preferably detected by optical measurement using a plurality of wavelengths.
It is preferable that the microchip of the present invention is used for immediate clinical site inspection. The present invention also relates to a measurement system using the above microchip.

Further, the present invention is a measuring method for measuring a plurality of types of objects to be measured using a microchip,
The microchip includes at least a reagent holding unit and a detection unit,
The reagent holding unit includes a plurality of types of test reagents corresponding to the plurality of types of objects to be measured,
The present invention also relates to a measurement method characterized in that a plurality of time courses for changes in detection values at the detection unit caused by a reaction between each of the test reagents and the corresponding object to be measured are all different.

  It is preferable to detect the detection value by optical measurement using a plurality of wavelengths.

  In the measurement using the microchip of the present invention, since the time course patterns of the detection values for the respective measurement objects are all different, it is possible to measure a plurality of types of objects to be measured with one detection unit. Further, even when a plurality of items are inspected, it is not always necessary to provide a plurality of optical cells, so that the structure of a microchip and a measurement system using the microchip can be simplified. In addition, since a plurality of items can be measured with a single detection unit, the amount of sample necessary for measurement can be reduced.

3 is a schematic graph for explaining the measurement method of the first embodiment. 6 is a schematic graph for explaining a measurement method according to Embodiment 2. 6 is a schematic graph for explaining a measurement method according to Embodiment 3. (A)-(d) is an upper surface schematic diagram of the microchip in each process of the measuring method of Example 1. FIG. 3 is a graph showing measurement results of Example 1. (A)-(d) is an upper surface schematic diagram of the microchip in each process of the measuring method of Example 2. FIG. 10 is a graph showing measurement results of Example 3. 10 is a graph showing another measurement result of Example 3. (A)-(e) is an upper surface schematic diagram of the microchip in each process of the measuring method of Example 4. FIG. 10 is a graph showing measurement results of Example 4. 10 is another graph showing the measurement results of Example 4. 10 is another graph showing the measurement results of Example 4. 6 is a schematic cross-sectional view of a test reagent used in the measurement method of Example 5. FIG. 10 is a schematic top view for explaining the effect of the measurement method of Example 5. FIG.

  The microchip of the present invention is a chip that can perform various chemical synthesis, inspection, analysis, and the like using a fluid circuit included in the microchip. The size of the microchip is not particularly limited, and can be, for example, about several cm to 10 cm in length and width and about several mm to several cm in thickness.

  The microchip is composed of, for example, a first substrate having a concave portion partitioned by a partition wall on at least one surface, and a second substrate bonded to at least the one surface of the first substrate. The The fluid circuit of the microchip includes a space constituted by the recess and the surface of the second substrate.

  Further, the microchip may be composed of a substrate having three or more layers. For example, the first substrate also has a recess on the surface opposite to the second substrate, and a third substrate similar to the second substrate is bonded to the recess side. Also good. In this case, the fluid circuit includes a first fluid circuit including a space formed by a recess provided on the second substrate side surface of the first substrate and the surface of the second substrate, and the first substrate. The second substrate has a two-layer structure composed of a second fluid circuit composed of a space formed by a recess provided on the third substrate side surface and the surface of the third substrate. Here, “two layers” means that fluid circuits are provided at two different positions in the thickness direction of the microchip. Such a two-layer fluid circuit can be connected by a through-hole penetrating the first substrate in the thickness direction.

  The method for bonding the substrates together is not particularly limited. For example, among the substrates to be bonded, at least one of the bonded surfaces of the substrates is melted and welded (welding method), and bonded using an adhesive. And the like. Examples of the welding method include a method of welding by heating the substrate; a method of irradiating light such as a laser and welding by heat generated during light absorption (laser welding); and a method of welding using ultrasonic waves. Can do.

  The material of each substrate constituting the microchip of the present invention is not particularly limited. For example, polyethylene terephthalate (PET), polyethylene naphthalate (PEN), polybutylene terephthalate (PBT), polymethyl methacrylate (PMMA), polycarbonate (PC), polystyrene (PS), polypropylene (PP), polyethylene (PE), polyarylate resin (PAR), acrylonitrile-butadiene-styrene resin (ABS), styrene-butadiene resin (styrene-butadiene copolymer), chloride Vinyl resin (PVC), polymethylpentene resin (PMP), polybutadiene resin (PBD), biodegradable polymer (BP), cycloolefin polymer (COP), polydimethylsiloxane (PDMS), polyacetal POM), polyamide (PA) organic materials such as; silicon, and glass, inorganic material such as quartz. Among these, it is preferable to use a resin in consideration of the ease of forming a fluid circuit.

  The method for forming the recesses (pattern grooves) and partition walls constituting the fluid circuit on the surface of the first substrate is not particularly limited, and injection molding using a mold having a transfer structure, imprinting, etc. Can be mentioned. The shapes of the recess and the partition are determined so as to obtain an appropriate fluid circuit structure.

  In the microchip, the fluid circuit performs an appropriate fluid treatment on the liquid in the fluid circuit (specimen, specific component in the specimen, liquid reagent, and a mixture or reaction liquid of two or more of these). In order to be able to carry out, various parts arranged at appropriate positions in the fluid circuit are provided, and these parts are appropriately connected through fine flow paths (connection flow paths). The part in the fluid circuit includes: a reagent holding part for holding a test reagent; a mixing part for mixing a test reagent and a specimen; a test or analysis of the obtained liquid mixture (for example, identification in a liquid mixture) And a detection unit for performing component detection). The microchip of the present invention includes at least a reagent holding unit and a detection unit. It is preferable that the number of detection units in one microchip is one.

  Here, the “specimen” is a substance that is introduced into the fluid circuit and is a target for testing or analysis performed by the microchip. For example, whole blood, plasma, serum, urine, nasal wipes, nasal suction Liquid, nasal discharge, throat swab, and sore throat. In addition, the “test reagent” is a reagent that processes a sample to be tested or analyzed by the microchip, or is mixed or reacted with the sample. Usually, the reagent of the fluid circuit is used in advance before using the microchip. Built in the holding part. It is preferable that at least one of the specimen and the test reagent is a liquid. The liquid mixture or reaction liquid finally obtained by mixing the sample and the test reagent, for example, irradiates the detection unit containing the liquid mixture with light and detects the intensity (transmittance) of the transmitted light. This method is used for optical measurement such as a method for measuring the absorption spectrum of the mixed liquid held in the detection unit, and inspection or analysis is performed.

  Various fluid treatments in the fluid circuit, such as mixing the sample and test reagent, and introducing the resulting mixture into the detection unit, for example, sequentially apply centrifugal force in the appropriate direction to the microchip. This can be done. Application of centrifugal force to the microchip can be performed by placing the microchip on a device (centrifuge) that can apply centrifugal force. A centrifuge device usually includes a rotatable rotor (rotor) and a rotatable stage disposed on the rotor. A centrifugal force in an arbitrary direction can be applied to the microchip by placing the microchip on the stage and rotating the stage to arbitrarily set the angle of the microchip with respect to the rotor.

  The mixed solution finally obtained by mixing the specimen and the test reagent is, for example, a method of detecting the intensity (transmittance) of the transmitted light by irradiating the detection unit containing the mixed solution with light; It is subjected to optical measurement such as a method of measuring the absorption spectrum of the mixed liquid held in the section, and inspection or analysis is performed.

  The microchip of the present invention is a microchip used for measuring a plurality of types of objects to be measured, and the reagent holding unit includes a plurality of types of test reagents corresponding to each of the plurality of types of objects to be measured. . The plurality of types of test reagents are selected such that a plurality of time courses regarding the change in the detection value at the detection unit caused by the reaction between each of the test reagents and the corresponding measurement object are all different. Here, the time course means a change in the detected value with the passage of time.

  A plurality of types of test reagents may be incorporated as a mixed solution of a plurality of types of test reagents in one reagent holding unit, or each test reagent may be built in a separate reagent holding unit.

  The microchip of the present invention and the measurement system using the microchip can be suitably used particularly for an immediate clinical site inspection. The point-of-care test (POCT) is a real-time test performed at a medical site using a small analyzer or a rapid diagnostic kit. For example, infectious disease tests such as influenza virus test, adenovirus test, troponin T test to confirm the presence or absence of myocardial infarction or myocardial injury, blood gas and blood cell count test to grasp the general condition, electrolytes and blood glucose level Examination is mentioned.

Hereinafter, the present invention will be described in detail with reference to embodiments.
(Embodiment 1)
FIG. 1 is a schematic graph for explaining the measurement method of the first embodiment.

  In FIG. 1, A1 to A3 are schematic graphs showing the time course of absorbance (detection value) for a mixed solution of the specimen including the object A to be measured and the test reagent 1. The concentration of the measurement object A in the sample increases in the order of A1, A2, and A3. This time course is a positive time course showing a tendency that the detected value increases with the passage of time.

  B1 to B3 are schematic graphs showing the time course of absorbance (detection value) for the mixed solution of the specimen including the object to be measured B and the test reagent 2. The concentration of the measurement object B in the sample increases in the order of B1, B2, and B3. This time course is also a positive time course showing a tendency for the detected value to increase with time.

  That is, in this embodiment, the test reagents 1 and 2 have a plurality of times for the change in the detection value at the detection unit caused by the reaction between each of the test reagents 1 and 2 and the corresponding objects A and B. Courses are all selected differently.

  In the present embodiment, the test reagents 1 and 2 are selected so that the time course of each of them is a positive time course. In the present invention, a plurality of time courses are thus provided. May be either a positive time course or a negative time course, and a plurality of time courses may be both a positive time course and a negative time course, as in Embodiment 2 described later. May be included.

  In this embodiment, since the reaction is slow in both the reaction between the object to be measured A and the test reagent 1 and the reaction between the object to be measured B and the test reagent 2, the time course of A1 to A3 and Each of the time courses B1 to B3 is a time course suitable for the rate method in which the object to be measured is detected based on the change speed of the detection value when a predetermined time has elapsed.

  By using a microchip equipped with such a plurality of types of test reagents in the reagent holding part, either the sample A or B to be measured is included in the sample due to the difference in the time course for the test reagents 1 and 2. It can be detected. In addition, if the measurement results such as A1 to A3 and B1 to B3 in FIG. 1 are obtained in advance by preliminary measurement and a calibration curve is created based on the measurement results, the objects A and B to be measured are obtained. It is also possible to measure the concentration and the like. Note that this embodiment is preferably used when the specimen normally contains only one of the objects A or B to be measured.

(Embodiment 2)
FIG. 2 is a schematic graph for explaining the measurement method of the second embodiment.

  In FIG. 2, A1 to A3 are schematic graphs showing the time course of absorbance (detection value) for the mixed solution of the specimen including the object to be measured A and the test reagent 1. The concentration of the measurement object A in the sample increases in the order of A1, A2, and A3. This time course is a positive time course showing a tendency that the detected value increases with the passage of time.

  On the other hand, B1 to B3 are schematic graphs showing the time course of the absorbance (detection value) for the mixed solution of the specimen including the object to be measured B and the test reagent 2. The concentration of the measurement object B in the sample increases in the order of B1, B2, and B3. This time course is a negative time course showing a tendency that the detected value decreases with the passage of time.

  That is, in this embodiment, the test reagents 1 and 2 have a plurality of times for the change in the detection value at the detection unit caused by the reaction between each of the test reagents 1 and 2 and the corresponding objects A and B. Courses are all selected differently. Further, the test reagents 1 and 2 are selected so that the plurality of time courses include both a positive time course and a negative time course. Such time courses can usually be obtained by selecting the type of test reagent.

  In the present embodiment, since the reaction is slow in both the reaction between the object to be measured A and the test reagent 1 and the reaction between the object to be measured B and the test reagent 2, the time course of A1 to A3 and Each of the time courses B1 to B3 is a time course suitable for the rate method in which the object to be measured is detected based on the change speed of the detection value when a predetermined time has elapsed.

  By using a microchip equipped with such a plurality of types of test reagents in the reagent holding part, either the sample A or B to be measured is included in the sample due to the difference in the time course for the test reagents 1 and 2. It can be detected. In the present embodiment, in particular, since the time courses for the test reagents 1 and 2 are positive and negative time courses, only the time course in the detection result is positive or negative, and the object to be measured is included in the sample. It is possible to determine whether A or B is included. In addition, if the measurement results such as A1 to A3 and B1 to B3 in FIG. 2 are obtained in advance by preliminary measurement and a calibration curve is created based on the measurement results, the measured objects A and B It is also possible to measure the concentration and the like. Note that this embodiment is preferably used when the specimen normally contains only one of the objects A or B to be measured.

(Embodiment 3)
FIG. 3 is a schematic graph for explaining the measurement method of the third embodiment.

  In FIG. 3, A1 to A3 are schematic graphs showing the time course of absorbance (detection value) for the mixed solution of the specimen including the object to be measured A and the test reagent 1. The concentration of the measurement object A in the sample increases in the order of A1, A2, and A3. This time course is a positive time course showing a tendency that the detected value increases with the passage of time. Further, the time course of A1 to A3 is such that the reaction rate between the object A to be measured and the test reagent 1 is fast, and the absorbance (detected value) is in an almost steady state at the initial stage. This is a time course suitable for the endpoint method in which the object to be measured is detected based on the amount of change in the detected value when a predetermined time has elapsed.

  B1 to B3 are schematic graphs showing the time course of absorbance (detection value) for the mixed solution of the specimen including the object to be measured B and the test reagent 2. The concentration of the measurement object B in the sample increases in the order of B1, B2, and B3. This time course is also a positive time course showing a tendency for the detected value to increase with time. Further, the time course of B1 to B3 is a rate at which the measurement object is detected based on the change rate of the detection value when a predetermined time has elapsed since the reaction between the measurement object B and the test reagent 2 is slow. It is a time course suitable for the law.

  In other words, in this embodiment, the test reagents 1 and 2 have a time course for the change in the detection value at the detection unit caused by the reaction between each of the test reagents 1 and 2 and the corresponding objects A and B. Both are positive time courses, but one is a time course suitable for the end point method and the other is a time course suitable for the rate method. Such time courses can usually be obtained by selecting the type of test reagent.

  When such a microchip equipped with a plurality of types of test reagents in the reagent holding unit is used, even if the time course of each test reagent is a positive (or negative) time course, it is included in the sample. The object to be measured can be easily identified. Further, if preliminary measurement results such as A1 to A3 and B1 to B3 in FIG. 3 are obtained in advance, and a calibration curve is created based on the measurement results, the objects A and B to be measured are obtained. It is also possible to measure the concentration and the like.

Example 1
An outline of fluid processing using the microchip 1 of the present embodiment will be described with reference to FIGS. 4A to 4D are schematic top views of the microchip in each step of the measurement method of Example 1. FIG.

(1) Mixing Step First, centrifugal force is applied downward (in the direction of the arrow in FIG. 4A) to the microchip 1 in the state shown in FIG. As a result, the test reagent 11 incorporated in the reagent holding unit 10 and the sample 21 introduced into the sample holding unit 20 are introduced into the mixing unit 30 and become the mixed solution 31 (FIG. 4B). The test reagent 11 is a mixed solution of a latex reagent to which the influenza A virus antibody is immobilized and a gold colloid reagent to which the influenza B virus antibody is immobilized.

(2) Detection part introduction process Next, centrifugal force is applied rightward (the direction of the arrow of Drawing 4 (b)). Thereby, the liquid mixture 31 is introduce | transduced into the detection part 40 through a connection flow path (FIG.4 (c)).

(3) Detection process The liquid mixture 31 with which the detection part was filled is used for an optical measurement, and a test | inspection and analysis are performed. For example, by irradiating light from the direction of the arrow shown in FIG. 4 (d) and measuring the transmitted light, a specific component in the liquid mixture is detected.

  In this example, nasal discharge (liquid discharged by biting the nose) collected from each of influenza A positive affected persons and influenza B positive affected persons, and an equal mixture of both were used as specimens. As a control, nasal discharge collected from healthy people who were negative for influenza A and B was used.

  As a test reagent for the influenza A virus, which is an object to be measured, a latex reagent on which an influenza A virus antibody was immobilized was used. As a test reagent for influenza B virus, a gold colloid reagent on which an influenza B virus antibody was immobilized was used. The wavelength of light used for the measurement was 505 nm, and three measurements were performed for each specimen.

  FIG. 5 is a graph showing the measurement results of Example 1. In FIG. 5, all the measurement results are displayed in an overlapping manner. As FIG. 5 shows, it turns out that influenza A positive, influenza B positive, both positive, and both negative can be discriminate | determined from the time course of a light absorbency. Here, the time course for the influenza A positive specimen is a positive time course, and the time course for the influenza B positive specimen is a negative time course. In general, it is considered that both influenza A and influenza B are not affected (both positive), but considering both positive cases, nasal discharge of influenza A and influenza B Measurements were also made on an equal amount of the mixed solution. As shown in FIG. 5, the time course of both positive samples has a characteristic that the rate of change in absorbance changes from positive to negative. On the other hand, the time course of a specimen that is positive only for either influenza A or influenza B has a characteristic that the rate of change in absorbance is always positive or always negative. Based on this time course difference, it is possible to discriminate between both positive specimens and a specimen positive only for either influenza A or influenza B.

(Example 2)
6A to 6D are schematic top views of the microchip in each step of the measurement method of Example 2. FIG. In this embodiment, as shown in FIG. 6A, the test reagents 11a and 11b are built in the separate reagent holding units 10a and 10b. Since the other points are the same as those in the first embodiment, description thereof is omitted. Such a form is effective when it is necessary to separate and hold a plurality of types of test reagents until measurement.

(Example 3)
In this example, optical measurement at the detection unit was performed using two wavelengths of 450 nm and 505 nm. The measurement was performed on an influenza A positive sample (nasal discharge of an influenza A affected person), an influenza B positive sample (nasal discharge of an influenza B affected person), and a negative sample (a healthy person's nasal discharge). Since the other points are the same as those in the first embodiment, description thereof is omitted.

FIG. 7 shows a graph of the absorbance (A ₄₅₀ ) of light having a wavelength of 450 nm. In addition, about the influenza B positive sample, the measurement result about 1/2 dilution liquid and 1/4 dilution liquid is also shown. FIG. 7 shows that only influenza A virus can be measured by optical measurement using light having a wavelength of 450 nm.

On the other hand, FIG. 8 is a graph of a value (A ₅₀₅ -a × A ₄₅₀ ) obtained by subtracting the absorbance (A ₄₅₀ ) × a (constant) of light having a wavelength of 450 nm from the absorbance (A ₅₀₅ ) of light having a wavelength of 505 nm. . Here, the constant a is 0.79. This constant a is the ratio of the light absorbance at a wavelength of 505 nm to the light absorbance at a wavelength of 450 nm for the reaction solution of the latex reagent and influenza A virus, and is a value inherent to the latex reagent. This ratio a is constant even if the concentration of influenza virus changes. In addition, about the influenza B positive sample, the measurement result about 1/2 dilution liquid and 1/4 dilution liquid is also shown. FIG. 8 shows that only influenza B virus can be measured by optical measurement using light with wavelengths of 450 nm and 505 nm. In this way, when measuring using a plurality of wavelengths, the individual measurement results (individual time courses) are separated from the overall measurement results (in which the individual time courses are combined), and the measured object The concentration can be examined.

Example 4
An outline of fluid processing using the microchip 1 of the present embodiment will be described with reference to FIGS. 9A to 9E are schematic top views of a microchip in each step of the measurement method of Example 4. FIG.

(1) First Mixing Step First, centrifugal force is applied to the microchip 1 in the state shown in FIG. 9A in the left direction (the direction of the arrow in FIG. 9A). Thereby, the test reagent 11a (latex reagent in which the influenza A virus antibody is immobilized) incorporated in the reagent holding unit 10a and the sample 21 introduced into the sample holding unit 20 are introduced into the mixing unit 30a and mixed. It becomes the liquid 31a (FIG. 9B).

(2) Second Mixing Step 60 seconds after the test reagent 11a and the specimen 21 are mixed, the microchip 1 in the state shown in FIG. 9B is directed downward (the arrow in FIG. 9B). Apply centrifugal force in the direction of As a result, the mixed solution 31a introduced into the mixture 30a and the test reagent 11b (gold colloid reagent with the influenza B virus antibody immobilized) incorporated in the reagent holding unit 10b are introduced into the mixing unit 30b and mixed. It becomes the liquid 31b (FIG.9 (c)).

(3) Detection part introduction process Next, centrifugal force is applied rightward (the direction of the arrow of Drawing 9 (c)). Thereby, the liquid mixture 31b is introduced into the detection part 40 through a connection flow path (FIG.9 (d)).

(4) Detection process The liquid mixture 31b with which the detection part was filled is used for an optical measurement, and a test | inspection and analysis are performed. For example, by irradiating light from the direction of the arrow shown in FIG. 9 (d) and measuring the transmitted light, the specific component in the mixed liquid is detected. In this example, the absorbance of light having a wavelength of 505 nm was measured.

  In this example, a liquid obtained by combining the nasal discharge of an influenza A affected person or a diluted liquid thereof and the nasal discharge of an influenza B affected person or a diluted liquid thereof was used as a specimen.

  As a test reagent for the influenza A virus, which is an object to be measured, a latex reagent on which an influenza A virus antibody was immobilized was used. As a test reagent for influenza B virus, a gold colloid reagent on which an influenza B virus antibody was immobilized was used. The wavelength of light used for the measurement was 505 nm, and measurement was performed once for each specimen.

  The measurement results are shown in FIG. In FIG. 10, A0 is a measurement result of only a nasal discharge of a healthy person, A1 is a measurement result of only a 1/2 dilution of a nasal discharge of an influenza B affected person, and A2 is only a nasal discharge of an influenza B affected person. It is a measurement result. E0 is the measurement result of only the nasal discharge of those suffering from influenza A, and D0, C0, and B0 are respectively the 1/2 dilution, 1/4 dilution, and 1/8 dilution of the nasal discharge of influenza A affected persons. It is a measurement result. E1 is a measurement result of an equal volume mixture of a nasal discharge of an influenza A affected person and a ½ dilution of a nasal discharge of an influenza B affected person. E2 is a nasal discharge of an influenza A affected person and an influenza B affected person. It is a measurement result of an equivalent liquid mixture.

  FIG. 11 is a graph obtained by extrapolating the graph from the measurement result shown in FIG. 10 to the time of mixing the latex reagent (test reagent 11a) to the time of mixing the gold colloid reagent (test reagent 11b). Thus, it can be seen that the influenza A virus can be measured based on the absorbance at 0 hours in FIG. 10 (the part indicated by the arrow in FIG. 10).

  “E1-E0” in FIG. 12 is a graph of the value obtained by subtracting the E0 absorbance from the E1 absorbance shown in FIG. 10, and “E2-E0” is the E2 absorbance shown in FIG. 10 minus the E0 absorbance. It is a graph of the measured value. In this way, it can be seen that influenza B virus can be measured.

(Example 5)
In this example, instead of the gold colloid reagent used in Example 1 (a colloidal dispersion of gold particles having influenza A virus antibody immobilized on the surface of gold particles), a two-layer structure as shown in FIG. A colloidal dispersion of particles in which antibodies are immobilized on the surface of the particles is used. In FIG. 13, a thin metal coating 51 is formed on the surface of a core particle 50 made of a material (resin or the like) having a specific gravity of about 1. An antibody 52 such as an influenza A virus antibody is immobilized on the surface of the metal coating 51.

  In a microchip that performs fluid processing using centrifugal force, when a metal colloid reagent is used as a test reagent, the specific gravity of the metal (for example, Au: 19.3, Pt: 21.45, Ag: 10.49) is water. Therefore, the metal 5 may be separated in the mixed solution 31 containing the colloidal gold reagent as shown in FIG. 14 due to the application of centrifugal force in the fluid processing.

  In contrast, in this example, the specific gravity of the particles in the reagent is approximately the same as the specific gravity of the core material 50, so that the antibody is immobilized in the liquid mixture in the biochip by applying centrifugal force in fluid processing. The phenomenon that separated particles are separated can be suppressed.

  Further, when the material having a specific gravity of about 1 is a resin, the core particle 50 made of the resin can be easily controlled in particle diameter by various known methods, so that the measurement sensitivity can be improved. Is possible. Examples of the resin include polyethylene (PE), polypropylene (PP), polystyrene (PS), ABS resin, AS resin, polycarbonate (PC), polyethylene terephthalate (PET), and polymethylpentene (PMP). Examples of the material of the metal coating 51 include gold, platinum, and silver.

  When a metal colloid reagent is used, measurement is performed using surface plasmon resonance due to interaction between light and free electrons existing on the metal surface. Also in the metal colloid reagent of this example, free electrons are present on the surface of the metal coating, and surface plasmon phenomenon appears, so that the characteristics necessary as an in vitro diagnostic reagent such as immune reaction are maintained. As described above, the metal colloid reagent of this example has a preferable characteristic that it does not separate even when a centrifugal force is applied while maintaining the characteristic as a test reagent. It can be suitably used for a microchip that performs the above.

  It should be understood that the embodiments and examples disclosed herein are illustrative and non-restrictive in every respect. The scope of the present invention is defined by the terms of the claims, rather than the description above, and is intended to include any modifications within the scope and meaning equivalent to the terms of the claims.

  1 Microchip, 10, 10a, 10b Reagent holding unit, 11, 11a, 11b Test reagent, 20, 20a, 20b Sample holding unit, 21, 21a, 21b Sample, 30, 30a, 30b Mixing unit, 31, 31a, 31b Mixed solution, 40 detector, 5 metal, 50 core particles, 51 metal coating, 52 antibody.

Claims (10)

A microchip used for measuring multiple types of objects to be measured by light irradiation,
Comprising at least a reagent holding part and a detection part,
The reagent holding unit includes a plurality of types of test reagents corresponding to the plurality of types of objects to be measured,
The light is irradiated to the detection unit at one type of wavelength,
As detected by the light irradiation at the same time , each of the test reagents and the detection value in the detection unit caused by the reaction between the object to be measured and the plurality of time courses about the change of all are different . The microchip, wherein the plurality of types of test reagents are selected .   The microchip according to claim 1, wherein the number of the detection units is one.   Both of the plurality of time courses are a positive time course in which the detected value tends to increase with the passage of time, and a negative time course in which the detected value tends to decrease with the passage of time. The microchip according to claim 1, comprising:   2. The micro time course according to claim 1, wherein all of the plurality of time courses are time courses suitable for a rate method of detecting the object to be measured based on a change speed of the detection value at a time when a predetermined time has elapsed. Chip.   The plurality of time courses are a time course suitable for a rate method for detecting the object to be measured based on a change speed of the detection value when a predetermined time has elapsed, and a predetermined time with respect to an initial value of the detection value 2. The microchip according to claim 1, comprising both a time course suitable for an end point method for detecting the object to be measured based on a change amount of the detection value at the time when elapses.   The microchip according to claim 1, wherein the plurality of types of test reagents include at least one latex reagent and at least one gold colloid reagent. The plurality of types of objects to be measured are influenza A virus and influenza B virus,
The microchip according to claim 3, wherein the plurality of types of test reagents are a latex reagent and a gold colloid reagent.   A measurement system using the microchip according to claim 1.   The measurement system according to claim 8, which is used for an immediate clinical examination. A measurement method for measuring a plurality of kinds of objects to be measured using a microchip by light irradiation,
The microchip includes at least a reagent holding unit and a detection unit,
The reagent holding unit includes a plurality of types of test reagents corresponding to the plurality of types of objects to be measured,
The light is irradiated to the detection unit at one type of wavelength,
As detected by the light irradiation at the same time , each of the test reagents and the detection value in the detection unit caused by the reaction between the object to be measured and the plurality of time courses about the change of all are different . Select multiple types of test reagents,
A measuring method comprising measuring the plurality of types of objects to be measured based on the detection values .

Images