JP2018054495A - Sensor chip for blood test, blood testing apparatus, and blood testing method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a sensor chip for blood tests capable of performing, in same flow passages, a quantitative measurement of a target substance and highly accurate measurement of a hematocrit value of the blood, and a blood testing apparatus and blood testing method using the sensor chip.SOLUTION: A sensor chip for blood tests according to the present invention includes: a first flow passage for passing blood; and a second flow passage located on an upstream or downstream side of the first flow passage and having the wider-width flow passage than that of the first flow passage. The blood testing apparatus according to the present invention includes: a sensor chip for blood tests; a light projector for applying light to the sensor chip for blood tests; and a measuring section for measuring a light quantity of light exited from the sensor chip for blood tests. Further, the blood testing method according to the present invention supplies test liquid including a target substance that is present in the blood to the first flow passage and the second flow passage using the sensor chip for blood tests, performs quantitative measurement of the target substance using the first flow passage, and measurement of a hematocrit value of the blood using the second flow passage.SELECTED DRAWING: Figure 3

Description

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

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

  As a means for acquiring information on a specific substance (hereinafter referred to as a target substance) present in blood, there is a measurement method using a sensor chip having a fine channel. A reaction system that reacts with the target substance is provided in the microchannel. 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 corresponding to the concentration of the target substance can be acquired.

  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 is greatly affected by the hematocrit value of the blood. The hematocrit value is a numerical value indicating the proportion of the volume of red blood cells in 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. A measurement method for correcting the concentration of a specific component in blood is provided.

Japanese Patent No. 4785611

  When measuring a hematocrit value by an optical method such as absorbance in a measurement method using a sensor chip having a fine flow path, 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 absorption 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 be as close as possible to the region where reaction is possible, and small so as to eliminate useless 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 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 flow path height is increased in order to increase the optical path length, the absorbance of the sample liquid increases according to Equation 1, but the measurement light is affected by absorption, scattering, and the like due to contaminants in the sample liquid. Since it becomes easy to receive, it is still not possible to accurately measure the hematocrit value.

  In particular, when a rare substance in the blood such as troponin is used as a target substance in the first place, even if the effect of the measurement accuracy described above appears remarkably and 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.

  Furthermore, since the invention as disclosed in Patent Document 1 requires a plurality of light sources having different wavelengths, there is a problem that the configuration of the measurement method or the measurement apparatus itself must be complicated.

  The sensor chip for blood test of the present invention includes a first flow path for flowing blood, and a second flow that is located upstream or downstream of the first flow path and has a wider flow path than the first flow path. And a road.

  The blood test apparatus of the present invention includes a first flow path for flowing blood, a second flow path positioned upstream or downstream of the first flow path and having a wider width than the first flow path. A blood test sensor chip, a light projecting unit for irradiating the blood test sensor chip with light, and a measurement unit for measuring 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 positioned upstream or downstream of the first flow path and having a wider width than the first flow path. A sample liquid containing a target substance present in the blood is supplied to the first flow path and the second flow path, and the first flow path is used to 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 perform quantitative measurement of a target substance in the same flow path and to accurately measure the hematocrit value of blood. Further, according to the present invention, it is possible to perform quantitative measurement of a target substance and measurement of blood hematocrit value by a simplified apparatus or method.

It is a schematic diagram which shows the structure of a measuring device. It is a block diagram of a control calculating part. It is sectional drawing of a sensor chip. It is a schematic diagram which shows the optical path of incident light and each to-be-measured light. It is a flowchart for demonstrating operation | movement of a measuring device. It is a graph which shows the correlation with the measurable range of the transmittance | permeability, and the optical path length of incident light.

  Hereinafter, an embodiment for carrying out the present invention will be described with reference to the drawings. In this embodiment, as a method for measuring the concentration of the target substance, the target substance is captured using an antigen-antibody reaction, a fluorescent label is applied to the captured target substance, and the fluorescence intensity emitted from the fluorescent label is measured. The surface plasmon resonance excitation enhanced fluorescence spectroscopy (SPFS) method is employed, but 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 for measuring the concentration of the target substance.

(Measurement device)
First, the configuration of the measurement apparatus 10 according to the present embodiment will be described.

  FIG. 1 is a schematic diagram illustrating the configuration of the measurement apparatus 10. The measuring device 10 uses troponin (Tn) present in blood as a target substance, measures its concentration by the SPFS method, and also measures the hematocrit (Hct) value of blood by the absorbance of the sample solution. However, other than troponin, for example, cystatin, D-dimer (D-dimer), BNP (B-type natriuretic peptide), CRP (C-reactive protein), etc. may be measured as the target substance. The sample solution refers to blood collected directly from the examinee, or a solution obtained by diluting it 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 measurement apparatus 10 main body.

  The light projecting unit 20 includes a light source (not shown), optical elements (not shown) for constructing an optical path for guiding 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. And a photodiode 21 for receiving the reflected light 44 emitted from the reflective surface 31a of the light.

  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 a laser diode, and any light source that can generate light having a wavelength within the above-described range may be used. For example, an LED capable of generating light having a wavelength of 200 to 900 nm may be used. When the light source can generate light having a wavelength of 500 to 700 nm, the light emitted from the light source includes excitation light for measuring the concentration of Tn and measurement light for measuring the Hct value of blood. Can be used as both. As a result, it is possible to measure the concentration of Tn and the Hct value of blood with a single light source, and the measurement apparatus can be simplified. However, two light sources capable of generating different wavelengths may be provided, and the light source to be incident may be selected depending on the type of measurement.

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

  The photodiode 21 is disposed on the optical path of the reflected light 44 and measures the amount of the reflected light 44. When measuring the concentration of Tn, the resonance angle that is the incident angle when the amount of the 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 attached 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 measurement unit 40 includes a fluorescence measurement unit, an absorbance measurement unit, and two measurement mechanisms. When measuring the concentration of Tn, the light to be measured 42 is emitted to the measurement unit 40, the light quantity is measured by the fluorescence measurement unit, and when measuring the Hct value, the light to be measured 42 is emitted to the measurement unit 40 and the absorbance is measured. 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 under measurement 42 when measuring the concentration of Tn, and measures the light quantity of the light under measurement 42. In this case, the measured light 42 is so-called surface plasmon excitation fluorescence (measured light 42a) emitted from a fluorescent label imparted to Tn. The amount of light 42a to be measured is used for specifying the density of Tn.

  The photomultiplier tube may also measure the amount of scattered light 43. In this case, the light quantity 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 light quantity of the scattered light 43 is maximized, 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 the scattered light 43.

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

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

  The conveyance unit 80 includes a holder that detachably holds the sensor chip 30 and a conveyance stage that is freely movable. When performing measurement, the sensor chip 30 is mounted on 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 measurement apparatus will be described later). .

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

  The syringe pump includes a syringe and a plunger that can reciprocate within 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 includes, for example, a robot arm, a two-axis stage, or a turntable that can move up and down.

  In addition, the liquid feeding unit 90 further includes means for detecting the position of the tip of the pipette tip, relatively adjusts the position of the pipette tip in the sensor tip 30, and the amount of remaining liquid in the sensor tip 30 is constant. It is preferable from the viewpoint of management.

  The control calculation unit 50 is a computer including a CPU, a RAM, and a storage device (ROM, hard disk, nonvolatile semiconductor memory, etc.). In the storage device, a readable / executable program is stored in the control arithmetic unit 50, and the control arithmetic unit 50 executes the program to control the operation of each unit described above to realize a desired function. .

  FIG. 2 is a block diagram of the control calculation unit 50. The control / calculation function of the control computation 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 transport 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.

  The CPU 51 controls the entire measurement and activates each control unit or calculation unit described later as necessary. The light projection control unit 52 irradiates a predetermined light source and makes it enter a predetermined position. The conveyance control unit 53 moves the conveyance stage of the conveyance unit 80 to a reaction position A that is away from the measurement optical path or a measurement position B that is located on the measurement optical path. The liquid feed drive control unit 54 moves the plunger of the syringe pump, and sucks or discharges a predetermined liquid at a predetermined amount at a predetermined speed. The liquid feeding 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 tip position of the pipette tip and finely adjusts it. The measurement control unit 57 responds depending on whether the amount of surface plasmon excitation fluorescence (measurement light 42a) is measured when measuring the concentration of Tn or the absorbance of the sample solution is measured when measuring the Hct value. Switch the measurement mechanism. The measurement calculation unit 58 performs processing for calculating the concentration of Tn from the amount of surface plasmon excitation fluorescence, processing for calculating the Hct value of blood from the absorbance of the sample solution, correction processing for other data, and the like.

  As described above, the concentration of Tn and the Hct value of blood can be performed with 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 column 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 member 32 includes a support member 32a and a prism 32b.

  The support member 32a is made of acrylic resin. However, the material of the support member 32 a is not limited to this, and any material that is transparent to the incident light 41 may be used. The prism 32 b is made of a dielectric that is transparent to the incident light 41. A metal thin film 32c is formed on the surface of the prism 32b on the channel 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, gold, silver, copper, aluminum, or an alloy thereof may be included. The thickness of the metal thin film 32c is not particularly limited, but is preferably in the range of 30 to 70 nm.

  The channel sheet 33 also serves as an adhesive that joins the bottom member 32 and the channel lid 35. Although not shown, the channel sheet 33 is formed with a channel groove that is an elongated opening for forming the microchannel 60. The channel groove has, for example, a width of 0.1 to 5 mm and a length of 10 to 50 mm.

  The channel lid 35 is a structure made of acrylic resin. However, the material of the channel lid 35 is not limited to this, and any material that is transparent to the incident light 41 and the measured light 42 may be used. Thereby, scattering is reduced when the incident light 41 and the measured light 42 pass through the flow path lid 35. The measurement light 42 is surface plasmon excitation fluorescence (measurement light 42a) when measuring the concentration of Tn, and incident light 41 (measurement light 42b) that has passed through the sample solution when measuring the Hct value. ). If a material having good moldability is used as the material of the flow path lid 35, a desired optical path length of the light to be measured 42b when measuring the Hct value can be ensured.

  The channel lid 35 is joined to the bottom surface member 32 via the channel sheet 33. When the channel lid 35 and the bottom surface member 32 are joined, the channel sheet 33 is sandwiched between the bottom surface of the channel lid 35 on the channel sheet 33 side and the surface of the bottom surface member 32 on the channel lid 35 side. The flow channel grooves formed in the flow channel sheet 33 become the fine flow channel 60. As will be described later, the height of the fine channel 60 varies depending on the structure of the bottom surface of the channel lid 35 on the channel sheet 33 side, and is, for example, 0.1 to 6 mm. A troponin trapping film 61 serving as a reaction field with Tn is provided in the fine channel 60. The troponin capture film 61 is formed by fixing a troponin capture antibody that reacts specifically with Tn to the 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 capture membrane 61 come into contact with each other, so that Tn reacts with the troponin capture antibody and is captured. And remains on the troponin trapping film 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 used to agitate the liquid when reciprocating the liquid in the channel. The mixing tank 37 is connected. The liquid refers to various reagents and sample liquids used for measurement, for example, sample liquid, measurement liquid, washing liquid, and fluorescent labeling liquid. An interface hermetic seal 36a is affixed to the opening of the interface 36 that is not connected to the fine flow path 60, and a mixing tank seal 37a is affixed to the opening of the mixing tank 37 that is not connected to the fine flow path 60. . The mixing tank seal 37a is provided with a vent hole 37b for ensuring air permeability.

  The interface hermetic seal 36a is a film made of polyurethane. However, the material of the interface hermetic seal 36a is not limited to this, and when the pipette tip is inserted into the interface 36, the pipette tip and the interface hermetic seal 36a are in close contact with each other, and the interface 36 is allowed to finely flow liquid. What is necessary is just to be able to seal to the extent which can be supplied in the path | route 60. FIG. For example, the material of the interface hermetic 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 channel 60 is provided on the bottom surface of the channel lid 35 on the interface 36 side. Thereby, in the location where the notch 38 is provided, the height of the fine channel 60 becomes larger than the location where the notch 38 is not provided.

  FIG. 4 is a schematic diagram showing optical paths of the incident light 41 and the measured light 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 32 b and reflected corresponding to the position of the troponin trapping film 61 formed in the microchannel 60. The light to be measured 42a is incident so as to hit the region on the surface 31a, 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 opposite to the channel lid 35 side of the support member 32a so as to pass through the fine channel 60 and the notch 38, and is measured. The light 42b is emitted to the measurement unit 40, and the amount of light is measured by the absorbance measurement unit. As a result, the optical path length of the incident light 41 when measuring the Hct value is increased by the amount by which the height of the fine channel 60 is increased by the notch 38.

  The height of the portion of the fine channel 60 that also serves as the optical path length of the incident light 41 when measuring the Hct value will be described in detail later. For example, the height of the fine channel 60 in the portion where the notch 38 is not provided. The height is preferably 0.1 to 1 mm, but 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 the Hct value is measured. Further, as shown in FIG. 3, the portion of the notch 38 adjacent to the bottom portion of the channel lid 35 where the notch 38 is not provided has a smooth structure with no corners. When the liquid is recovered from the fine channel 60, the liquid can be prevented from adhering to the bottom surface of the channel lid 35 and remaining in the fine channel 60.

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

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

(Operation of measuring device)
Next, the operation of the measurement apparatus 10 according to this embodiment will be described. FIG. 6 is a flowchart for explaining the operation of the measurement apparatus 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 storage portion of the reagent chip.

  Next, the inside of the fine channel 60 is washed (step S102). Specifically, the measurement liquid is supplied into the fine channel 60 and reciprocated in the fine channel 60. Since the measurement liquid also serves as a cleaning function, the inside of the fine channel 60 is thereby cleaned, and a protective layer is applied to the troponin capturing film 61 for maintaining the capturing ability of the troponin capturing antibody for a long period of time. The protective layer is removed. However, the above-described microchannel 60 may be cleaned using a cleaning liquid instead of the measurement liquid. Moreover, when the protective layer mentioned above is not apply | coated to the troponin capture film | membrane 61, and the inside of the microchannel 60 and the troponin capture film | membrane 61 are clean, it is not necessary to wash | clean.

  Next, enhancement measurement is performed by scanning the enhancement angle while the measurement liquid is filled in the fine channel 60 without collecting the measurement liquid (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 angle of the incident light 41 when measuring the concentration of Tn is used as the enhancement angle that is the incident angle when the amount of the scattered light 43 is maximized. It is specified as the 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 the reflected light 44 is minimized, is the incident angle θ of the incident light 41 at the time of measurement. You may specify as.

  Next, without recovering the measurement liquid, fluorescent blank measurement is performed to measure the fluorescence intensity when Tn is not present while the measurement liquid is filled in the fine channel 60 (step S104). Specifically, the incident light 41 is incident so that it corresponds to the region on the reflection surface 31a of the bottom surface member 32 corresponding to the position of the troponin capturing film 61 and the incident angle θ becomes the enhancement angle specified in step S103. Then, the amount of light to be measured 42a is measured by the fluorescence measurement unit of the measurement unit 40. After the measurement of the fluorescent blank, 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 transferred to the liquid storage part that stores the waste liquid of the reagent chip.

  Next, a primary reaction for binding Tn and the troponin capture antibody is performed (step S105). Specifically, a sample solution for measuring the Tn concentration is supplied into the fine channel 60 and reciprocated in the fine channel 60. Since Tn is present in the sample solution for measuring the Tn concentration, when the sample solution is filled in the fine channel 60, Tn comes into contact with the troponin capture antibody present in the troponin capture film 61 and performs a specific reaction. It is raised and trapped, and remains on the troponin trapping film 61. After a sufficient time has elapsed for the reaction, the sample liquid is recovered from the fine channel 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 nonspecifically adsorbed Tn, impurities, etc. in the fine channel 60, the inside of the fine channel 60 is washed (step S106). Specifically, the cleaning liquid is supplied into the fine channel 60 and reciprocated in the fine channel 60. After completion of the cleaning, the cleaning liquid is collected from the fine channel 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 for applying a fluorescent label to Tn captured by the troponin capturing film 61 is performed (step S107). Specifically, the fluorescent labeling liquid previously stored in the liquid storage part of the reagent chip is supplied into the microchannel 60 and reciprocated in the microchannel 60. When the fluorescent labeling liquid is filled in the fine flow path 60, the fluorescent labeling liquid and the troponin capturing film 61 come into contact with each other, and the fluorescent substance for labeling present in the fluorescent labeling liquid and Tn captured by the troponin capturing antibody are combined. Then, a fluorescent label is imparted to Tn captured by the troponin capture antibody. After a sufficient time has elapsed for the reaction, the fluorescent labeling liquid is recovered from the fine channel 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 inside of the fine channel 60 is washed in order to remove the labeling fluorescent substance and impurities adsorbed nonspecifically in the fine channel 60 (step S108). Specifically, the cleaning liquid is supplied into the fine channel 60 and reciprocated in the fine channel 60. After completion of the cleaning, the cleaning liquid is collected from the fine channel 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 measurement liquid is supplied into the fine channel 60, and fluorescence signal measurement is performed to measure a fluorescence signal emitted from Tn captured by the troponin capture film 61 (step S109). Specifically, the incident light 41 strikes a region on the reflecting surface 31a of the bottom surface member 32 corresponding to the position of the troponin capturing film 61, and the incident angle θ becomes the enhancement angle specified in step S103. Make it incident. Incident incident light 41 is reflected by the reflecting surface 31a of the bottom member 32, but when reflected, an evanescent wave is generated and penetrates from the reflecting surface 31a to the metal thin film 32c side. The electric field of the penetrated evanescent wave resonates with the surface plasmon of the metal thin film 32c, and excites the fluorescent label attached to Tn captured by the troponin capturing film 61. When excited, the fluorescent label emits surface plasmon excitation fluorescence (measurement light 42a). The control calculation unit 50 causes the fluorescence measurement unit of the measurement unit 40 to measure the light amount of the measured light 42a, and specifies the concentration of Tn based on the measured light amount of the measured light 42a.

  After completion of the fluorescence signal measurement, an absorbance blank measurement is performed to measure the amount of incident light 41 that has passed through the optical path when the sample liquid is not present, without collecting the measurement liquid, without collecting the measurement liquid. (Step S110). By performing this absorbance blank measurement, it is possible to measure only the absorbance change derived from Hct. Specifically, the incident light 41 passes through the fine channel 60 and the notch 38 from the surface opposite to the channel lid 35 side of the support member 32a, and the incident angle θ becomes 0 degree. Then, the absorbance measurement unit of the measurement unit 40 measures the light amount of the incident light 41 that has passed through the measurement liquid existing on the optical path. 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 channel 60. After completion of the absorbance blank measurement, the measurement liquid supplied into the microchannel 60 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, a hemolytic agent is added to and mixed with the sample liquid for Hct value measurement prepared in advance and stored in the liquid storage part of the reagent chip (step S111). By adding the hemolytic agent to the sample solution, blood cell components in the blood are dissolved and the concentration distribution becomes more uniform, so that noise in absorbance measurement is reduced.

  Next, the sample solution for measuring the Hct value to which the hemolytic agent is added is supplied into the fine channel 60, and absorbance signal measurement is performed to measure the absorbance of the sample solution (step S112). Specifically, the incident light 41 passes through the fine channel 60 and the notch 38 from the surface opposite to the channel lid 35 side of the support member 32a, and the incident angle θ becomes 0 degree. So that it is 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 channel 60. The incident incident light 41 passes through the sample liquid existing on the optical path and exits to the measuring unit 40. The control calculation unit 50 causes the absorbance measurement unit of the measurement unit 40 to measure the amount of light that has passed through and emitted from the sample solution (measurement light 42b), and based on the measured light amount of the measurement light 42b, the Hct value of blood Is identified. After completion of the absorbance signal measurement, the sample liquid supplied into the microchannel 60 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. The sensor chip 30 is discarded.

  Next, the control calculation unit 50 calculates the accurate value of the Tn concentration based on the measurement result of the fluorescence blank measurement and the absorbance blank measurement, and the Tn concentration and Hct value obtained by the fluorescence signal measurement and the absorbance signal measurement. Post-processing to calculate (step S113) is performed, and the measurement is terminated.

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

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

(Relationship between optical path length of incident light and sample solution concentration when measuring Hct value)
A notch 38 is provided, and the height of the fine channel 60 in the portion that also serves as the optical path length of the incident light 41 when measuring the Hct value is the height of the sample liquid for measuring the Hct value, as will be described below. Depending on the concentration, the absorbance resolution can be adjusted to be optimal.

As described above, the Hct value is specified based on the absorbance of the sample solution. When measuring the Hct value, the light quantity of the measured light 42b is actually measured, and the ratio (I ₁ / I ₀ ) between the measured light 42b intensity and the incident light 41 intensity is calculated according to the above-described equation 1. The absorbance A of the liquid is calculated. Note that the ratio (I ₁ / I ₀ ) between the measured light 42 b intensity and the incident light 41 intensity is the transmittance T of the sample liquid. Therefore, if the transmittance T of the sample solution can be measured with high accuracy, the accuracy of the absorbance A obtained can be improved, and the Hct value can be measured with high accuracy.

  FIG. 6 is a graph showing the correlation between the transmittance measurable range (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 specimens having known Hct values distributed over the normal range of Hct values of human blood at a predetermined dilution ratio. When a plurality of sample liquids 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 individual transmittances obtained by measuring a plurality of sample solutions obtained by diluting at the same dilution rate with different optical path lengths forms a curve of the same dilution rate in FIG.

  If the transmittance measurable range (Tmax-Tmin) is large, the accuracy of the absorbance A obtained by measurement, and hence the accuracy of the Hct value, will improve, and the Hct value will be measured accurately without modifying existing equipment. Is possible. That is, the transmittance measurable range (Tmax−Tmin) is one of the indexes reflecting the resolution of the absorbance A obtained by 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 of transmittance (Tmax-Tmin) is 15% or more at each dilution factor. It represents the product C · l (minimum product C · lmin and maximum product C · lmax) of each of them and the concentration C (reciprocal of the dilution factor) of the sample solution. According to 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 the conditions under which the absorbance A can be measured under conditions where the transmittance measurable range (Tmax−Tmin) is 15% or more. The lower limit and the upper limit of the product C · l, which are indices including two factors, the sample solution concentration C and the optical path length l, are shown. That is, if the optical path length l is set so that the product C · l is not less than the minimum product C · lmin and not more than the maximum product C · lmax at the same dilution factor, the transmittance measurable range (Tmax−Tmin) is 15 The absorbance A of the sample solution can be measured so as to be at least%, and the Hct value can be accurately measured.

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

  On the other hand, as can be seen from FIG. 6, the measurable range (Tmax−Tmin) increases as the optical path length l increases. However, the measurable range (Tmax−Tmin) becomes maximum at each dilution factor, and thus the absorbance A There is an optimum optical path length that optimizes the resolution of each. Table 2 shows the optimum optical path length when the dilution factor is 1.5 times, 3 times, and 6 times. At these dilution magnifications, the optical path length l of the incident light 41 is set to each optimum optical path length shown in Table 2 (specifically, when the dilution magnification is 1.5 times, 175 μm, and when the dilution magnification is 3 times) If it is 350 μm and the dilution factor is 6 times, 700 μm), 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.

DESCRIPTION OF SYMBOLS 10 Measuring apparatus 20 Light projection part 30 Sensor chip 40 Measurement part 50 Control calculating part 80 Conveyance part 90 Liquid feeding part 32 Bottom member 32a Support member 32b Prism 32c Metal thin film 33 Channel sheet 35 Channel cover 38 Notch 60 Fine channel 61 Troponin capture film 41 Incident light 42 Light to be measured 43 Scattered light 44 Reflected light

Claims (20)

A first flow path for flowing blood;
A second channel that is located upstream or downstream of the first channel and has a wider channel than the first channel;
A blood test sensor chip comprising: Performing a first measurement on the blood in the first flow path to obtain first information about the blood;
2. The blood test sensor chip according to claim 1, wherein a second measurement is performed on the blood in the second flow path to acquire second information related to the blood. The first information is a concentration of a target substance present in the blood,
The blood test sensor chip according to claim 2, wherein the second information is a hematocrit value of the blood.   The blood test sensor chip according to any one of claims 1 to 3, wherein the first channel and the second channel have the same bottom surface.   The sensor chip for blood test according to any one of claims 1 to 4, wherein the sensor chip is made of a material that transmits light having a wavelength of 200 to 900 nm.   6. The blood test sensor chip according to claim 5, comprising an acrylic resin.   7. 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. 7. Blood test sensor chip.   8. The reaction system according to claim 1, wherein a reaction system that reacts with a target substance existing in the blood and retains the target substance is provided on a bottom surface of the first flow path. 9. Blood test sensor chip. A blood test sensor chip comprising: 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 for irradiating light to the blood test sensor chip;
A measuring unit for measuring the amount of light emitted from the blood test sensor chip;
A blood test apparatus characterized by comprising: A control unit for controlling operations of the light projecting unit and the measuring unit;
The blood test apparatus according to claim 9, wherein the control unit causes the light projecting unit to irradiate the light so that the light passes through a width of the second flow path.   The said control part switches the operation | movement of the said light projection part so that the said light may pass through the width | variety of the said 2nd flow path, or the said light hits the said 1st flow path. Item 11. The blood test apparatus according to Item 10. The measurement unit measures the amount of the light that has passed through the width of the second flow path,
The blood test according to any one of claims 9 to 11, wherein the control unit measures a hematocrit value of the blood based on a light amount of the light that has passed through a width of the second flow path. apparatus.   10. The blood test sensor chip is provided with a reaction system for reacting with a target substance present in the blood and retaining the target substance on a bottom surface of the first flow path. The blood test apparatus according to any one of 12 above.   The blood test apparatus according to claim 13, wherein the control unit performs concentration of the target substance based on a reaction result of the reaction system. A blood test sensor chip comprising: 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,
Supplying a sample liquid containing a target substance present in the blood to the first flow path and the second flow path;
A blood test method, wherein the target substance is quantitatively measured using the first flow path, and the hematocrit value of the blood is measured using the second flow path.   The hematocrit value of the blood is measured by irradiating the blood test sensor chip 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 15.   The concentration of the target substance is performed based on a reaction result of the reaction system by providing a reaction system that reacts with the target substance and retains the target substance on the bottom surface of the first flow path. The blood test method according to 15 or 16. The sample solution is obtained by diluting blood collected directly from the examinee,
The width of the second flow path and the blood concentration in the sample liquid have the relationship shown in the following formula (1) when the blood concentration in the sample liquid is 0.0417 or more and 0.6667 or less. The blood test method according to claim 15, wherein the blood test method is provided.
26 <C · l <156 (1)
In the above formula (1), C is the concentration of the blood in the sample liquid, that is, the reciprocal of the dilution ratio of the blood for obtaining the sample liquid, 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, when the concentration of the blood in the sample solution is 0.6667, the width of the second channel is set to 175 μm,
When the dilution ratio of the blood for obtaining the sample solution is 3 times, that is, when the concentration of the blood in the sample solution is 0.3333, the width of the second flow path is 350 μm,
When the dilution rate of the blood for obtaining the sample solution is 6 times, that is, when the concentration of the blood in the sample solution is 0.1667, the width of the second flow path is set to 700 μm. Item 19. The blood test method according to Item 18.   The blood test method according to any one of claims 15 to 19, wherein the target substance is quantitatively measured before measuring the hematocrit value of the blood.

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