JP6911318B2 - Blood test sensor chip, blood test device and blood test method - Google Patents

Description

The present invention relates to a blood test sensor chip used for a blood test, and a blood test device and a blood test method using the sensor chip. Specifically, a blood test sensor chip that quantitatively measures a specific substance present in blood and also measures the hematocrit value of blood, and a blood test device and blood test method using the sensor chip. It is about.

A blood test that quantitatively grasps information on a specific substance (for example, protein, glucose, etc.) present in blood by measuring the concentration of the substance is important in the diagnosis and treatment of various diseases. For example, since troponin present in the cytoplasm of a cardiomyocyte deviates into the blood due to necrosis of the cardiomyocyte, grasping the troponin concentration in the blood is of great significance in early detection of myocardial infarction.

As a means for acquiring information on a specific substance (hereinafter referred to as a target substance) existing in blood, there is a measurement method using a sensor chip having a fine flow path. A reaction system that reacts with the target substance is provided in the microchannel, and when a blood-derived sample solution or the like is supplied into the microchannel and comes into contact with the reaction system, the target substance and the reaction system The reaction proceeds. Based on the result of the reaction, information according to the concentration of the target substance and the like can be obtained.

However, when whole blood containing blood cells is used as a sample solution, the detection result of information according to the concentration of the target substance and the like is greatly affected by the hematocrit value of blood. The hematocrit value is a numerical value indicating the ratio of the volume of red blood cells to the blood. Therefore, in order to accurately detect information such as the concentration of the target substance, it is necessary to correct the detection result based on the hematocrit value, and the hematocrit value must be accurately grasped.

Therefore, Patent Document 1 measures the concentration of a specific component in blood in the same flow path, measures a correction value based on the hematocrit value of blood by an optical method, and based on the measured correction value. It provides a measurement method for correcting the concentration of a specific component in blood.

Japanese Patent No. 4785611

In a measurement method using a sensor chip having a fine flow path, when measuring the hematocrit value by an optical method such as absorbance, it is necessary to detect the measurement light that has passed through the sample solution, so the optical path length of the measurement light is fine. It must be limited by the dimensions of the flow path.

Then, according to the following formula 1, the absorbance A of the sample solution can be expressed by the product of the molar extinction coefficient ε of the sample solution, the concentration C of the sample solution, and the optical path length l. That is, under a constant molar extinction coefficient ε and a constant concentration C, the absorbance A of the sample solution decreases as the optical path l becomes shorter. In Equation 1, A is the absorbance of the sample solution, I ₁ is the transmitted light intensity, I ₀ is the incident light intensity, ε is the molar extinction coefficient of the sample solution, C is the concentration of the sample solution, and l is the optical path length.
A = -log (I ₁ / I ₀ ) = ε ・ C ・ l (1)

From the viewpoint of concentrating the target substance in the reaction system and improving the reaction efficiency, it is desirable that the height of the flow path is made as uniform as possible in the reactionable region and reduced in order to eliminate wasted space. However, as in the invention disclosed in Patent Document 1, when the reaction system is provided on the bottom surface of the flow path and the height of the flow path is the optical path length of the measurement light, the height of the flow path is reduced. Then, the optical path of the measurement light is also shortened, and the absorbance of the sample solution is reduced as described above, so that the measurement accuracy of the hematocrit value is lowered.

On the other hand, if the height of the flow path is increased in an attempt to increase the optical path length, the measured light is affected by absorption and scattering by impurities in the sample solution, even though the absorbance of the sample solution is increased according to Equation 1. It is still not possible to measure the hematocrit value accurately because it is easily received.

In particular, when a substance such as troponin, which is rare in the blood, is targeted as a target substance, the effect of the above-mentioned measurement accuracy is remarkable, and even if the hematocrit value can be measured, it has sufficient accuracy. Therefore, it is not possible to obtain accurate information such as the concentration of the target substance by correcting the obtained hematocrit value.

Further, the invention as disclosed in Patent Document 1 requires a plurality of light sources having different wavelengths, so that there is a problem that the measuring method or the configuration of the measuring device itself has to be complicated.

The sensor chip for a blood test of the present invention has a first flow path for flowing blood and a second flow path located upstream or downstream of the first flow path and having a wider flow path than the first flow path. It is characterized by having a road.

The blood test apparatus of the present invention includes a first flow path for flowing blood and a second flow path located upstream or downstream of the first flow path and having a wider flow path than the first flow path. It is characterized by having a blood test sensor chip having, a light projecting unit that irradiates the blood test sensor chip with light, and a measuring unit that measures the amount of light emitted from the blood test sensor chip. And.

The blood test method of the present invention includes a first flow path for flowing blood and a second flow path located upstream or downstream of the first flow path and having a wider flow path than the first flow path. A sample solution containing a target substance present in the blood is supplied to the first flow path and the second flow path by using a blood test sensor chip having It is characterized in that the target substance is quantitatively measured and the hematocrit value of the blood is measured using the second flow path.

According to the present invention, it is possible to quantitatively measure a target substance and accurately measure a hematocrit value of blood in the same flow path. Further, according to the present invention, it is possible to perform quantitative measurement of a target substance and measurement of a hematocrit value of blood by a simplified device or method.

It is a schematic diagram which shows the structure of the measuring apparatus. It is a block diagram of a control calculation part. It is sectional drawing of the sensor chip. It is a schematic diagram which shows the optical path of the incident light and each light to be measured. It is a flowchart for demonstrating operation of a measuring apparatus. It is a graph which shows the correlation between the measurable range of the transmittance and the optical path length of the incident light.

Hereinafter, one embodiment for carrying out the present invention will be described with reference to the drawings. In this embodiment, as a method of measuring the concentration of the target substance, the target substance is captured by using an antigen-antibody reaction, a fluorescent label is attached to the captured target substance, and the fluorescence intensity emitted from the fluorescent label is measured. Although the surface plasmon resonance excitation enhanced fluorescence spectroscopy (SPFS) method is adopted, the present invention is not limited to this method. For example, a method such as surface plasmon resonance (SPR) or fluorescence immunoassay may be used to measure the concentration of the target substance.

(Measuring device)
First, the configuration of the measuring device 10 according to the present embodiment will be described.

FIG. 1 is a schematic view showing the configuration of the measuring device 10. The measuring device 10 targets troponin (Tn) present in blood as a target substance, measures the concentration thereof by the SPFS method, and also measures the hematocrit (Hct) value of blood by the absorbance of the sample solution. However, as the target substance, for example, cystatin, D-dimer, BNP (B-type natriuretic peptide), CRP (C-reactive protein) and the like may be measured in addition to troponin. The sample solution refers to blood collected directly from the examinee or a solution obtained by diluting the sample solution with a buffer solution or the like to a desired concentration.

As shown in FIG. 1, the measuring device 10 includes a light projecting unit 20, a measuring unit 40, a control calculation unit 50, a transport unit 80, and a liquid feeding unit 90. At the time of measurement, the sensor chip 30 is attached to the main body of the measuring device 10.

The light projecting unit 20 includes a light source (not shown), each optical element (not shown) for constructing an optical path for guiding the incident light 41 emitted from the light source to a predetermined position on the reflection surface 31a of the sensor chip 30, and a sensor chip. It is composed of a photodiode 21 for receiving the reflected light 44 emitted from the reflecting surface 31a of the 30.

The light source is a laser diode capable of generating light having a wavelength of 200 to 900 nm, and a light source capable of generating light having a wavelength of 500 to 700 nm is particularly preferable. However, the light source is not limited to the laser diode, and may be any light source capable of generating light having a wavelength within the above range. For example, it may be an LED capable of generating light having a wavelength of 200 to 900 nm. When the light source is capable of generating light having a wavelength of 500 to 700 nm, the light emitted from the light source is the excitation light for measuring the Tn concentration and the measurement light for measuring the Hct value of blood. Can also be used as. This makes it possible to measure the Tn concentration and the Hct value of blood with one light source, and it is possible to simplify the measuring device. However, it may be configured so that it is provided with two light sources capable of generating different wavelengths and the light source to be incident can be selected depending on the type of measurement.

Although not shown, each optical element is located in a region on the reflective surface 31a of the sensor chip 30 corresponding to the position of the troponin capturing film 61 described later when measuring the Tn concentration, and when measuring the Hct value, the sensor chip. An optical path for guiding the incident light 41 so as to be incident at a predetermined incident angle θ is formed in the opening 38 of the 30 (details of the structure of the sensor chip 30 will be described later). However, this is a case where the location where the incident light 41 hits is controlled by switching the optical path. Each optical element may form only one optical path, and the sensor chip 30 may be moved to switch the location where the incident light 41 hits without switching the optical path depending on the type of measurement.

The photodiode 21 is arranged on the optical path of the reflected light 44 and measures the amount of light of the reflected light 44. When measuring the density of Tn, the resonance angle, which is the incident angle when the amount of reflected light 44 is minimized, is specified as the incident angle θ of the incident light 41 at the time of measurement. In this case, the incident light 41 functions as so-called excitation light that excites the fluorescent label given to Tn. When the incident angle θ of the incident light 41 (excitation light) is not specified by the amount of the reflected light 44, the photodiode 21 may be omitted and replaced with a light absorber or the like.

Although not shown, the measuring unit 40 includes a fluorescence measuring unit, an absorbance measuring unit, and two measuring mechanisms. When measuring the concentration of Tn, the light 42 to be measured is emitted to the measuring unit 40, the amount of light is measured by the fluorescence measuring unit, and when measuring the Hct value, the light 42 to be measured is emitted to the measuring unit 40 to measure the absorbance. The amount of light is measured by the unit.

The fluorescence measuring unit includes a photomultiplier tube as a light receiving element. The photomultiplier tube is arranged on the optical path of the light 42 to be measured when measuring the concentration of Tn, and measures the amount of light of the light 42 to be measured. In this case, the light 42 to be measured is so-called surface plasmon excitation fluorescence (light 42a to be measured) emitted from the fluorescent label given to Tn. The amount of light to be measured 42a is used to specify the concentration of Tn.

The photomultiplier tube may also measure the amount of scattered light 43. In this case, the amount of light of the scattered light 43 is used to specify the incident angle θ of the incident light 41 when measuring the concentration of Tn, and the enhancement angle θr, which is the incident angle when the amount of light of the scattered light 43 is maximized, is It is specified as the incident angle θ of the incident light 41 at the time of measurement. When the incident angle θ of the incident light 41 is specified by the photodiode 21 as described above, it is not necessary to measure the amount of scattered light 43.

The absorbance measuring unit includes a photomultiplier tube as a light receiving element. However, CMOS (complementary metal-oxide-semiconductor), a photodiode, or the like may be used as the light receiving element of the absorbance measuring unit. The photomultiplier tube is arranged on the optical path of the light 42 to be measured when measuring the Hct value, and measures the amount of light of the light 42 to be measured. In this case, the light 42 to be measured is the incident light 41 (light 42b to be measured) that has passed through the sample liquid. The amount of light to be measured 42b is used to identify the Hct value of blood.

When a photomultiplier tube or a photodiode is used as the light receiving element of the absorbance measuring unit, the absorbance measuring unit may be configured to receive light using the photomultiplier tube or the photodiode 21 of the fluorescence measuring unit. .. In either case, it is not necessary to prepare two measuring mechanisms.

The transport unit 80 includes a holder that holds the sensor chip 30 in a detachable manner and a transport stage that can be freely moved. When performing the measurement, the sensor chip 30 is attached to the holder. The transport stage moves the sensor chip 30 mounted on the holder and arranges it at the reaction position A away from the measurement optical path or at the measurement position B located on the measurement optical path (details of the operation of the measuring device will be described later). ..

The liquid feeding unit 90 includes a syringe pump, a driving mechanism for driving the syringe pump, and a moving mechanism for moving a pipette tip that temporarily holds a liquid such as a sample liquid to a predetermined position.

The syringe pump consists of a syringe and a plunger capable of reciprocating in the syringe. A pipette tip is attached to the tip of the syringe. The liquid is quantitatively sucked or discharged by the reciprocating motion of the plunger. The drive mechanism is a means for reciprocating the plunger, and includes, for example, a stepping motor. The moving mechanism is a means for moving the pipette tip attached to the tip of the syringe, and comprises, for example, a robot arm, a biaxial stage, or a turntable that can move up and down.

Further, the liquid feeding unit 90 further has a means for detecting the position of the tip of the pipette tip, thereby relatively adjusting the position of the pipette tip on the sensor tip 30 and keeping the amount of residual liquid in the sensor tip 30 constant. It is preferable from the viewpoint of management.

The control calculation unit 50 is a computer including a CPU, a RAM, a storage device (ROM, a hard disk, a non-volatile semiconductor memory, etc.). The storage device stores a program that can be read and executed by the control calculation unit 50, and the control calculation unit 50 controls the operation of each of the above-described units by executing the program to realize a desired function. ..

FIG. 2 is a block diagram of the control calculation unit 50. The control / calculation function of the control calculation unit 50 will be described with reference to FIG.

As shown in FIG. 2, the control calculation unit 50 includes a CPU 51, a light projection control unit 52, a transfer control unit 53, a liquid feed drive control unit 54, a liquid feed movement control unit 55, and a liquid feed position control unit. 56, a measurement control unit 57, and a measurement calculation unit 58 are provided.

The CPU 51 controls the entire measurement, and operates each control unit or calculation unit described later as necessary. The light projection control unit 52 irradiates a predetermined light source and incidents the light at a predetermined position. The transport control unit 53 moves the transport stage of the transport unit 80 to the reaction position A away from the measurement optical path or the measurement position B located on the measurement optical path. The liquid feed drive control unit 54 moves the plunger of the syringe pump to suck or discharge a predetermined liquid at a predetermined amount and at a predetermined speed. The liquid feed movement control unit 55 moves the pipette tip attached to the tip of the syringe to a predetermined position. The liquid feeding position control unit 56 detects the position of the tip of the pipette tip and fine-tunes it. The measurement control unit 57 responds depending on whether the surface plasmon excitation fluorescence (light 42a to be measured) is measured when measuring the Tn concentration or the absorbance of the sample solution is measured when measuring the Hct value. Switch the measurement mechanism to be used. The measurement calculation unit 58 performs a process of calculating the Tn concentration from the amount of light of surface plasmon excitation fluorescence, a process of calculating the Hct value of blood from the absorbance of the sample solution, a process of correcting other data, and the like.

As described above, the Tn concentration and the Hct value of blood can be determined by a simplified device.

(Sensor chip)
Next, the structure of the sensor chip 30 according to the present embodiment will be described.

FIG. 3 is a cross-sectional view of the sensor chip 30. As shown in FIG. 3, the sensor chip 30 includes a bottom surface member 32, a flow path sheet 33, and a flow path lid 35.

The bottom member 32 is a prism having a trapezoidal bottom surface. However, the shape of the bottom surface member 32 is not limited to this, and can be changed as appropriate. As shown in FIG. 3, the bottom surface member 32 includes a support member 32a and a prism 32b.

The support member 32a is made of an acrylic resin. However, the material of the support member 32a is not limited to this, and may be any material that is transparent to the incident light 41. The prism 32b is made of a dielectric material that is transparent to the incident light 41. A metal thin film 32c is formed on the surface of the prism 32b on the flow path lid 35 side. The surface of the prism 32b corresponding to the region where the metal thin film 32c is formed is referred to as a reflective surface 31a.

The metal thin film 32c is a gold thin film. However, the material of the metal thin film 32c is not limited to this, and may be any metal that causes surface plasmon resonance. For example, it may contain gold, silver, copper, aluminum, or an alloy thereof. The film thickness of the metal thin film 32c is not particularly limited, but is preferably in the range of 30 to 70 nm.

The flow path sheet 33 also functions as an adhesive for joining the bottom surface member 32 and the flow path lid 35. Although not shown, the flow path sheet 33 is formed with a flow path groove, which is an elongated opening for forming the fine flow path 60. The flow path groove has, for example, a width of 0.1 to 5 mm and a length of 10 to 50 mm.

The flow path lid 35 is a structure made of acrylic resin. However, the material of the flow path lid 35 is not limited to this, and may be any material that is transparent to the incident light 41 and the light to be measured 42. As a result, scattering is reduced when the incident light 41 and the light to be measured 42 pass through the flow path lid 35. The light 42 to be measured is surface plasmon-excited fluorescence (light 42a to be measured) when measuring the concentration of Tn, and the incident light 41 (light 42b to be measured) that has passed through the sample solution when measuring the Hct value. ). Further, if a material having good moldability is used as the material of the flow path lid 35, it is possible to secure a desired optical path length of the light to be measured 42b when measuring the Hct value.

The flow path lid 35 is joined to the bottom surface member 32 via the flow path sheet 33. When the flow path lid 35 and the bottom surface member 32 are joined, the flow path sheet 33 is sandwiched between the bottom surface of the flow path lid 35 on the flow path sheet 33 side and the surface of the bottom surface member 32 on the flow path lid 35 side. The flow path groove formed in the flow path sheet 33 becomes the fine flow path 60. As will be described later, the height of the fine flow path 60 varies depending on the structure of the bottom surface of the flow path lid 35 on the flow path sheet 33 side, and is, for example, 0.1 to 6 mm. A troponin trapping membrane 61 that serves as a reaction field with Tn is provided in the microchannel 60. The troponin trapping membrane 61 is formed by immobilizing a troponin trapping antibody that specifically reacts with Tn on a metal thin film 32c. When measuring the concentration of Tn, if a sample solution containing Tn is supplied into the microchannel 60, the sample solution and the troponin capturing membrane 61 come into contact with each other, so that Tn is captured by reacting with the troponin capturing antibody. And stays on the troponin trapping membrane 61.

One end of the microchannel 60 is connected to an interface 36 for inserting a pipette tip, and the other end of the microchannel 60 is to allow the liquid to be agitated as it reciprocates in the flow path. It is connected to the mixing tank 37 of the above. The liquid refers to various reagents, sample liquids, etc. used for measurement, and is, for example, a sample liquid, a measurement liquid, a cleaning liquid, and a fluorescent labeling liquid. The interface sealing seal 36a is attached to the opening of the interface 36 that is not connected to the fine flow path 60, and the mixing tank seal 37a is attached to the opening that is not connected to the fine flow path 60 of the mixing tank 37. .. The mixing tank seal 37a is provided with a ventilation hole 37b for ensuring ventilation.

The interface sealing seal 36a is a film made of polyurethane. However, the material of the interface sealing seal 36a is not limited to this, and when the pipette tip is inserted into the interface 36, the pipette tip and the interface sealing seal 36a are in close contact with each other, and the liquid flows through the interface 36. Anything that can be sealed to the extent that it can be supplied into the road 60 may be used. For example, the material of the interface sealed seal 36a is low density polyethylene (LDPE), linear low density polyethylene (LLDPE), medium density polyethylene (MDPE), nylon, unstretched polypropylene (CPP), ethylene-vinyl alcohol copolymer. (EVOH), silicone, polyvinyl alcohol (PVA), polyvinyl chloride (PVC) and the like may be used.

A notch 38 for increasing the height of the fine flow path 60 is provided on the bottom surface of the flow path lid 35 on the interface 36 side. As a result, the height of the fine flow path 60 becomes larger in the portion where the notch 38 is provided than in the portion where the notch 38 is not provided.

FIG. 4 is a schematic view showing the optical paths of the incident light 41 and the light to be measured 42. As shown in FIG. 4, when measuring the concentration of Tn, the incident light 41 is incident on an incident surface (not shown) of the prism 32b, and is reflected corresponding to the position of the troponin capturing film 61 formed in the fine flow path 60. The light is incident so as to hit the region on the surface 31a, the light 42a to be measured is emitted to the measuring unit 40, and the amount of light is measured by the fluorescence measuring unit. On the other hand, when measuring the Hct value, the incident light 41 is incident from the surface of the support member 32a opposite to the flow path lid 35 side so as to pass through the fine flow path 60 and the notch 38, and is measured. The light 42b is emitted to the measuring unit 40, and the amount of light is measured by the absorbance measuring unit. As a result, the optical path length of the incident light 41 when measuring the Hct value becomes longer by the amount that the height of the fine flow path 60 is increased by the notch 38.

The height of the microchannel 60 of the portion that also serves as the optical path length of the incident light 41 when measuring the Hct value will be described in detail later, but for example, the microchannel 60 of the portion where the notch 38 is not provided. The height of the light is 0.1 to 1 mm, whereas it is preferably more than 0.1 mm and 6 mm or less. However, the location where the notch 38 is provided is not limited to this, and can be appropriately changed depending on the direction of the incident light 41 when measuring the Hct value. Further, as shown in FIG. 3, if the portion of the notch 38 adjacent to the portion of the bottom surface of the flow path lid 35 in which the notch 38 is not provided has a smooth structure having no corners, the sample liquid or the like can be used. When the liquid is recovered from the fine flow path 60, it is possible to prevent the liquid from adhering to the bottom surface of the flow path lid 35 and remaining in the fine flow path 60.

Further, the sensor chip 30 is detachably configured on a reagent chip (not shown) having one or more liquid storage portions. When performing the measurement, the sensor chip 30 is attached to the reagent chip, and each reagent chip is attached to the holder of the transport unit 80. However, the sensor chip 30 does not necessarily have to be integrated with the reagent chip, and may be prepared separately from the reagent chip so that only the sensor chip 30 is mounted on the holder of the transport unit 80. Further, the sensor chip 30 or the reagent chip may have a function of accommodating the pipette tip.

Each liquid container of the reagent chip contains a sample containing a target substance (troponin), a diluting solution for diluting the sample, a sample solution actually used for measurement, and measurement for maintaining the activity of an antibody or the like. The above-mentioned used liquid, a washing liquid for washing impurities, a fluorescent labeling liquid for imparting a fluorescent label to a target substance (troponin), a hemolytic agent for dissolving blood cell components in blood, and the above-mentioned used ones. Each liquid, waste liquid, is stored. The sample is blood collected directly from the examinee. The diluent comprises BSA (bovine serum albumin), Antifoam SI, NaN3, CMD (carboxymethyl-dextran), HAMA (human anti-mouse antibodies) inhibitor, and PBST (phosphate buffered saline with Tween 20). The sample solution is a sample diluted with a diluent. The measuring solution comprises BSA, Antifoam SI, NaN ₃ , Tween 20, and PBS (phosphate buffered saline). The cleaning solution consists of Antifoam SI, NaN ₃ , and PBST. The fluorescent labeling solution consists of PBST, a secondary antibody labeled with CF660.

(Operation of measuring device)
Next, the operation of the measuring device 10 according to the present embodiment will be described. FIG. 6 is a flowchart for explaining the operation of the measuring device 10.

First, a sample solution for measuring the Tn concentration and a sample solution for measuring the Hct value are prepared (step S101). Specifically, the sample is diluted to a desired concentration by using the sample and the diluent stored in advance in the liquid container of the reagent chip.

Next, the inside of the fine flow path 60 is washed (step S102). Specifically, the measuring liquid is supplied into the fine flow path 60 and reciprocates in the fine flow path 60. Since the measurement solution also has a cleaning function, the inside of the microchannel 60 is cleaned, and the troponin trapping membrane 61 is coated with a protective layer for maintaining the trapping ability of the troponin trapping antibody for a long period of time. If so, the protective layer is removed. However, instead of the measuring liquid, a cleaning liquid may be used to clean the inside of the fine flow path 60 described above. Further, when the protective layer described above is not applied to the troponin trapping film 61 and the inside of the fine flow path 60 and the troponin trapping film 61 are clean, it is not always necessary to perform cleaning.

Next, without collecting the measurement liquid, the enhancement measurement is performed by scanning the enhancement angle while the measurement liquid is filled in the fine flow path 60 (step S103). At this time, the sensor chip 30 is arranged at the measurement position B. Specifically, the incident light 41 is scanned while changing the incident angle θ, and the incident light 41 is incident when the increasing angle, which is the incident angle when the amount of scattered light 43 is maximized, is measured and the Tn concentration is measured. Specify as an angle θ. However, as described above, the incident light 41 is scanned while changing the incident angle θ, and the resonance angle, which is the incident angle when the amount of reflected light 44 is minimized, is the incident angle θ of the incident light 41 at the time of measurement. It may be specified as.

Next, without collecting the measurement liquid, a fluorescence blank measurement is performed to measure the fluorescence intensity when Tn does not exist while the measurement liquid is filled in the fine flow path 60 (step S104). Specifically, the incident light 41 is incident so as to hit the region on the reflecting surface 31a of the bottom member 32 corresponding to the position of the troponin capturing film 61 and the incident angle θ is the enhancement angle specified in step S103. Then, the light amount of the light to be measured 42a is measured by the fluorescence measuring unit of the measuring unit 40. After the fluorescence blank measurement is completed, the measurement liquid supplied into the fine flow path 60 is recovered from the fine flow path 60 under the control of the control calculation unit 50, and is transferred to the liquid storage unit that stores the waste liquid of the reagent chip.

Next, a primary reaction for binding Tn to a troponin-capturing antibody is carried out (step S105). Specifically, the sample liquid for measuring the Tn concentration is supplied into the microchannel 60 and reciprocated in the microchannel 60. Since Tn is present in the sample solution for measuring the Tn concentration, when the sample solution is filled in the microchannel 60, Tn comes into contact with the troponin trapping antibody present in the troponin trapping membrane 61 and causes a specific reaction. It is raised and captured and stays on the troponin capture membrane 61. After a sufficient time has elapsed for the reaction, the sample liquid is recovered from the microchannel 60 under the control of the control calculation unit 50 and transferred to the liquid storage unit that stores the waste liquid of the reagent chip.

Next, in order to remove Tn and impurities non-specifically adsorbed in the fine flow path 60, the inside of the fine flow path 60 is washed (step S106). Specifically, the cleaning liquid is supplied into the fine flow path 60 and reciprocates in the fine flow path 60. After the cleaning is completed, the cleaning liquid is collected from the fine flow path 60 under the control of the control calculation unit 50 and transferred to the liquid storage unit that stores the waste liquid of the reagent chip.

Next, a secondary reaction of imparting a fluorescent label to Tn captured by the troponin trapping membrane 61 is carried out (step S107). Specifically, the fluorescent labeling liquid previously stored in the liquid storage portion of the reagent chip is supplied into the microchannel 60 and reciprocated in the microchannel 60. When the microchannel 60 is filled with the fluorescent labeling solution, the fluorescent labeling solution and the troponin trapping film 61 come into contact with each other, and the fluorescent substance for labeling present in the fluorescent labeling solution and Tn captured by the troponin trapping antibody bind to each other. Then, the Tn captured by the troponin-capturing antibody is given a fluorescent label. After a sufficient time has elapsed for the reaction, the fluorescent labeling liquid is recovered from the microchannel 60 under the control of the control calculation unit 50 and transferred to the liquid storage unit that stores the waste liquid of the reagent chip.

Next, in order to remove the fluorescent substance for labeling, impurities, and the like that are non-specifically adsorbed in the fine flow path 60, the inside of the fine flow path 60 is washed (step S108). Specifically, the cleaning liquid is supplied into the fine flow path 60 and reciprocates in the fine flow path 60. After the cleaning is completed, the cleaning liquid is collected from the fine flow path 60 under the control of the control calculation unit 50 and transferred to the liquid storage unit that stores the waste liquid of the reagent chip.

Next, the measurement liquid is supplied into the microchannel 60, and fluorescence signal measurement is performed to measure the fluorescence signal emitted from Tn captured by the troponin trapping membrane 61 (step S109). Specifically, the incident light 41 hits the region on the reflecting surface 31a of the bottom member 32 corresponding to the position of the troponin capturing film 61, and the incident angle θ is the enhancement angle specified in step S103. Make it incident. The incident light 41 is reflected by the reflecting surface 31a of the bottom surface member 32, but when reflected, an evanescent wave is generated from the reflecting surface 31a to the metal thin film 32c side and permeates. The electric field of the permeated evanescent wave resonates with the surface plasmon of the metal thin film 32c and is enhanced to excite the fluorescent label attached to Tn captured by the troponin trapping film 61. When the fluorescent label is excited, it emits surface plasmon excitation fluorescence (light to be measured 42a). The control calculation unit 50 causes the fluorescence measurement unit of the measurement unit 40 to measure the amount of light 42a to be measured, and specifies the concentration of Tn based on the measured amount of light 42a to be measured.

After the fluorescence signal measurement is completed, the absorbance blank measurement is performed to measure the amount of incident light 41 passing through the optical path when the sample solution is not present while the measurement solution is filled in the fine flow path 60 without collecting the measurement solution. (Step S110). By performing this absorbance blank measurement, it is possible to measure only the change in absorbance derived from Hct. Specifically, the incident light 41 passes through the fine flow path 60 and the notch 38 from the surface of the support member 32a opposite to the flow path lid 35 side, and the incident angle θ is 0 degrees. The light amount of the incident light 41 that has passed through the measuring liquid existing on the optical path is measured by the absorbance measuring unit of the measuring unit 40. However, the incident angle in this case is not particularly limited as long as it is less than the critical angle at the interface between the support member 32a and the fine flow path 60. After the absorbance blank measurement is completed, the measurement liquid supplied into the fine flow path 60 is recovered from the fine flow path 60 under the control of the control calculation unit 50, and is transferred to the liquid storage unit that stores the waste liquid of the reagent chip.

Next, a hemolytic agent is added to the sample solution for measuring the Hct value, which is prepared in advance and stored in the liquid container of the reagent chip, and mixed (step S111). By adding the hemolytic agent to the sample solution, the blood cell component in the blood is dissolved and the concentration distribution becomes more uniform, so that the noise in the absorbance measurement is reduced.

Next, a sample solution for measuring the Hct value to which a hemolytic agent is added is supplied into the microchannel 60, and the absorbance signal is measured to measure the absorbance of the sample solution (step S112). Specifically, the incident light 41 passes through the fine flow path 60 and the notch 38 from the surface of the support member 32a opposite to the flow path lid 35 side, and the incident angle θ is 0 degrees. To make it incident. However, the incident angle in this case is not particularly limited as long as it is less than the critical angle at the interface between the support member 32a and the fine flow path 60. The incident light 41 passes through the sample liquid existing on the optical path and is emitted to the measuring unit 40. The control calculation unit 50 causes the absorbance measuring unit of the measuring unit 40 to measure the light amount of the light (measured light 42b) emitted through the sample solution, and the Hct value of blood is based on the measured light amount of the measured light 42b. To identify. After the measurement of the absorbance signal is completed, the sample liquid supplied into the fine flow path 60 is collected from the fine flow path 60 under the control of the control calculation unit 50, and is transferred to the liquid storage unit that stores the waste liquid of the reagent chip. The sensor chip 30 is discarded.

Next, the control calculation unit 50 determines the accurate value of the Tn concentration based on the measurement results of the fluorescence blank measurement and the absorbance blank measurement, and the Tn concentration and the Hct value obtained by the fluorescence signal measurement and the absorbance signal measurement. The post-processing (step S113) for calculation is performed, and the measurement is completed.

Further, in the present embodiment, by measuring the Tn concentration before measuring the Hct value, Tn reacts with the troponin-capturing antibody at the time of measuring the Hct value, and the measurement accuracy of the Tn concentration measurement performed thereafter is performed. Can be prevented from being lowered. However, the measuring method according to the present invention is not limited to this order, and the Hct value may be measured before measuring the Tn concentration as long as it can prevent the Tn from reacting with the troponin-capturing antibody.

As described above, since the optical path length of the incident light 41 when measuring the Hct value is increased by providing the notch 38, the Tn concentration is measured in the same flow path and the Hct value of blood is measured accurately. It is possible to accurately measure the Tn concentration by a simple method such as fluorescence light amount measurement and absorbance measurement.

(Relationship between the optical path length of the incident light and the sample solution concentration when measuring the Hct value)
As described below, the height of the fine flow path 60 at the portion where the notch 38 is provided and which is also the optical path length of the incident light 41 when measuring the Hct value is the sample liquid for measuring the Hct value. The absorbance resolution can be adjusted to be optimal according to the concentration.

As described above, the Hct value is specified based on the absorbance of the sample solution. When measuring the Hct value, the amount of light of the light to be measured 42b is actually measured, and the ratio (I ₁ / I ₀ ) of the intensity of the light to be measured 42b to the intensity of the incident light 41 is calculated by the above-mentioned equation 1 to obtain a sample. The absorbance A of the liquid is calculated. _{The ratio (I 1} / I ₀ ) of the intensity of the light to be measured 42b and the intensity of the incident light 41 is the transmittance T of the sample liquid. Therefore, if the transmittance T of the sample liquid can be measured with high accuracy, the accuracy of the obtained absorbance A will be improved, and the Hct value can be measured with high accuracy.

FIG. 6 is a graph showing the correlation between the measurable range of transmittance (Tmax-Tmin) and the optical path length of the incident light 41.

The measurable range of transmittance (Tmax-Tmin) is obtained by diluting a plurality of samples having a known Hct value distributed over a normal range of the Hct value of human blood at a predetermined dilution ratio. When a plurality of sample solutions are measured with different optical path lengths, it means the difference between the maximum transmittance Tmax and the minimum transmittance Tmin obtained by measuring with the same optical path length. The measurable range (Tmax-Tmin) of the individual transmittances obtained by measuring a plurality of sample solutions obtained by diluting at the same dilution ratio with different optical path lengths forms the curve of the same dilution ratio in FIG.

If the measurable range (Tmax-Tmin) of the transmittance is large, the accuracy of the absorbance A obtained by the measurement and the accuracy of the Hct value are improved, and the Hct value can be measured accurately without modifying the existing equipment. Is possible. That is, the measurable range of transmittance (Tmax-Tmin) is one of the indexes reflecting the resolution of the absorbance A obtained by the measurement.

Table 1 shows the maximum value (maximum optical path length) and the minimum value (minimum optical path length) of the optical path length when the measurable range (Tmax-Tmin) of the transmittance is 15% or more at each dilution ratio. It represents the respective product C · l (minimum product C · lmin and maximum product C · lmax) of them and the concentration C (reciprocal of the dilution ratio) of the sample solution. According to the above-mentioned equation 1, the absorbance A of the sample solution can be expressed by the product of the molar extinction coefficient ε of the sample solution, the concentration C of the sample solution, and the optical path length l. The absorbance A of the sample solution is proportional to the product C · l of the concentration C of the sample solution and the optical path length l. Therefore, at the same dilution ratio, the minimum product C. lmin and the maximum product C. lmax are conditions in which the absorbance A can be measured under the condition that the measurable range (Tmax-Tmin) of the transmittance is 15% or more. , The lower limit and the upper limit of the product C · l, which is an index including the two factors of the sample liquid concentration C and the optical path length l, are shown, respectively. That is, if the optical path length l is set so that the product C · l is equal to or more than the minimum product C · lmin and equal to or less than the maximum product C · lmax at the same dilution ratio, the measurable range of transmittance (Tmax-Tmin) is 15. The absorbance A of the sample solution can be measured so as to be% or more, and the Hct value can be measured with high accuracy.

Further, as can be seen from Table 1, the intersection of the product C ・ l range in which the measurable range (Tmax-Tmin) at each dilution ratio is 15% or more is the maximum value in each minimum product C ・ lmin. It is defined by 26 and 156, which is the minimum value in each maximum product C · lmax. If the optical path length l is set within the range of this common portion, that is, the product C · l is 26 or more and 156 or less, the sample is diluted at a dilution ratio of 1.5 times or more and 24 times or less. Even if the prepared sample solution is used as the sample solution, the absorbance A of the sample solution can be measured so that the measurable range (Tmax-Tmin) of the transmittance is 15% or more, and the Hct value can be measured accurately. It becomes possible to do. However, the specific numerical values of each product C and l are not strictly limited, and the same effect can be obtained even if there is some error.

On the other hand, as can be seen from FIG. 6, the measurable range (Tmax-Tmin) increases as the optical path length l increases, but under each dilution ratio, the measurable range (Tmax-Tmin) becomes maximum, and thus the absorbance A. There is an optimum optical path length for which the resolution of is optimal. Table 2 shows the optimum optical path lengths when the dilution ratios are 1.5 times, 3 times, and 6 times. At these dilution ratios, the optical path length l of the incident light 41 is set to the optimum optical path lengths shown in Table 2 (specifically, 175 μm when the dilution ratio is 1.5 times, and 175 μm when the dilution ratio is 3 times). If it is 350 μm and 700 μm when the dilution ratio is 6 times), the absorbance A can be measured with the maximum resolution obtained in that environment, and the accuracy of the Hct value can be ensured. However, the specific numerical value of the optimum optical path length is not strictly limited, and the same effect can be obtained even if there is some error.

10 Measuring device 20 Floodlight 30 Sensor chip 40 Measuring unit 50 Control calculation unit 80 Transport unit 90 Liquid transfer unit 32 Bottom member 32a Support member 32b Prism 32c Metal thin film 33 Flow path sheet 35 Flow path lid 38 Notch 60 Fine flow path 61 Troponin capture film 41 Incident light 42 Measured light 43 Scattered light 44 Reflected light

Claims (21)

The first flow path for blood flow and
A second flow path located upstream or downstream of the first flow path and having a wider flow path than the first flow path,
With a mixing tank connected to the first flow path or one end of the second flow path,
Have a,
A sensor chip for a blood test, wherein the first flow path and the second flow path are continuous. The first flow path for blood flow and
A second flow path located upstream or downstream of the first flow path and having a notch so that the height of the flow path is higher than that of the first flow path.
A sensor chip for a blood test characterized by having. The first measurement is performed on the blood in the first flow path, and the first information about the blood is acquired.
The sensor chip for a blood test according to claim 1 or 2, wherein the second measurement is performed on the blood in the second flow path and the second information about the blood is acquired. The first information is the concentration of the target substance present in the blood.
The sensor chip for a blood test according to claim 3, wherein the second information is a hematocrit value of the blood. The sensor chip for a blood test according to any one of claims 1 to 4, wherein the first flow path and the second flow path have the same flat surface. The sensor chip for a blood test according to any one of claims 1 to 5, wherein the sensor chip is made of a material that transmits light having a wavelength of 200 to 900 nm. The sensor chip for a blood test according to claim 6, which is made of an acrylic resin. The invention according to any one of claims 1 to 7, wherein the width of the first flow path is 0.1 to 1 mm, and the width of the second flow path is more than 0.1 mm and 6 mm or less. Sensor chip for blood tests. The invention according to any one of claims 1 to 8, wherein a reaction system that reacts with the target substance existing in the blood and retains the target substance is provided on the bottom surface of the first flow path. Sensor chip for blood tests. A blood test sensor chip having a first flow path for flowing blood and a second flow path located upstream or downstream of the first flow path and having a wider flow path than the first flow path. When,
A light projecting unit that irradiates the blood test sensor chip with light,
A measuring unit that measures the amount of light emitted from the blood test sensor chip, and
A control unit that controls the operation of the light projecting unit and
Have,
The first flow path and the second flow path are continuous,
The control unit is a blood test device characterized in that the light projecting unit is irradiated with the light so that the light passes through the width of the second flow path. The blood test apparatus according to claim 10, wherein the control unit further controls the operation of the measurement unit. The control unit is characterized in that the operation of the light projecting unit is switched so that the light passes through the width of the second flow path or the light hits the first flow path. Item 10. The blood test apparatus according to Item 10. The measuring unit measures the amount of light that has passed through the width of the second flow path, and measures the amount of light.
The blood test according to any one of claims 10 to 12, wherein the control unit measures the hematocrit value of the blood based on the amount of light passing through the width of the second flow path. Device. 10. The blood test sensor chip is characterized in that a reaction system that reacts with a target substance existing in the blood and retains the target substance is provided on the bottom surface of the first flow path. 13. The blood test apparatus according to any one of 13. The blood test apparatus according to claim 14, wherein the control unit measures the concentration of the target substance based on the reaction result of the reaction system. A blood test sensor chip having a first flow path for flowing blood and a second flow path located upstream or downstream of the first flow path and having a wider flow path than the first flow path. Using,
A sample solution containing the target substance present in the blood is supplied to the first flow path and the second flow path.
A blood test method characterized by quantitatively measuring the target substance using the first flow path and measuring the hematocrit value of the blood using the second flow path. A claim characterized in that the hematocrit value of the blood is measured by irradiating the sensor chip for a blood test with light so as to pass through the width of the second flow path and measuring the absorbance of the sample solution. Item 16. The blood test method according to item 16. The claim is characterized in that a reaction system that reacts with the target substance and retains the target substance is provided on the bottom surface of the first flow path, and the concentration of the target substance is determined based on the reaction result of the reaction system. The blood test method according to 16 or 17. The sample solution was obtained by diluting blood collected directly from the examinee.
The width of the second flow path and the concentration of the blood in the sample solution have a relationship represented by the following formula (1) when the concentration of the blood in the sample solution is 0.0417 or more and 0.6667 or less. The blood test method according to any one of claims 16 to 18, characterized in that there is.
26 <C ・ l <156 (1)
In the above formula (1), C is the concentration of the blood in the sample solution, that is, the inverse of the dilution ratio of the blood for obtaining the sample solution, and l is the width of the second flow path. The unit is μm. When the dilution ratio of the blood for obtaining the sample solution is 1.5 times, that is, the concentration of the blood in the sample solution is 0.6667, the width of the second flow path is set to 175 μm.
When the dilution ratio of the blood for obtaining the sample solution is 3 times, that is, the concentration of the blood in the sample solution is 0.3333, the width of the second flow path is set to 350 μm.
When the dilution ratio of the blood for obtaining the sample solution is 6 times, that is, the concentration of the blood in the sample solution is 0.1667, the width of the second flow path is 700 μm. Item 19. The blood test method according to item 19. The blood test method according to any one of claims 16 to 20, wherein the target substance is quantitatively measured before measuring the hematocrit value of the blood.

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